Ursolic Acid-Based Oleolite for Hyperpigmentary Disorders: Molecular Mechanisms and Clinical Efficacy

Ursolic Acid-Based Oleolite for Hyperpigmentary Disorders: Molecular Mechanisms and Clinical Efficacy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ursolic Acid-Based Oleolite for Hyperpigmentary Disorders: Molecular Mechanisms and Clinical Efficacy Maria Maisto, Vincenzo Piccolo, Adua Marzocchi, Lucia Ricci, Benedetta Romano, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7028178/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Skin-lightening molecules have gained widespread attention for their relevance in both cosmetic applications and the treatment of dermatological disorders. Hyperpigmentation and skin spots can be counteracted by suppressing oxidative stress, inhibiting tyrosinase activity, or regulating the expression of tyrosinase-related proteins (TYRP-1 and TYRP-2) and the microphthalmia-associated transcription factor (MITF). Among natural compounds, ursolic acid (UA), a triterpene primarily found in apple peel was investigated for its potential depigmenting properties. In this study, UA was extracted in oil form from Annurca apple (AAO) to formulate a standardized oleolite (784.40 ± 7.58 mg/mL) and evaluate its efficacy through in vitro and clinical models. AAO exhibited tyrosinase inhibitory activity and significantly reduced melanin content in A375 melanoma cells, accompanied by decreased expression of TYRP-1, TYRP-2, and MITF, as well as modulation of oxidative stress markers. These effects were confirmed in a randomized, double-blind, placebo-controlled clinical trial, in which a topical formulation containing 2.5% AAO significantly improved skin hyperpigmentation compared to placebo. Overall, the results highlight AAO as a promising natural agent for managing skin hyperpigmentation through multiple mechanisms, suggesting its potential utility in both cosmetic and dermatological settings. Biological sciences/Biochemistry Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Health care Health sciences/Medical research ursolic acid hyperpigmentation melanogenesis skin wellness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Melanin is the most important biological pigment in humans; it is responsible for the pigmentation of skin, eyes, and hair 1 . It is produced by melanocytes in specific sub-cellular lysosome-like structures called melanosomes. Physiologically, melanin synthesis and production protect human skin from UV radiation; however, its abnormal synthesis could lead to several skin disorders, including hyperpigmentation, melasma, and age spots. Specifically, melanogenesis is a multi-step process involving melanin synthesis and transport, and the subsequent release of melanosomes. Chemically, the melanogenesis process begins with the oxidation of tyrosine to dopaquinone, which is catalyzed by the enzyme tyrosinase. Subsequently, the dopaquinone is converted into dopachrome by an auto-oxidation reaction 2 . The L-Dopa produced is itself a tyrosinase substrate and is oxidized again to dopaquinone, which undergoes further oxidative polymerization steps leading to the melanin formation 3 , 4 . Several molecular players are involved in regulating the melanogenesis process 1 . Specifically, UV-irradiation induces the synthesis of alpha-melanocyte-stimulating hormone (α-MSH) in melanocytes, which activates the melanocortin 1 receptor, leading to increased expression of microphthalmia-associated transcription factor (MITF) through activation of the cyclic adenosine monophosphate pathway. The MITF produced is a key protein that controls the synthesis of several melanogenesis-regulating protein factors, including tyrosinase and tyrosinase-related proteins 1 and 2 (TYRP-1 and TYRP-2) 5 , 6 . Due to their pivotal role in melanin production, tyrosinase, TYRP-1, and TYRP-2 are generally recognized as the most important molecular targets for active agents with skin whitening and depigmenting potentials. Regarding oxidative balance, melanocytes are characterized by higher levels of reactive oxygen species (ROS) than other cell types, such as fibroblasts 7 . This difference could be related to their higher rate of melanin production, which releases high levels of ROS as a collateral product, leading to an alteration in the redox potential of melanocytes. In this regard, several skin pigmentation disorders such as vitiligo have been reported to be positively related to an alteration of cellular defense enzymes with antioxidant activity, including Nrf2-related enzymes such as the catalytic and modulatory subunits of γ-glutamylcysteine ligase (GCLC and GCLM, respectively) as well as the enzyme heme oxygenase (HMOX-1) 8 – 10 . Given the multifactorial nature of skin pigmentation disorders, there is great interest in identifying compounds with double potential: i) antioxidant, and ii) ability to modulate melanogenesis at different molecular levels. In this regard, increasing attention has been paid to the identification of natural and food-derived compounds as effective inhibitors of tyrosinase 11 . Among natural lipophilic compounds, ursolic acid (UA), a triterpene acid, has shown valuable in vitro tyrosinase inhibitory activity (IC 50 : 170.2 µM) 12 . This molecule reached a valuable concentration in several medical plant species 13 and especially in apple peel (14.3 mg/g DW) 13 . Interestingly, a comparative study conducted on both commercial and traditional Italian apple cultivars showed that Annurca apple (AA) was richer in UA (9.09 mg/g DW) and total terpenic content (14.19 mg/g DW) than other examined varieties [11]. Specifically, AA is the only apple cultivar native to southern Italy that has been registered by the European Council as a product with a Protected Geographical Indication (PGI) [Commission Regulation (EC) No.417/2006]. While the nutraceutical potential of the polyphenolic fraction of AA has been widely documented for its beneficial effects in the management of plasma cholesterol levels in humans 15 – 19 and on the regulation of glycemia 20 , the potential health-promoting application of AA triterpenoid fraction is less well explored. In light of these considerations, in our recent study, we demonstrated that Annurca apple oil-based formulation (AAO) with standardized UA concentration (736 µg/mL) exerts valuable skin antiaging activity and favourable skin permeation features 21 , 22 . Given these considerations, the main objectives of the present manuscript were to investigate the potential application of AAO as an active ingredient for the preparation of novel topical depigmenting formulations. Firstly, the in vitro inhibitory effect of AAO on tyrosinase was investigated. Next, we investigated the ability of AAO to reduce melanin production and transcriptionally modulate the expression of melanogenesis-regulating protein factors in human melanoma cell lines, as well as to modulate oxidative stress responses. Based on these promising in vitro results, AAO was tested in a randomized, double-blind, placebo-controlled, two parallel-arm clinical trials, conducted on 40 subjects with hyperpigmentation and diagnosed photo-damage, in order to investigate its effects on the main clinical skin pigmentation-related parameters, i.e., UV and brown spots, melanin index, and skin lightening markers (Individual Typology Angle, ITA° and luminescence, L*). 2. Results 2.1. Tyrosinase Inhibition Assay The tyrosinase enzyme plays a pivotal role in human skin pigmentation, thus its inhibition represents a potential treatment for hyperpigmentation-related disorders. To establish AAO as a potential whitening ingredient, the in vitro inhibition of tyrosinase activity was evaluated. As reported in Figure 1 the AAO has shown a valuable capacity to inhibit tyrosinase activity in a concentration-dependent manner, with an IC 50 of 286.42 mg/mL. To confirm that UA was the main molecule responsible for tyrosinase inhibition, the pure UA was tested, resulting in IC 50 of 26.82 µg/mL (Figure 2B), which is approximately ten times more active than the AAO, as expected. Additionally, as a positive control we used kojic acid (KA), a fungal metabolite, with strong chelating properties for copper at the active site of the tyrosinase enzyme 23 . As expected, the KA IC 50 (0.59 µg/mL) is the lowest calculated value in the present study (Figure 2A), and it was in line with the tyrosinase inhibitory activity calculated by others, who reported KA IC 50 values ranged from 1-10 µg/mL 24 . 2.2 AAO did not affect the proliferation of human melanoma cells To better characterize the anti-melanogenic effects of AAO, we decided to use a human melanoma cell line known to display an altered melanogenesis process. First, we evaluated whether AAO exerts a cytotoxic effect by using A375 human melanoma cells. Cells were treated with increased concentrations of AAO (0, 0.01, 0.1, 1, 10, and 100 µg/mL) for 72 h, and cell viability was evaluated by performing an MTT assay. As shown in Figure 3A, we found that the concentration of 100 µg/mL impaired A375 cell viability. Conversely, the pure UA reduced the proliferation of A375 melanoma cells already at the concentration of 10 µg/mL (Figure 3B). Thus, we selected 30 µg/mL and 3 µg/mL as the concentrations for the next experiments for AAO and UA, respectively. 2.3 AAO reduced melanin production and the expression of melanogenesis-related genes Next, we examined the effect of AAO and UA on melanin production in A375 human melanoma cells stimulated with α-MSH, known to trigger the melanin synthesis processes. As expected, the MSH treatment significantly increased the melanin production compared to A375 control cells. Importantly, as shown in Figure 4A, we observed that both AAO (30 µg/mL) and UA (3 µg/mL) significantly reduced melanin production in MSH-stimulated A375 cells after 72 h of treatment. Moreover, in line with the findings described above, AAO reduced the mRNA expression levels of tyrosinase enzyme (TYR) and its related proteins (TYRP-1 and TYRP-2) (Figure 4B) in MSH-stimulated A375 human melanoma cells. We also found that AAO suppressed the mRNA expression level of MITF, the most important transcription factor involved in melanogenesis and melanoma progression (Figure 4C). Taken together, these data demonstrated that AAO reduced melanin production by modulating the expression levels of the key players involved in melanogenesis. 2.4 AAO modulation of ROS production Melanogenesis and oxidative stress are intertwined in a feedback loop: while melanin production generates free radicals, the reactive species can further stimulate melanogenesis 25 . Therefore, we investigated the ability of AAO to modulate oxidative stress in A375 human melanoma cells by evaluating the production of ROS and the expression of the key antioxidant enzymes. First, we analyzed ROS production by using the 2',7'-dichlorofluorescin (DCF) probe and inducing oxidative damage with Fenton’s reagent in order to increase ROS production. As shown in Figures 5A and B, the preincubation with both AAO and UA significantly reduced ROS production, suggesting their potential antioxidant protective role. To corroborate these data, we analyzed the expression of three important antioxidant enzymes: GCLC, GCLM, and HMOX-1 in AAO- and UA-treated A375 cells and stimulated with Fenton’s reagent. Our results showed that treatment with AAO significantly increased the expression of all the enzymes tested, whereas UA induced an up-regulation of the HMOX-1 enzyme (Figure 5C). In addition, we also evaluated the contribution of oxidative damage to the expression of the melanogenesis-related proteins MITF and TYR. As expected, the expression of both MITF and TYR was increased in oxidative stress conditions compared to A375 control cells. Interestingly, the preincubation with AAO (30 mg/mL) or UA (3 mg/mL) drastically reduced their expression, confirming their modulation also under strong oxidative unbalance (Figure 5D). Finally, to address putative crosstalk between melanogenesis and oxidative stress, we also investigated ROS production in MSH-stimulated A375 cells. As shown in Figures 5E and F, MSH increased ROS production while both AAO and UA reduced the production of ROS. Collectively, these results demonstrated that both AAO and UA exert antioxidant effects by reducing ROS production and modulating the expression of antioxidant enzymes. Moreover, both the antioxidant effects and the modulation of melanogenesis of AAO could be interconnected, given the potential crosstalk between these processes. 2.5. Clinical data 2.5.1. Demographic data During the recruitment process, out of 48 participants enrolled, 6 subjects dropped out (3 were lost to follow-up and 3 subjects underwent aesthetic procedures), leaving a total of 42 females. The demographic characteristics of the participants are shown in Table 1. The majority of participants were 45-55 years old (30 subjects, 71.4%). Overall, 66.7% of the participants had a Fitzpatrick skin type II, and 33.3% had a Fitzpatrick skin type III. 2.5.2. Effects of AAO-based topical formulation on pigmentation-related skin parameters The key parameters used to evaluate AAO’s efficacy on photo-damage were the Individual Typology Angle (ITA°), L* luminance, and the melanin index (MI). ITA° classifies skin pigmentation based on colorimetry 26 , and is calculated using the formula: Where L* represents luminance (ranging from 0 for black to 100 for white) and b* ranges from yellow (-b*) to blue (+b*). Higher ITA° values indicate lighter skin and greater lightening from treatment. The average percentage changes in ITA° and L* at baseline (D0), day 14 (D14), and day 28 (D28) are shown in Figure 6A and 6B, while MI changes are depicted in Figure 6C. Results showed no significant differences between groups at baseline. However, L*, ITA°, and MI significantly improved in the AAO group during and after treatment ( p < 0.05). These improvements were supported by VISIA 7 th analysis, demonstrating AAO’s whitening and brightening effects. AAO also significantly reduced UV and brown spots compared to baseline and placebo by day 14 (Figures 7 and 8). Neither treatment caused irritation or desquamation. 3. Discussion Skin-lightening molecules have found a large distribution worldwide for their relevant application for both cosmetic and clinical disorders. Their use is widely diffused in cosmetic practice to improve skin appearance, but also in medical therapy for the treatment of hyperpigmented skin disorders such as melasma, café-au-lait spots, and solar lentigo 27 . The formation of skin spots has been mainly associated with an over-activation of the melanin synthesis, making the regulation of this biosynthetic pathway a major target of lightening agents. In particular, melanogenesis was controlled by several molecular modulators such as MITF, TYR, TYRP-1, and TYRP-2. To identify a novel and natural approach for the management of hyperpigmentation disorders, in our previously published work, we have optimized the formulation of UA oil extract from annurca apple (AAO) as a functional ingredient for topical formulation 22 . Specifically, we have reported that the AAO prepared with a UA concentration of 736 mg/mL, has shown a valuable in vitro antioxidant potential (evaluated using DPPH, ABTS, and FRAP assays, resulting in the antiradical potential of 16.63 ± 0.22, 5.90 ± 0.49, and 21.72 ± 0.68 µmol Trolox equivalent/g, respectively) and favorable in vitro skin permeation parameters. Particularly, after 4 h of AAO application, UA reached a maximum contraction in the dermal layer, without reaching the bloodstream compartment. This last feature was highly required for the preparation of topical formulation with local activity 28 . Thus, in order to investigate the potential depigmenting potential of the formulated AAO, we evaluated its inhibitory potential on tyrosinase activity using an enzymatic assay. Our results show that the formulated AAO was able to inhibit the tyrosinase activity in vitro with a calculated IC 50 of 286.42 mg/mL. This promising result could be mainly due to its relevant UA content, which has shown to be a noticeable tyrosinase inhibitor agent, with a calculated IC 50 of 26.8 mg/mL (0.026 mM), a value in perfect agreement with results previously described by others 29 . Once established the inhibitory activity of AAO in an in vitro enzymatic system, we moved to investigate the effects of AAO on melanin production in A375 human melanoma cell line. The results obtained showed that AAO (30 mg/mL), compared to UA (3 mg/mL), was able to cause a significant reduction ( p < 0.0001 vs CTR) in melanin production in A375 melanoma cells stimulated with α-MSH. A similar trend in terms of melanin content has already been observed in the murine melanoma cells B16F10 after treatment with UA (3-10 mg/mL), resulting in efficient inhibition of melanin production 30 . To further investigate the effective molecular mechanism of action involved in the regulation of melanin synthesis observed, the effects of AAO on TYR, TYRP-1, TYRP-2, and MITF mRNA transcription levels were investigated. Our results indicate that AAO treatment resulted in a significant reduction in mRNA expression levels of all the melanogenic modulators studied. Furthermore, these results were also observed in the Fenton-stressed cell model, in which the induced oxidative stress was able to increase the expression of TYR and MITF at mRNA expression levels. These data corroborate the AAO’s ability to downregulate the expression of melanogenic modulators in both basal and stressed conditions in the A375 human melanoma cell line. Interestingly, the implication of UA or UA-containing extracts in the management of these melanogenic factors has not yet been described. Therefore, this work represents the first attempt to establish the effects of UA and UA-based products on the control of the melanogenesis process. Indeed, among all the triterpenic acids, only betulinic acid has been described to exert an anti-melanogenic activity by inhibiting TYR, TYRP-1, and TYRP-2 mRNA expression through the inactivation of cAMP response element binding protein (CREB) and MITF in isobutylmethylxanthine-stimulated B16F10 cells 31 . Given the complex and bivalent relationship between melanogenesis and cellular oxidative stress, in which increased melanin synthesis could generate high levels of reactive oxygen species in melanosomes and a subsequent increase in oxidative stress could further stimulate melanogenesis, coupled with the fact that melanin itself has played a central role in modulating oxidative homeostasis at the skin level 29,30 , the ability of AAO to modulate oxidative cell system was evaluated. Specifically, oxidative stress is defined as an imbalance between the production of reactive oxygen and nitrogen species (ROS/RNS) and the physiological human antioxidant defenses at the cellular level. It has been positively correlated with the development of age-related disorders, including pathological conditions and aesthetic alterations, such as skin wrinkles and spots. Since the in vitro antioxidant activity of AAO was already demonstrated in our previous work 21 , its ability to contrast ROS production through stimulation of cell defense enzymes, i.e. enzymes of the glutathione biosynthesis pathway, including GCLC, GCLM, and HMOX-1, was further investigated. Our results show that AAO was able to induce a significant ( p < 0.05) increase in mRNA expression of GCLC and especially of HMOX-1 (3-fold increased vs CTR, p < 0.001) in A375 melanoma cells. The observed beneficial effect of AAO on HMOX-1 mRNA expression could likely be related to its valuable UA content, whose regulatory activity in transcriptional upregulation of HMOX-1 mRNA expression was previously reported in JB6 P+ murine cells 31 . In addition, while no relevant modulatory effect on the expression of GCLC or GCLM has been attributed to UA, the observed upregulation of these enzymes could be related to a synergic activity of the polyphenolic AAO content 32,33 . Moreover, to corroborate the AAO antioxidant activity at the cellular level, the ability of AAO and UA to modulate mitochondrial activity, given their key role as important sources of ROS, was further investigated. Mitotracker Green and TMRM are cationic probes that allow the measurement of mitochondrial mass and membrane potential since they are sequestered in the matrix space of mitochondria and released once mitochondria experience change in the membrane 32,33 .Our findings demonstrated that AAO and UA reduced mitochondrial ROS generation by modulating mitochondrial mass and mitochondrial membrane potential as demonstrated for other natural compounds such as resveratrol and quercetin 34 . Encouraged by the achieved promising in vitro results, a double-blind, placebo-controlled, 2-parallel-arm instrumental clinical trial was performed on 40 subjects with hyperpigmentation and diagnosed photo-damage, in order to investigate AAO-based topical formulation effects on the main clinical skin pigmentation-related parameters, i.e., UV spots, brown spots, and melanin index. Clinically, both types of skin spots are characterized by an increased production and accumulation of melanin in the skin, but they differ in the origin of their formation. While brown spots, also known as age spots or liver spots, are usually caused by a combination of several factors such as aging, genetics, or other causes, including hormonal changes, skin lesions such as hyperpigmentation, freckles, lentigines, and melasma. In contrast, UV spots are mainly caused by damage from the sun's UV exposure. Our results indicate that, after 28 days of treatment, a significative reduction of spot score for both UV (-6.4% vs baseline; p < 0.001) and brown spots (-4.1% vs baseline; p < 0.001) was registered, accompanied by a valuable reduction of their skin melanin index (-10.2 vs baseline; p < 0.001). Similar results were reported in a patent, describing that phosphorylated triterpenic acids and/or their salts formulated in a topical preparation used for cosmetics and dermatological purposes have shown a relevant anti-inflammatory action and melanin production downregulation in the skin. Additionally, our results are consistent with those reported by the few pieces of evidence related to the beneficial effects of UA-based topical formulations. These studies describe the effectiveness of UA in protecting against ultraviolet absorbers and highlight its potential to counteract skin pigmentation induced by UV irradiation 35,36 . Finally, an improvement in the color complexion and skin brightness (ITA°: +12.41% vs baseline; p < 0.001, and L*: +3.1% vs baseline p < 0.001) was also registered. 4. Conclusion The triterpenic fraction of AAO, primarily composed of ursolic acid (UA), demonstrated significant inhibition of tyrosinase activity in an in vitro model. In A375 melanoma cells, AAO down-regulated key melanogenesis modulators (TYR, TYRP-1, TYRP-2, MITF) at the transcriptional level, both in α-MSH and Fenton-stimulated models. Given the link between hyperpigmentation and oxidative stress, AAO's antioxidant potential was also explored. Results showed AAO up-regulated antioxidant enzyme expression while reducing MITF and TYR levels under oxidative stress. These findings were supported by the clinical trial, further confirming AAO's potential as a functional ingredient in depigmenting topical formulations. 5. Materials and Methods 5.1. Reagents All chemicals, reagents, and standards used were analytical or LC-MS grade reagents. The water was treated in a Milli-Q water purification system (Millipore, Bedford, Burlington, MA, USA) before use. Sunflower oil was purchased in a local market. Ursolic acid (purity ≥ 98.5% HPLC), phloridzin (purity ≥ 99% HPLC), kojic acid (purity ≥ 98.5% HPLC), L-tyrosine (purity ≥ 98% HPLC), tyrosinase from mushroom (activity ≥ 1000 U/mg), human melanoma cells lines A375, MTT, TRI-Reagent, Nanodrop were purchased from Sigma-Aldrich (Milan, Italy). DMEM medium, heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptomycin, HEPES buffer were purchased from Gibco (New York, NY, USA). The iScript Reverse Transcription Supermix for RT-qPCR was purchased from Bio-Rad (Milan, Italy). H 2 DCFDHA, MitoTracker Green and TMRM were purchased from Thermo Fisher Waltham (MA, USA). 5.2. Oleolite preparation Annurca apple fruits (Malus pumila Miller cv Annurca) (about 100 g each) were collected in October 2023, Valle di Maddaloni (Caserta, Italy), when the fruits had just been harvested (green peel). The fruits were reddened for about 30 days following the typical treatment 37 . After this time, the apples were washed and sliced for freeze-drying. The oleolite preparation was performed according to the conditions optimized in our previously published article 22 to achieve the maximum UA concentration of 784.40 ± 7.58 mg/mL. Specifically, the ratio of freeze-dried AA to sunflower oil was 1:4, and the mixture was stirred at 68.85 ◦C for 63 h. Then, the AAO was subjected to a specific extraction protocol for the isolation of its triterpenic fraction, according to our published protocols. The extraction was performed with ethyl acetate. The fractions obtained were first dried using a rotary evaporator, when the solvents used were completely removed, the residues obtained were suspended in water, frozen at -80°, and freeze-dried (for 24 h, -69 °C at 0.096 mbar). The resulting powder was ground (IKA A11 analytical mill) to obtain a homogeneous powder representative of the triterpenic AAO composition 22 . 5.3. Tyrosinase inhibition assay The tyrosinase inhibition assay was performed according to the modified dopachrome method 38 . Practically, the inhibition of the enzyme was assessed by monitoring the enzymatic metabolization of L-tyrosine substrate according to the HPLC method described. According to the used protocol, 70 µL of different concentrations of the samples were added to a 96-well microplate. Then, 100 µL of 1 mM L-tyrosine and 30 µL of mushroom tyrosinase (500 U/mL), both of them prepared in 50 mM sodium phosphate buffer pH 6.5, were added, mixed well, and incubated at room temperature for 30 min. After this incubation time, the enzymatic reaction was stopped by the addition to each sample of 100 µL of methanol. The analyzed standards and the extracts were dissolved in 100 % v/v DMSO and diluted with 50 mM sodium phosphate buffer pH 6.5 to obtain a final concentration of 1% v/v DMSO in the enzymatic mixture. Kojic acid was used as a positive control, while the control was performed by replacing the sample with an equal volume of buffer. In the HPLC assay, enzymatic activity was evaluated as a decrease in the chromatographic L-tyrosine peak area for the tyrosinase reaction of the enzyme. The L-tyrosine peak area was normalized to the 1 mM L-tyrosine peak area. The inhibitory effect was expressed as the concentration required to inhibit L-tyrosine metabolism by 50% (IC 50 ). 5.3.1 HPLC-FLD method for L-tyrosine analysis A Jasco Extrema LC-4000 HPLC system (Jasco Inc., Easton, MD, USA), coupled with an autosampler, a binary solvent pump, and a fluorescence detector (FLD), was used for the analysis. The chromatographic analysis was performed according to 39 , with slight modifications. Elution was performed on a Luna HILIC column (150 mm x 3.0, 5 µm; Phenomenex, Torrance, CA, USA). The mobile phases were an ammonium acetate buffer, 5 mM, containing 0.1% formic acid (A) and acetonitrile (B). The elution gradient was performed under the following conditions: 0-5 min, isocratic on 90% phase B; 5-17 min, linear gradient from 90 to 70% B; 17-27 min, isocratic with 90% B; 24-29 min, isocratic with 60% B for column reconditioning. The separation parameters were as follows: column temperature was set at 35 °C, injection volume was 20 µl, and flow rate was set at 1 mL/min. The quantification of tyrosine was performed with an excitation wavelength of 272 nm and an emission wavelength of 312 nm. 5.4. Cell culture The human melanoma cells lines A375 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 10 mM HEPES buffer and grown at 37°C in a humidified incubator under 5% CO 2 . 5.5. MTT assay A375 cells (10 3 cells/well) were plated on a 96-well plate and treated with AAO and UA (0.01 to 100 μg/mL) for 72 hr. Next, 25 μL of MTT was added and incubated for 3 hr at 37°C. Thereafter, the dark blue crystals developed, were solubilized with DMSO and the absorbance was measured at 545 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific, MA, USA). 5.6. Measurement of Melanin Content A375 cells (3x10 5 /well) were plated in a 6-well plate and treated with AAO (30 µg/mL) and UA (3 µg/mL). After 72 hours, cells were lysed in NaOH 1N and incubated for 90 min at 37°C, then centrifuged for 10 min at 10,000 rpm. The optical density (OD) of supernatant was measured at 450 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). 5.7. RNA extraction and qPCR analysis A375 melanoma cells were treated with AAO and UA (30 µg/mL and 3 µg/mL, respectively) for 6 hours. Thereafter, total RNA was extracted by using TRI-Reagent, according to the manufacturer's instructions. After quantification with Nanodrop, 1 µg of total RNA was reverse-transcribed by using iScript Reverse Transcription Supermix for RT-qPCR. qPCR analysis was performed with the Bio-Rad CFX384 real-time PCR detection system (Bio-Rad, Milan, Italy) by using the following primers: TYR (Gene ID: 7299) 5’- GCACAGATGAGTACATGGGAGG -3’; 5’- CTGATGGCTGTTGTACTCCTCC -3’ TYRP-1 (Gene ID: 7306) 5’- TCTCAATGGCGAGTGGTCTGTG-3’; 5’- CCTGTGGTTCAGGAAGAC-GTTG-3’ TYRP-2 (Gene ID: 1638) 5’- CTCAGACCAACTTGGCTACAGC-3’; 5’- CAACCAAAGCCACCAG-TGTTCC-3’ MITF (Gene ID: 4286) 5’- GGCTTGATGGATCCTGCTTTGC-3’; 5’- GAAGGTTGGCTGGACAG-GAGTT-3’ GCLC (Gene ID: 2729) 5’-GTTGGGGTTTGTCCTCTCCC-3’; 5’-GGGGTGACGAGGTGGAGTA-3’ GCLM (Gene ID: 2730) 5’-AGGAGCTTCGGGACTGTATCC-3’; 5’-GGGACATGGTGCATTCCAAAA-3’ HMOX-1(Gene ID: 3162) 5’-GCCGTGTAGATATGGTACAAGGA-3’; 5’-AAGCCGAGAATGCTGAGTTCA-3’ The 2ΔDCt formula was used for the analysis and ribosomal protein S16 (RPS16) was used as a housekeeping gene. 5.8. Flow cytometry analysis A375 were treated for 48 hours with AAO and UA (30 µg/mL and 3 µg/mL, respectively). Thereafter, ROS production was evaluated by using the H 2 DCFDHA probe. Likewise, mitochondrial mass and membrane potential were evaluated by using Mito-Tracker Green and TMRM probes as previously described. Samples were acquired on a BriCyte E6 flow cytometer (Mindray Medical Italy S.r.l., Milan, Italy), and data were analyzed with FlowJo software (TreeStar V.10; Carrboro, NC, USA). 5.9. Clinical trial The study was a 4-week, monocentric, randomized, double-blinded, placebo-controlled clinical trial assessing the safety and efficacy of an AAO-based topical formulation versus placebo in adults with moderate-to-severe hyperpigmentation. Conducted from February 2022 to December 2022 at the RD Cosmetics Lab, University of Naples Federico II, it adhered to UNI EN ISO 9001 standards for efficacy studies. The trial included a 15-day screening period, a 7-day washout, and a 4-week treatment phase. The study protocol (Unique Protocol ID: EAAO22G01) was revised by the Institutional Review Board of the Pharmacy Department and recorded on clinicaltrials.gov (ID: NCT07040345). The study was carried out according to ICH GCP, the Declaration of Helsinki and the SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation (12th revision). The informed consent was obtained from all participants, and the study followed CONSORT guidelines. The study flow is depicted in Figure 9. 5.9.1. Study population and randomization Eligible participants were women aged 40 to 65 with diagnosed photodamage and hyperpigmentation. A target of 40 participants was set based on power calculations, with 48 participants recruited to account for a 20% loss to follow-up. Full eligibility criteria are detailed in the Supporting Information. Exclusion criteria included the use of treatments for hyperpigmentation or topical retinoids and allergies to the formulation ingredients. Participants were enrolled at their second laboratory visit, sequentially numbered, and randomized in blocks of 20 at a 1:1 ratio to receive either the active treatment (w/o emulsion with 2.5% w/w AAO) or a placebo. Both treatments were identical in appearance and packaging. Blinding was maintained for both participants and observers. 5.9.2. Treatment Participants applied 2 fingertip units of the allocated test product, whose composition is reported in Table 2. In particular, Group A received the active treatment with 2.5% AAO, whereas Group B received the placebo. The lab’s technicians instructed to apply the assigned product twice daily, at least 12 hours apart, for 28 days. Training on product application was provided, with the first application supervised. Baseline measurements, including independent instrumental analysis and photographs, were conducted at the initial visit. Participants were reviewed at days 14 and 28, with pigmentation changes assessed using the melanin index (Mexameter MX 18) and skin luminance measured through L* and ITA° values (Skin-Colorimeter CL 400). Brown and UV spots resulting from photodamage were analyzed using VISIA 7th technology with RBX analysis 40 . Skin acceptability was previously tested with an occlusive patch test. 5.10. Statistical Analysis GraphPad Prism software version 9 (San Diego, CA, USA) was used for statistical analysis. t-test and ANOVA tests were used for comparison of two groups or multiple groups, respectively. The data were shown as mean percentage variation (%) ± SEM. A p-value < 0.05 was considered statistically significant and was labelled with *; p-values < 0.01, 0.001, or 0.0001 were labelled with **, ***, or ****, respectively. Abbreviations AA: Annurca apple AAO: Annurca apple oil CREB: cAMP response element binding protein DCF: 2',7'-dichlorofluorescin DMEM: Dulbecco’s modified Eagle’s medium FLD: fluorescence detector GCLC: γ-glutamylcysteine ligase HMOX-1: enzyme heme oxygenase ITA: Individual Typology Angle KA: kojic acid MI: melanin index MITF: microphthalmia-associated transcription factor OD: optical density PGI: Protected Geographical Indication ROS: reactive oxygen species TYR: tyrosinase TYRP: tyrosinase-related protein UA: ursolic acid α-MSH: alpha-melanocyte-stimulating hormone Declarations Author Contributions: Conceptualization: Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), and Ritamaria Di Lorenzo (R.D.L.); Data Curation: Ritamaria Di Lorenzo (R.D.L.), Maria Maisto (M.M.), and Giuseppe Ercolano (G.E.); Formal Analysis: Lucia Ricci (L.R.), Vincenzo Piccolo (V.P.), Daniela Claudia Maresca (C.M.), Benedetta Romano (B.R.), and Adua Marzocchi (A.D.); Investigation: Ritamaria Di Lorenzo (R.D.L.), Lucia Ricci (L.R.), Vincenzo Piccolo (V.P.), Daniela Claudia Maresca (C.M.), Benedetta Romano (B.R.), and Adua Marzocchi (A.D.); Methodology: Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), and Ritamaria Di Lorenzo (R.D.L.); Resources: Sonia Laneri (S.L.), Angela Ianaro (A.I.), and Giuseppe Ercolano (G.E.); Supervision: Ritamaria Di Lorenzo (R.D.L.), Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), Sonia Laneri (S.L.), and Angela Ianaro (A.I.); Writing – Original Draft Preparation: Ritamaria Di Lorenzo (R.D.L.), Maria Maisto (M.M.), and Giuseppe Ercolano (G.E.); Writing – Review & Editing: Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), and Ritamaria Di Lorenzo (R.D.L.). Data availability statement: The authors confirm that the data supporting the findings of this study are available within the article. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request. Competing Interests Statement: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article. Funding : This research received no external funding. References D’Mello, S. A. N., Finlay, G. J., Baguley, B. C. & Askarian-Amiri, M. E. Signaling pathways in melanogenesis. Int J Mol Sci 17 , 1–18 (2016). Du, D. et al. Real-time fluorometric monitoring of monophenolase activity using a matrix-matched calibration curve. doi:10.1007/s00216-020. Guo, N. et al. Continuous Fluorometric Method for Determining the Monophenolase Activity of Tyrosinase on L-Tyrosine, through Quenching L-DOPA Fluorescence by Borate. Anal Chem 92 , 5780–5786 (2020). Zhang, L. et al. Synchronous fluorometric method for continuous assay of monophenolase activity. Spectrochim Acta A Mol Biomol Spectrosc 252 , (2021). Nguyen, N. T. & Fisher, D. E. MITF and UV responses in skin: From pigmentation to addiction. Pigment Cell Melanoma Res 32 , 224–236 (2019). Park, H. J. et al. Ursolic acid inhibits pigmentation by increasing melanosomal autophagy in B16F1 cells. Biochem Biophys Res Commun 531 , 209–214 (2020). Jenkins, N. C. & Grossman, D. Role of melanin in melanocyte dysregulation of reactive oxygen species. Biomed Res Int 2013 , (2013). Qiu, L., Song, Z. & Setaluri, V. Oxidative stress and vitiligo: The Nrf2-ARE signaling connection. Journal of Investigative Dermatology 134 , 2074–2076 (2014). Xuan, Y., Yang, Y., Xiang, L. & Zhang, C. The Role of Oxidative Stress in the Pathogenesis of Vitiligo: A Culprit for Melanocyte Death. Oxid Med Cell Longev 2022 , (2022). Chang, W. L. & Ko, C. H. The Role of Oxidative Stress in Vitiligo: An Update on Its Pathogenesis and Therapeutic Implications. Cells 12 , 1–17 (2023). Di Lorenzo, R. et al. Phenylalanine Butyramide : A Butyrate Derivative as a Novel Inhibitor of Tyrosinase. Int J Mol Sci 25 , 7310 (2024). Bayrakçeken Güven, Z. et al. Food plant with antioxidant, tyrosinase inhibitory and antimelanoma activity: Prunus mahaleb L. Food Biosci 48 , 101804 (2022). López-Hortas, L., Pérez-Larrán, P., González-Muñoz, M. J., Falqué, E. & Domínguez, H. Recent developments on the extraction and application of ursolic acid. A review. Food Research International 103 , 130–149 (2018). Nkuimi Wandjou, J. G. et al. Comprehensive characterization of phytochemicals and biological activities of the Italian ancient apple ‘Mela Rosa dei Monti Sibillini’. Food Research International 137 , 109422 (2020). Tenore, G. C., Campiglia, P., Stiuso, P., Ritieni, A. & Novellino, E. Nutraceutical potential of polyphenolic fractions from Annurca apple (M. pumila Miller cv Annurca). Food Chem 140 , 614–622 (2013). Tenore, G. C. et al. Effects of Annurca apple polyphenols on lipid metabolism in HepG2 cell lines: A source of nutraceuticals potentially indicated for the metabolic syndrome. Food Research International 63 , 252–257 (2014). Tenore, G. C. et al. A Healthy Balance of Plasma Cholesterol by a Novel Annurca Apple-Based Nutraceutical Formulation: Results of a Randomized Trial. J Med Food 20 , 288–300 (2017). Riccio, G. et al. WNT inhibitory activity of malus pumila miller cv annurca and malus domestica cv limoncella apple extracts on human colon-rectal cells carrying familial adenomatous polyposis mutations. Nutrients 9 , (2017). Tenore, G. C. et al. A nutraceutical formulation based on Annurca apple polyphenolic extract is effective on intestinal cholesterol absorption: A randomised, placebo-controlled, crossover study. PharmaNutrition 6 , 85–94 (2018). Maisto, M. et al. Optimization of Phlorizin Extraction from Annurca Apple Tree Leaves Using Response Surface Methodology. Antioxidants 11 , 1–15 (2022). Di Lorenzo, R. Di et al. Annurca Apple Oleolite as Functional Ingredient for the Formulation of Cosmetics with Skin-Antiaging Activity. International journal of molecural science 25 , 1677 (2024). Maisto, M. et al. Optimization of Ursolic Acid Extraction in Oil from Annurca Apple to Obtain Oleolytes with Potential Cosmeceutical Application. Antioxidants 12 , 224 (2023). Nile, S. H., Nile, A., Liu, J., Kim, D. H. & Kai, G. Exploitation of apple pomace towards extraction of triterpenic acids, antioxidant potential, cytotoxic effects, and inhibition of clinically important enzymes. Food and Chemical Toxicology 131 , 110563 (2019). Neeley, E. et al. Variations in IC50 values with purity of mushroom tyrosinase. Int J Mol Sci 10 , 3811–3823 (2009). Kaminski, K., Kazimierczak, U. & Kolenda, T. Oxidative stress in melanogenesis and melanoma development. Wspolczesna Onkologia 26 , 1–7 (2022). Krutmann, J. et al. Photoprotection for people with skin of colour: needs and strategies. British Journal of Dermatology 188 , 168–175 (2023). Gillbro, J. M. & Olsson, M. J. The melanogenesis and mechanisms of skin-lightening agents - Existing and new approaches. Int J Cosmet Sci 33 , 210–221 (2011). Di Lorenzo, R. et al. Phenylalanine butyramide is a new cosmetic ingredient with soothing and anti-reddening potential. Molecules 26 , 1–12 (2021). Nile, S. H., Nile, A., Liu, J., Kim, D. H. & Kai, G. Exploitation of apple pomace towards extraction of triterpenic acids, antioxidant potential, cytotoxic effects, and inhibition of clinically important enzymes. Food and Chemical Toxicology 131 , 110563 (2019). Zhang, L. et al. Synchronous fluorometric method for continuous assay of monophenolase activity. Spectrochim Acta A Mol Biomol Spectrosc 252 , 119486 (2021). Jin, K. S., Oh, Y. N., Hyun, S. K., Kwon, H. J. & Kim, B. W. Betulinic acid isolated from Vitis amurensis root inhibits 3-isobutyl-1-methylxanthine induced melanogenesis via the regulation of MEK/ERK and PI3K/Akt pathways in B16F10 cells. Food and Chemical Toxicology 68 , 38–43 (2014). Kachadourian, R. et al. A synthetic chalcone as a potent inducer of glutathione biosynthesis. J Med Chem 55 , 1382–1388 (2012). Yang, Y. C. et al. Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress. Free Radic Biol Med 51 , 2073–2081 (2011). Gibellini, L. et al. Natural Compounds Modulating Mitochondrial Functions. Evidence-based Complementary and Alternative Medicine 2015 , (2015). Fukuda, S. Skin external preparation suitable for preventing sunburn. (2010). Fukuda, Y. Skin external preparation. (2010). Lo Scalzo, R., Testoni, A. & Genna, A. ‘Annurca’ apple fruit, a southern Italy apple cultivar: Textural properties and aroma composition. Food Chem 73 , 333–343 (2001). Kolakul, P. & Sripanidkulchai, B. Phytochemicals and anti-aging potentials of the extracts from Lagerstroemia speciosa and Lagerstroemia floribunda. Ind Crops Prod 109 , 707–716 (2017). Pan, Y., Li, J., Li, X., Chen, J. & Bai, G. Determination of free amino acids in isatidis radix by HILIC-UPLC-MS/MS. Bull Korean Chem Soc 35 , 197–203 (2014). Zawodny, P., Stój, E., Kulig, P., Skonieczna-żydecka, K. & Sieńko, J. VISIA Skin Analysis System as a Tool to Evaluate the Reduction of Pigmented Skin and Vascular Lesions Using the 532 Nm Laser. Clin Cosmet Investig Dermatol 15 , 2187–2195 (2022). Tables Table 1. Demographic characteristics of the study population. Table 2. List of ingredients of AAO-based formulation (INCI name). Phase Ingredients Functions % w/w A Glyceryl-Stearate and PEG-100 Stearate Emulsifier 4.00 A Cetearyl Alcohol Consistency factor 0.50 A Cetyl Ricinoleate Emollient/Consistency factor 2.00 A Caprylic/Capric Triglycerides Emollient 10.00 B Aqua Solvent qs 1 to 100 B Sodium Gluconate Chelating agent 0.10 B Carbomer Viscosizing agent 0.35 C Aqua Solvent 3.00 C Phenoxyethanol (and) Ethylhexylglycerin Preservative 0.90 C AAO Active ingredient 2.50 C NaOH 20% sol. pH modifier Qs to pH 5.0 1 quantity sufficient. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 12 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviewers agreed at journal 10 Aug, 2025 Reviews received at journal 02 Aug, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviewers invited by journal 22 Jul, 2025 Editor assigned by journal 22 Jul, 2025 Editor invited by journal 22 Jul, 2025 Submission checks completed at journal 15 Jul, 2025 First submitted to journal 15 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7028178","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489433728,"identity":"26fa16e0-2af7-40ba-999d-5f5d85ce995d","order_by":0,"name":"Maria Maisto","email":"","orcid":"","institution":"ChimNutra labs","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Maisto","suffix":""},{"id":489433729,"identity":"902e31b4-aab3-40ad-b688-96325206142b","order_by":1,"name":"Vincenzo Piccolo","email":"","orcid":"","institution":"ChimNutra labs","correspondingAuthor":false,"prefix":"","firstName":"Vincenzo","middleName":"","lastName":"Piccolo","suffix":""},{"id":489433730,"identity":"e37f5cb2-3906-44f8-ba55-42c65eb0c261","order_by":2,"name":"Adua Marzocchi","email":"","orcid":"","institution":"ChimNutra labs","correspondingAuthor":false,"prefix":"","firstName":"Adua","middleName":"","lastName":"Marzocchi","suffix":""},{"id":489433732,"identity":"9f43c53c-0615-45b4-8d78-ebe2252aff91","order_by":3,"name":"Lucia Ricci","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"","lastName":"Ricci","suffix":""},{"id":489433734,"identity":"ab677441-87f5-4711-854a-ca6bb6a9540a","order_by":4,"name":"Benedetta Romano","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Benedetta","middleName":"","lastName":"Romano","suffix":""},{"id":489433737,"identity":"3a2e9a6d-f8e6-4b0d-80ca-fd1e0f1f88b1","order_by":5,"name":"Daniela Claudia Marasca","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"Claudia","lastName":"Marasca","suffix":""},{"id":489433738,"identity":"48f22d4c-e5fc-4ee1-990b-f3ca2050d1ff","order_by":6,"name":"Ritamaria Di Lorenzo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYDACZgjFA8QGBx5AOIxAWgJEN2DXwoykJQEqZgDV0ohDD5xlwADVwiYBFcFqjXk7/8FPN2ruyfA3MG88kFBxT86c//Cxat4dFnlAkfYHWLTIHGZmls45VswjcYCt4EDCmWJjy4Zjabd5z0gUSxzA7jAJoF+kc9gSeBgO8BgcSGxLSNxwsMfsNm+bRGIDbi3Mv3P+JfDIQ7XUbzjMY1YM0jIftxY26dy2BKB6iJYEg2M8ZswgLRtwazGzzu1L4DE8DPZLguGGM2zJknOBWjYeZmycgU0L/8HHt3O+JdjLHW/e/OFDRYK8wfnDBz+8batLnHe8/cEHLFoQgJkIkVEwCkbBKBgFRAIAQ0piKzUQwBgAAAAASUVORK5CYII=","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":true,"prefix":"","firstName":"Ritamaria","middleName":"Di","lastName":"Lorenzo","suffix":""},{"id":489433739,"identity":"dd465e97-ebb7-4dbc-bc69-602330dca893","order_by":7,"name":"Sonia Laneri","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Sonia","middleName":"","lastName":"Laneri","suffix":""},{"id":489433740,"identity":"cd4d36ae-da37-461b-b674-342e245f1f2a","order_by":8,"name":"Giuseppe Ercolano","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Ercolano","suffix":""},{"id":489433743,"identity":"9c2d70c1-cf03-41f5-a7a3-90089e8ac165","order_by":9,"name":"Angela Ianaro","email":"","orcid":"","institution":"University of Naples Federico II","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Ianaro","suffix":""}],"badges":[],"createdAt":"2025-07-02 10:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7028178/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7028178/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-29398-0","type":"published","date":"2025-12-12T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87512161,"identity":"8edebdd6-73e7-4279-a9ce-bacc3f3e22f7","added_by":"auto","created_at":"2025-07-24 15:51:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55149,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of tyrosinase enzyme (%) by AAO calculated as IC\u003csub\u003e50\u003c/sub\u003e. Values represent the mean ± of three replicates analysed.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/54e52198b8933f6ffb9b7031.png"},{"id":87512159,"identity":"06325c8e-7cd7-43e7-9d81-a2bf4fa28ddd","added_by":"auto","created_at":"2025-07-24 15:51:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105051,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of tyrosinase enzyme (%) by Kojic acid (A) and UA (B) calculated as IC\u003csub\u003e50\u003c/sub\u003e. Values represent the mean ± of three replicates analyzed.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/327b75b73258ec6e43bd13c5.png"},{"id":87513819,"identity":"ce736aa0-2813-405c-843f-a44647ba094b","added_by":"auto","created_at":"2025-07-24 16:07:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":242047,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect of AAO (A) and UA (B) on A375 human melanoma cells after 72h evaluated by the MTT assay. Data were shown as mean ± SEM of at least three independent experiments (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001 vs CTR).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/eaac18f0b2921eec2b14a97c.png"},{"id":87513294,"identity":"48224195-9a70-420f-aaae-567520f5eb76","added_by":"auto","created_at":"2025-07-24 15:59:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":288792,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Melanin content in A375 melanoma cells upon 72 h of treatment with AAO (30 µg/mL) and UA (3 µg/mL) and in the presence of MSH. Expression of TYR, TYRP-1, TYRP-2 (B) and MITF (C) assessed with qPCR in A375 cells upon 6 h of treatment with AAO (30 µg/mL) and UA (3 µg/mL) and in the presence of MSH. Data were shown as mean ± SEM of at least three independent experiments (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, and **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs MSH; °\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, °°\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 vs CTR).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/98df096cb76e9a3829ef5a26.png"},{"id":87513298,"identity":"095a7803-d9c9-4013-9470-daa419029ed1","added_by":"auto","created_at":"2025-07-24 15:59:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":433486,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative example of flow cytometry analysis of DCF-DHA in A375 untreated (black histograms) and after AAO (30 μg/mL) and UA (3 μg/mL) treatment (red and orange histogram respectively) in the presence of Fenton's reagent (green histogram) for 48 h and (B) quantification in terms of mean fluorescence intensity (MFI). (C) Expression of GCLC, GCLM, HMOX and MITF and TYR (D) assessed by qPCR in A375 cells upon 6 h of treatment with AAO (30 µg/mL) and UA (3 µg/mL) and in the presence of Fenton's reagent. (E) Representative example of flow cytometry analysis of DCF-DHA in A375 untreated (black histograms) and after AAO (30 μg/mL) and UA (3 μg/mL) treatment (red and orange histogram respectively) in the presence of MSH (blue histogram) for 48 h and (F) quantification in terms of mean fluorescence intensity (MFI). Data were shown as mean ± SEM of at least three independent experiments (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs Fenton or MSH; °° \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, °°° \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 vs CTR).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/e74e31bafe9c63c1a53c0eee.png"},{"id":87512167,"identity":"6cf148d5-4de8-4688-aeed-b3228bee7389","added_by":"auto","created_at":"2025-07-24 15:51:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":303676,"visible":true,"origin":"","legend":"\u003cp\u003eA) L*, B) ITA° and C) melanin content percentage variation after treatment with AAO or placebo, respectively, at each follow-up session (D14 and D28). The baseline data served as the reference value for each analysis. Data were shown as mean ± SEM (n=20). (** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs D0, \u003csup\u003e$$$\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs placebo).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/17c45cd732c38fbabcbb0f82.png"},{"id":87514690,"identity":"a7f5374f-a276-4429-ab41-5de4e7ca006f","added_by":"auto","created_at":"2025-07-24 16:15:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":252279,"visible":true,"origin":"","legend":"\u003cp\u003eSkin luminosity and spots visibility after treatment with AAO or placebo, respectively, at each follow-up session (D14 and D28). The baseline data served as the reference for each analysis.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/5d948c3ec8507da59d4967ca.png"},{"id":87513310,"identity":"4f8aaffa-809f-44fa-a526-1aad8603e98e","added_by":"auto","created_at":"2025-07-24 15:59:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":17424255,"visible":true,"origin":"","legend":"\u003cp\u003eThe UV and brown spots score percentage variation after treatment with AAO or placebo, respectively, at each follow-up session (D14 and D28). The baseline data served as the reference value for each analysis. Data were shown as mean ± SEM (n=20). (*\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, vs D0, \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e$$$\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e placebo).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/dc80ea54494728538fecec6e.png"},{"id":87513296,"identity":"72e8c1da-b9cf-43cd-8e07-db46157f29e7","added_by":"auto","created_at":"2025-07-24 15:59:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":220020,"visible":true,"origin":"","legend":"\u003cp\u003eTrial Flowchart.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/3df43db1a2ab1294eddc18f2.png"},{"id":98243508,"identity":"549f2346-8945-47fb-8199-b66c3ceecb3e","added_by":"auto","created_at":"2025-12-15 16:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20461724,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7028178/v1/03a38579-43c6-4825-9488-b8a0c09b25a7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ursolic Acid-Based Oleolite for Hyperpigmentary Disorders: Molecular Mechanisms and Clinical Efficacy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMelanin is the most important biological pigment in humans; it is responsible for the pigmentation of skin, eyes, and hair \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is produced by melanocytes in specific sub-cellular lysosome-like structures called melanosomes. Physiologically, melanin synthesis and production protect human skin from UV radiation; however, its abnormal synthesis could lead to several skin disorders, including hyperpigmentation, melasma, and age spots. Specifically, melanogenesis is a multi-step process involving melanin synthesis and transport, and the subsequent release of melanosomes. Chemically, the melanogenesis process begins with the oxidation of tyrosine to dopaquinone, which is catalyzed by the enzyme tyrosinase. Subsequently, the dopaquinone is converted into dopachrome by an auto-oxidation reaction \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The L-Dopa produced is itself a tyrosinase substrate and is oxidized again to dopaquinone, which undergoes further oxidative polymerization steps leading to the melanin formation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Several molecular players are involved in regulating the melanogenesis process \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Specifically, UV-irradiation induces the synthesis of alpha-melanocyte-stimulating hormone (α-MSH) in melanocytes, which activates the melanocortin 1 receptor, leading to increased expression of microphthalmia-associated transcription factor (MITF) through activation of the cyclic adenosine monophosphate pathway. The MITF produced is a key protein that controls the synthesis of several melanogenesis-regulating protein factors, including tyrosinase and tyrosinase-related proteins 1 and 2 (TYRP-1 and TYRP-2) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Due to their pivotal role in melanin production, tyrosinase, TYRP-1, and TYRP-2 are generally recognized as the most important molecular targets for active agents with skin whitening and depigmenting potentials. Regarding oxidative balance, melanocytes are characterized by higher levels of reactive oxygen species (ROS) than other cell types, such as fibroblasts \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This difference could be related to their higher rate of melanin production, which releases high levels of ROS as a collateral product, leading to an alteration in the redox potential of melanocytes. In this regard, several skin pigmentation disorders such as vitiligo have been reported to be positively related to an alteration of cellular defense enzymes with antioxidant activity, including Nrf2-related enzymes such as the catalytic and modulatory subunits of γ-glutamylcysteine ligase (GCLC and GCLM, respectively) as well as the enzyme heme oxygenase (HMOX-1) \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Given the multifactorial nature of skin pigmentation disorders, there is great interest in identifying compounds with double potential: i) antioxidant, and ii) ability to modulate melanogenesis at different molecular levels.\u003c/p\u003e\u003cp\u003eIn this regard, increasing attention has been paid to the identification of natural and food-derived compounds as effective inhibitors of tyrosinase\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among natural lipophilic compounds, ursolic acid (UA), a triterpene acid, has shown valuable \u003cem\u003ein vitro\u003c/em\u003e tyrosinase inhibitory activity (IC\u003csub\u003e50\u003c/sub\u003e: 170.2 \u0026micro;M) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This molecule reached a valuable concentration in several medical plant species \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and especially in apple peel (14.3 mg/g DW) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInterestingly, a comparative study conducted on both commercial and traditional Italian apple cultivars showed that Annurca apple (AA) was richer in UA (9.09 mg/g DW) and total terpenic content (14.19 mg/g DW) than other examined varieties [11]. Specifically, AA is the only apple cultivar native to southern Italy that has been registered by the European Council as a product with a Protected Geographical Indication (PGI) [Commission Regulation (EC) No.417/2006]. While the nutraceutical potential of the polyphenolic fraction of AA has been widely documented for its beneficial effects in the management of plasma cholesterol levels in humans \u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and on the regulation of glycemia \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, the potential health-promoting application of AA triterpenoid fraction is less well explored. In light of these considerations, in our recent study, we demonstrated that Annurca apple oil-based formulation (AAO) with standardized UA concentration (736 \u0026micro;g/mL) exerts valuable skin antiaging activity and favourable skin permeation features \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven these considerations, the main objectives of the present manuscript were to investigate the potential application of AAO as an active ingredient for the preparation of novel topical depigmenting formulations. Firstly, the \u003cem\u003ein vitro\u003c/em\u003e inhibitory effect of AAO on tyrosinase was investigated. Next, we investigated the ability of AAO to reduce melanin production and transcriptionally modulate the expression of melanogenesis-regulating protein factors in human melanoma cell lines, as well as to modulate oxidative stress responses. Based on these promising \u003cem\u003ein vitro\u003c/em\u003e results, AAO was tested in a randomized, double-blind, placebo-controlled, two parallel-arm clinical trials, conducted on 40 subjects with hyperpigmentation and diagnosed photo-damage, in order to investigate its effects on the main clinical skin pigmentation-related parameters, i.e., UV and brown spots, melanin index, and skin lightening markers (Individual Typology Angle, ITA\u0026deg; and luminescence, L*).\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e2.1. Tyrosinase Inhibition Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe tyrosinase enzyme plays a pivotal role in human skin pigmentation, thus its inhibition represents a potential treatment for hyperpigmentation-related disorders. To establish AAO as a potential whitening ingredient, the \u003cem\u003ein vitro\u003c/em\u003e inhibition of tyrosinase activity was evaluated. As reported in Figure 1 the AAO has shown a valuable capacity to inhibit tyrosinase activity in a concentration-dependent manner, with an IC\u003csub\u003e50\u003c/sub\u003e of 286.42 mg/mL. To confirm that UA was the main molecule responsible for tyrosinase inhibition, the pure UA was tested, resulting in IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003eof\u003csub\u003e\u0026nbsp;\u003c/sub\u003e26.82 \u0026micro;g/mL (Figure 2B), which is approximately ten times more active than the AAO, as expected. Additionally, as a positive control we used kojic acid (KA), a fungal metabolite, with strong chelating properties for copper at the active site of the tyrosinase enzyme \u003csup\u003e23\u003c/sup\u003e. As expected, the KA IC\u003csub\u003e50\u003c/sub\u003e (0.59 \u0026micro;g/mL) is the lowest calculated value in the present study (Figure 2A), and it was in line with the tyrosinase inhibitory activity calculated by others, who reported KA IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalues ranged from 1-10 \u0026micro;g/mL \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e2.2 AAO did not affect the proliferation of human melanoma cells\u003c/p\u003e\n\u003cp\u003eTo better characterize the anti-melanogenic effects of AAO, we decided to use a human melanoma cell line known to display an altered melanogenesis process. First, we evaluated whether AAO exerts a cytotoxic effect by using A375 human melanoma cells. Cells were treated with increased concentrations of AAO (0, 0.01, 0.1, 1, 10, and 100 \u0026micro;g/mL) for 72 h, and cell viability was evaluated by performing an MTT assay. As shown in Figure 3A, we found that the concentration of 100 \u0026micro;g/mL impaired A375 cell viability. Conversely, the pure UA reduced the proliferation of A375 melanoma cells already at the concentration of 10 \u0026micro;g/mL (Figure 3B). Thus, we selected 30 \u0026micro;g/mL and 3 \u0026micro;g/mL as the concentrations for the next experiments for AAO and UA, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;2.3 AAO reduced melanin production and the expression of melanogenesis-related genes\u003c/p\u003e\n\u003cp\u003eNext, we examined the effect of AAO and UA on melanin production in A375 human melanoma cells stimulated with \u0026alpha;-MSH, known to trigger the melanin synthesis processes. As expected, the MSH treatment significantly increased the melanin production compared to A375 control cells. Importantly, as shown in Figure 4A, we observed that both AAO (30 \u0026micro;g/mL) and UA (3 \u0026micro;g/mL) significantly reduced melanin production in MSH-stimulated A375 cells after 72 h of treatment. Moreover, in line with the findings described above, AAO reduced the mRNA expression levels of tyrosinase enzyme (TYR) and its related proteins (TYRP-1 and TYRP-2) (Figure 4B) in MSH-stimulated A375 human melanoma cells. \u0026nbsp;We also found that AAO suppressed the mRNA expression level of MITF, the most important transcription factor involved in melanogenesis and melanoma progression (Figure 4C). Taken together, these data demonstrated that AAO reduced melanin production by modulating the expression levels of the key players involved in melanogenesis.\u003c/p\u003e\n\u003cp\u003e2.4 AAO modulation of ROS production\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMelanogenesis and oxidative stress are intertwined in a feedback loop: while melanin production generates free radicals, the reactive species can further stimulate melanogenesis \u003csup\u003e25\u003c/sup\u003e. Therefore, we investigated the ability of AAO to modulate oxidative stress in A375 human melanoma cells by evaluating the production of ROS and the expression of the key antioxidant enzymes. First, we analyzed ROS production by using the 2\u0026apos;,7\u0026apos;-dichlorofluorescin (DCF) probe and inducing oxidative damage with Fenton\u0026rsquo;s reagent in order to increase ROS production. As shown in Figures 5A and B, the preincubation with both AAO and UA significantly reduced ROS production, suggesting their potential antioxidant protective role. To corroborate these data, we analyzed the expression of three important antioxidant enzymes: GCLC, GCLM, and HMOX-1 in AAO- and UA-treated A375 cells and stimulated with Fenton\u0026rsquo;s reagent. Our results showed that treatment with AAO significantly increased the expression of all the enzymes tested, whereas UA induced an up-regulation of the HMOX-1 enzyme (Figure 5C). In addition, we also evaluated the contribution of oxidative damage to \u0026nbsp;the expression of the melanogenesis-related proteins MITF and TYR. As expected, the expression of both MITF and TYR was increased in oxidative stress conditions compared to A375 control cells. Interestingly, the preincubation with AAO (30 mg/mL) or UA (3 mg/mL) drastically reduced their expression, confirming their modulation also under strong oxidative unbalance (Figure 5D). Finally, to address putative crosstalk between melanogenesis and oxidative stress, we also investigated ROS production in MSH-stimulated A375 cells. As shown in Figures 5E and F, MSH increased ROS production while both AAO and UA reduced the production of ROS. Collectively, these results demonstrated that both AAO and UA exert antioxidant effects by reducing ROS production and modulating the expression of antioxidant enzymes. Moreover, both the antioxidant effects and the modulation of melanogenesis of AAO could be interconnected, given the potential crosstalk between these processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Clinical data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.5.1. Demographic data\u003c/p\u003e\n\u003cp\u003eDuring the recruitment process, out of 48 participants enrolled, 6 subjects dropped out (3 were lost to follow-up and 3 subjects underwent aesthetic procedures), leaving a total of 42 females. The demographic characteristics of the participants are shown in Table 1. The majority of participants were 45-55 years old (30 subjects, 71.4%). Overall, 66.7% of the participants had a Fitzpatrick skin type II, and 33.3% had a Fitzpatrick skin type III.\u003c/p\u003e\n\u003cp\u003e2.5.2. Effects of AAO-based topical formulation on pigmentation-related skin parameters\u003c/p\u003e\n\u003cp\u003eThe key parameters used to evaluate AAO\u0026rsquo;s efficacy on photo-damage were the Individual Typology Angle (ITA\u0026deg;), L* luminance, and the melanin index (MI). ITA\u0026deg; classifies skin pigmentation based on colorimetry\u0026nbsp;\u003csup\u003e26\u003c/sup\u003e, and is calculated using the formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"257\" height=\"18\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere L* represents luminance (ranging from 0 for black to 100 for white) and b* ranges from yellow (-b*) to blue (+b*). Higher ITA\u0026deg; values indicate lighter skin and greater lightening from treatment. The average percentage changes in ITA\u0026deg; and L* at baseline (D0), day 14 (D14), and day 28 (D28) are shown in Figure 6A and 6B, while MI changes are depicted in Figure 6C.\u003c/p\u003e\n\u003cp\u003eResults showed no significant differences between groups at baseline. However, L*, ITA\u0026deg;, and MI significantly improved in the AAO group during and after treatment (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). These improvements were supported by VISIA 7\u003csup\u003eth\u003c/sup\u003e analysis, demonstrating AAO\u0026rsquo;s whitening and brightening effects. AAO also significantly reduced UV and brown spots compared to baseline and placebo by day 14 (Figures 7 and 8). Neither treatment caused irritation or desquamation.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eSkin-lightening molecules have found a large distribution worldwide for their relevant application for both cosmetic and clinical disorders. Their use is widely diffused in cosmetic practice to improve skin appearance, but also in medical therapy for the treatment of hyperpigmented skin disorders such as melasma, café-au-lait spots, and solar lentigo \u003csup\u003e27\u003c/sup\u003e. The formation of skin spots has been mainly associated with an over-activation of the melanin synthesis, making the regulation of this biosynthetic pathway a major target of lightening agents. In particular, melanogenesis was controlled by several molecular modulators such as MITF, TYR, TYRP-1, and TYRP-2. To identify a novel and natural approach for the management of hyperpigmentation disorders, in our previously published work, we have optimized the formulation of UA oil extract from annurca apple (AAO) as a functional ingredient for topical formulation \u003csup\u003e22\u003c/sup\u003e. Specifically, we have reported that the AAO prepared with a UA concentration of 736 mg/mL, has shown a valuable \u003cem\u003ein vitro\u003c/em\u003e antioxidant potential (evaluated using DPPH, ABTS, and FRAP assays, resulting in the antiradical potential of 16.63 ± 0.22, 5.90 ± 0.49, and 21.72 ± 0.68 µmol Trolox equivalent/g, respectively) and favorable \u003cem\u003ein vitro\u003c/em\u003e skin permeation parameters. Particularly, after 4 h of AAO application, UA reached a maximum contraction in the dermal layer, without reaching the bloodstream compartment. This last feature was highly required for the preparation of topical formulation with local activity \u003csup\u003e28\u003c/sup\u003e. Thus, in order to investigate the potential depigmenting potential of the formulated AAO, we evaluated its inhibitory potential on tyrosinase activity using an enzymatic assay. Our results show that the formulated AAO was able to inhibit the tyrosinase activity \u003cem\u003ein vitro\u003c/em\u003e with a calculated IC\u003csub\u003e50\u003c/sub\u003e of 286.42 mg/mL. This promising result could be mainly due to its relevant UA content, which has shown to be a noticeable tyrosinase inhibitor agent, with a calculated IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003eof 26.8 mg/mL (0.026 mM), a value in perfect agreement with results previously described by others \u003csup\u003e29\u003c/sup\u003e. Once established the inhibitory activity of AAO in an \u003cem\u003ein vitro\u003c/em\u003e enzymatic system, we moved to investigate the effects of AAO on melanin production in A375 human melanoma cell line.\u003c/p\u003e\n\u003cp\u003eThe results obtained showed that AAO (30 mg/mL), compared to UA (3 mg/mL), was able to cause a significant reduction (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001\u003cem\u003e\u0026nbsp;vs\u003c/em\u003e CTR) in melanin production in A375 melanoma cells stimulated with α-MSH. A similar trend in terms of melanin content has already been observed in the murine melanoma cells B16F10 after treatment with UA (3-10 mg/mL), resulting in efficient inhibition of melanin production \u003csup\u003e30\u003c/sup\u003e.\u0026nbsp;To further investigate the effective molecular mechanism of action involved in the regulation of melanin synthesis observed, the effects of AAO on TYR, TYRP-1, TYRP-2, and MITF mRNA transcription levels were investigated. Our results indicate that AAO treatment resulted in a significant reduction in mRNA expression levels of all the melanogenic modulators studied. Furthermore, these results were also observed in the Fenton-stressed cell model, in which the induced oxidative stress was able to increase the expression of TYR and MITF at mRNA expression levels. These data corroborate the AAO’s ability to downregulate the expression of melanogenic modulators in both basal and stressed conditions in the A375 human melanoma cell line. Interestingly, the implication of UA or UA-containing extracts in the management of these melanogenic factors has not yet been described. Therefore, this work represents the first attempt to establish the effects of UA and UA-based products on the control of the melanogenesis process. Indeed, among all the triterpenic acids, only betulinic acid has been described to exert an anti-melanogenic activity by inhibiting TYR, TYRP-1, and TYRP-2 mRNA expression through the inactivation of cAMP response element binding protein (CREB) and MITF in isobutylmethylxanthine-stimulated B16F10 cells \u003csup\u003e31\u003c/sup\u003e. Given the complex and bivalent relationship between melanogenesis and cellular oxidative stress, in which increased melanin synthesis could generate high levels of reactive oxygen species in melanosomes and a subsequent increase in oxidative stress could further stimulate melanogenesis, coupled with the fact that melanin itself has played a central role in modulating oxidative homeostasis at the skin level \u003csup\u003e29,30\u003c/sup\u003e, the ability of AAO to modulate oxidative cell system was evaluated. Specifically, oxidative stress is defined as an imbalance between the production of reactive oxygen and nitrogen species (ROS/RNS) and the physiological human antioxidant defenses at the cellular level. It has been positively correlated with the development of age-related disorders, including pathological conditions and aesthetic alterations, such as skin wrinkles and spots. Since the \u003cem\u003ein vitro\u003c/em\u003e antioxidant activity of AAO was already demonstrated in our previous work \u003csup\u003e21\u003c/sup\u003e, its ability to contrast ROS production through stimulation of cell defense enzymes, i.e. enzymes of the glutathione biosynthesis pathway, including GCLC, GCLM, and HMOX-1, was further investigated. Our results show that AAO was able to induce a significant (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) increase in mRNA expression of GCLC and especially of HMOX-1 (3-fold increased vs CTR, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) in A375 melanoma cells. The observed beneficial effect of AAO on HMOX-1 mRNA expression could likely be related to its valuable UA content, whose regulatory activity in transcriptional upregulation of HMOX-1 mRNA expression was previously reported in JB6 P+ murine cells \u003csup\u003e31\u003c/sup\u003e. In addition, while no relevant modulatory effect on the expression of GCLC or GCLM has been attributed to UA, the observed upregulation of these enzymes could be related to a synergic activity of the polyphenolic AAO content \u003csup\u003e32,33\u003c/sup\u003e. Moreover, to corroborate the AAO antioxidant activity at the cellular level, the ability of AAO and UA to modulate mitochondrial activity, given their key role as important sources of ROS, was further investigated. Mitotracker Green and TMRM are cationic probes that allow the measurement of mitochondrial mass and membrane potential since they are sequestered in the matrix space of mitochondria and released once mitochondria experience change in the membrane \u003csup\u003e32,33\u003c/sup\u003e.Our findings demonstrated that AAO and UA reduced mitochondrial ROS generation by modulating mitochondrial mass and mitochondrial membrane potential as demonstrated for other natural compounds such as resveratrol and quercetin \u003csup\u003e34\u003c/sup\u003e. Encouraged by the achieved promising in vitro results, a double-blind, placebo-controlled, 2-parallel-arm instrumental clinical trial was performed on 40 subjects with hyperpigmentation and diagnosed photo-damage, in order to investigate AAO-based topical formulation effects on the main clinical skin pigmentation-related parameters, i.e., UV spots, brown spots, and melanin index. Clinically, both types of skin spots are characterized by an increased production and accumulation of melanin in the skin, but they differ in the origin of their formation. While brown spots, also known as age spots or liver spots, are usually caused by a combination of several factors such as aging, genetics, or other causes, including hormonal changes, skin lesions such as hyperpigmentation, freckles, lentigines, and melasma. In contrast, UV spots are mainly caused by damage from the sun's UV exposure. Our results indicate that, after 28 days of treatment, a significative reduction of spot score for both UV (-6.4% vs baseline; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and brown spots (-4.1% vs baseline; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) was registered, accompanied by a valuable reduction of their skin melanin index (-10.2 vs baseline; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Similar results were reported in a patent, describing that phosphorylated triterpenic acids and/or their salts formulated in a topical preparation used for cosmetics and dermatological purposes have shown a relevant anti-inflammatory action and melanin production downregulation in the skin. Additionally, our results are consistent with those reported by the few pieces of evidence related to the beneficial effects of UA-based topical formulations. These studies describe the effectiveness of UA in protecting against ultraviolet absorbers and highlight its potential to counteract skin pigmentation induced by UV irradiation \u003csup\u003e35,36\u003c/sup\u003e. Finally, an improvement in the color complexion and skin brightness (ITA°: +12.41% vs baseline; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and L*: \u0026nbsp;+3.1% vs baseline \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) was also registered.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe triterpenic fraction of AAO, primarily composed of ursolic acid (UA), demonstrated significant inhibition of tyrosinase activity in an in vitro model. In A375 melanoma cells, AAO down-regulated key melanogenesis modulators (TYR, TYRP-1, TYRP-2, MITF) at the transcriptional level, both in α-MSH and Fenton-stimulated models. Given the link between hyperpigmentation and oxidative stress, AAO's antioxidant potential was also explored. Results showed AAO up-regulated antioxidant enzyme expression while reducing MITF and TYR levels under oxidative stress. These findings were supported by the clinical trial, further confirming AAO's potential as a functional ingredient in depigmenting topical formulations.\u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cp\u003e5.1. Reagents\u003c/p\u003e\n\u003cp\u003eAll chemicals, reagents, and standards used were analytical or LC-MS grade reagents. The water was treated in a Milli-Q water purification system (Millipore, Bedford, Burlington, MA, USA) before use. Sunflower oil was purchased in a local market. Ursolic acid (purity \u0026ge; 98.5% HPLC), phloridzin (purity \u0026ge; 99% HPLC), kojic acid (purity \u0026ge; 98.5% HPLC), L-tyrosine (purity \u0026ge; 98% HPLC), tyrosinase from mushroom (activity \u0026ge; 1000 U/mg), human melanoma cells lines A375, MTT, TRI-Reagent, Nanodrop were purchased from Sigma-Aldrich (Milan, Italy). DMEM medium, heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptomycin, HEPES buffer were purchased from Gibco (New York, NY, USA). The iScript Reverse Transcription Supermix for RT-qPCR was purchased from Bio-Rad (Milan, Italy). H\u003csub\u003e2\u003c/sub\u003eDCFDHA, MitoTracker Green and TMRM were purchased from Thermo Fisher Waltham (MA, USA).\u003c/p\u003e\n\u003cp\u003e5.2. Oleolite preparation\u003c/p\u003e\n\u003cp\u003eAnnurca apple fruits (Malus pumila Miller cv Annurca) (about 100 g each) were collected in October 2023, Valle di Maddaloni (Caserta, Italy), when the fruits had just been harvested (green peel). The fruits were reddened for about 30 days following the typical treatment \u003csup\u003e37\u003c/sup\u003e. After this time, the apples were washed and sliced for freeze-drying. The oleolite preparation was performed according to the conditions optimized in our previously published article \u003csup\u003e22\u003c/sup\u003e to achieve the maximum UA concentration of 784.40 \u0026plusmn; 7.58\u0026nbsp;mg/mL. Specifically, the ratio of freeze-dried AA to sunflower oil was 1:4, and the mixture was stirred at 68.85 ◦C for 63 h. Then, the AAO was subjected to a specific extraction protocol for the isolation of its triterpenic fraction, according to our published protocols. The extraction was performed with ethyl acetate. The fractions obtained were first dried using a rotary evaporator, when the solvents used were completely removed, the residues obtained were suspended in water, frozen at -80\u0026deg;, and freeze-dried (for 24 h, -69 \u0026deg;C at 0.096 mbar). The resulting powder was ground (IKA A11 analytical mill) to obtain a homogeneous powder representative of the triterpenic AAO composition \u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e5.3. Tyrosinase inhibition assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe tyrosinase inhibition assay was performed according to the modified dopachrome method \u003csup\u003e38\u003c/sup\u003e. Practically, the inhibition of the enzyme was assessed by monitoring the enzymatic metabolization of L-tyrosine substrate according to the HPLC method described. According to the used protocol, 70 \u0026micro;L of different concentrations of the samples were added to a 96-well microplate. Then, 100 \u0026micro;L of 1 mM L-tyrosine and 30 \u0026micro;L of mushroom tyrosinase (500 U/mL), both of them prepared in 50 mM sodium phosphate buffer pH 6.5, were added, mixed well, and incubated at room temperature for 30 min. After this incubation time, the enzymatic reaction was stopped by the addition to each sample of 100 \u0026micro;L of methanol. The analyzed standards and the extracts were dissolved in 100 % v/v DMSO and diluted with 50 mM sodium phosphate buffer pH 6.5 to obtain a final concentration of 1% v/v DMSO in the enzymatic mixture. Kojic acid was used as a positive control, while the control was performed by replacing the sample with an equal volume of buffer. In the HPLC assay, enzymatic activity was evaluated as a decrease in the chromatographic L-tyrosine peak area for the tyrosinase reaction of the enzyme. The L-tyrosine peak area was normalized to the 1 mM L-tyrosine peak area. The inhibitory effect was expressed as the concentration required to inhibit L-tyrosine metabolism by 50% (IC\u003csub\u003e50\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003e5.3.1 HPLC-FLD method for L-tyrosine analysis\u003c/p\u003e\n\u003cp\u003eA Jasco Extrema LC-4000 HPLC system (Jasco Inc., Easton, MD, USA), coupled with an autosampler, a binary solvent pump, and a fluorescence detector (FLD), was used for the analysis. The chromatographic analysis was performed according to \u003csup\u003e39\u003c/sup\u003e, with slight modifications. Elution was performed on a Luna HILIC column (150 mm x 3.0, 5 \u0026micro;m; Phenomenex, Torrance, CA, USA). The mobile phases were an ammonium acetate buffer, 5 mM, containing 0.1% formic acid (A) and acetonitrile (B). The elution gradient was performed under the following conditions: 0-5 min, isocratic on 90% phase B; 5-17 min, linear gradient from 90 to 70% B; 17-27 min, isocratic with 90% B; 24-29 min, isocratic with 60% B for column reconditioning. The separation parameters were as follows: column temperature was set at 35 \u0026deg;C, injection volume was 20 \u0026micro;l, and flow rate was set at 1 mL/min. The quantification of tyrosine was performed with an excitation wavelength of 272 nm and an emission wavelength of 312 nm.\u003c/p\u003e\n\u003cp\u003e5.4. Cell culture\u003c/p\u003e\n\u003cp\u003eThe human melanoma cells lines A375 were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin and 10 mM HEPES buffer and grown at 37\u0026deg;C in a humidified incubator under 5% CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5.5. MTT assay\u003c/p\u003e\n\u003cp\u003eA375 cells (10\u003csup\u003e3\u003c/sup\u003e cells/well) were plated on a 96-well plate and treated with AAO and UA (0.01 to 100 \u0026mu;g/mL) for 72 hr. Next, 25 \u0026mu;L of MTT was added and incubated for 3 hr at 37\u0026deg;C. Thereafter, the dark blue crystals developed, were solubilized with DMSO and the absorbance was measured at 545 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific, MA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5.6. Measurement of Melanin Content\u003c/p\u003e\n\u003cp\u003eA375 cells (3x10\u003csup\u003e5\u003c/sup\u003e/well) were plated in a 6-well plate and treated with AAO (30 \u0026micro;g/mL) and UA (3 \u0026micro;g/mL). After 72 hours, cells were lysed in NaOH 1N and incubated for 90 min at 37\u0026deg;C, then centrifuged for 10 min at 10,000 rpm. The optical density (OD) of supernatant was measured at 450 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e5.7. RNA extraction and qPCR analysis\u003c/p\u003e\n\u003cp\u003eA375 melanoma cells were treated with AAO and UA (30 \u0026micro;g/mL and 3 \u0026micro;g/mL, respectively) for 6 hours. Thereafter, total RNA was extracted by using TRI-Reagent, according to the manufacturer\u0026apos;s instructions. After quantification with Nanodrop, 1 \u0026micro;g of total RNA was reverse-transcribed by using iScript Reverse Transcription Supermix for RT-qPCR. qPCR analysis was performed with the Bio-Rad CFX384 real-time PCR detection system (Bio-Rad, Milan, Italy) by using the following primers:\u003c/p\u003e\n\u003cp\u003eTYR (Gene ID: 7299)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;- GCACAGATGAGTACATGGGAGG -3\u0026rsquo;; 5\u0026rsquo;- CTGATGGCTGTTGTACTCCTCC -3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eTYRP-1 (Gene ID: 7306)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;- TCTCAATGGCGAGTGGTCTGTG-3\u0026rsquo;; 5\u0026rsquo;- CCTGTGGTTCAGGAAGAC-GTTG-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eTYRP-2 (Gene ID: 1638)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;- CTCAGACCAACTTGGCTACAGC-3\u0026rsquo;; 5\u0026rsquo;- CAACCAAAGCCACCAG-TGTTCC-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eMITF (Gene ID: 4286)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;- GGCTTGATGGATCCTGCTTTGC-3\u0026rsquo;; 5\u0026rsquo;- GAAGGTTGGCTGGACAG-GAGTT-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eGCLC (Gene ID: 2729)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;-GTTGGGGTTTGTCCTCTCCC-3\u0026rsquo;; 5\u0026rsquo;-GGGGTGACGAGGTGGAGTA-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eGCLM (Gene ID: 2730)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;-AGGAGCTTCGGGACTGTATCC-3\u0026rsquo;; 5\u0026rsquo;-GGGACATGGTGCATTCCAAAA-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eHMOX-1(Gene ID: 3162)\u003c/p\u003e\n\u003cp\u003e5\u0026rsquo;-GCCGTGTAGATATGGTACAAGGA-3\u0026rsquo;; 5\u0026rsquo;-AAGCCGAGAATGCTGAGTTCA-3\u0026rsquo;\u003c/p\u003e\n\u003cp\u003eThe 2\u0026Delta;DCt formula was used for the analysis and ribosomal protein S16 (RPS16) was used as a housekeeping gene.\u003c/p\u003e\n\u003cp\u003e5.8. Flow cytometry analysis\u003c/p\u003e\n\u003cp\u003eA375 were treated for 48 hours with AAO and UA (30 \u0026micro;g/mL and 3 \u0026micro;g/mL, respectively). Thereafter, ROS production was evaluated by using the H\u003csub\u003e2\u003c/sub\u003eDCFDHA probe. Likewise, mitochondrial mass and membrane potential were evaluated by using Mito-Tracker Green and TMRM probes as previously described. Samples were acquired on a BriCyte E6 flow cytometer (Mindray Medical Italy S.r.l., Milan, Italy), and data were analyzed with FlowJo software (TreeStar V.10; Carrboro, NC, USA).\u003c/p\u003e\n\u003cp\u003e5.9. Clinical trial\u003c/p\u003e\n\u003cp\u003eThe study was a 4-week, monocentric, randomized, double-blinded, placebo-controlled clinical trial assessing the safety and efficacy of an AAO-based topical formulation versus placebo in adults with moderate-to-severe hyperpigmentation. Conducted from February 2022 to December 2022 at the RD Cosmetics Lab, University of Naples Federico II, it adhered to UNI EN ISO 9001 standards for efficacy studies. The trial included a 15-day screening period, a 7-day washout, and a 4-week treatment phase. The study protocol (Unique Protocol ID: EAAO22G01) was revised by the Institutional Review Board of the Pharmacy Department and recorded on clinicaltrials.gov (ID: NCT07040345). The study was carried out according to ICH GCP, the Declaration of Helsinki and the SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation (12th revision). The informed consent was obtained from all participants, and the study followed CONSORT guidelines. The study flow is depicted in Figure 9.\u003c/p\u003e\n\u003cp\u003e5.9.1. Study population and randomization\u003c/p\u003e\n\u003cp\u003eEligible participants were women aged 40 to 65 with diagnosed photodamage and hyperpigmentation. A target of 40 participants was set based on power calculations, with 48 participants recruited to account for a 20% loss to follow-up. Full eligibility criteria are detailed in the Supporting Information. Exclusion criteria included the use of treatments for hyperpigmentation or topical retinoids and allergies to the formulation ingredients. Participants were enrolled at their second laboratory visit, sequentially numbered, and randomized in blocks of 20 at a 1:1 ratio to receive either the active treatment (w/o emulsion with 2.5%\u003csub\u003ew/w\u003c/sub\u003e AAO) or a placebo. Both treatments were identical in appearance and packaging. Blinding was maintained for both participants and observers.\u003c/p\u003e\n\u003cp\u003e5.9.2. Treatment\u003c/p\u003e\n\u003cp\u003eParticipants applied 2 fingertip units of the allocated test product, whose composition is reported in Table 2. In particular, Group A received the active treatment with 2.5% AAO, whereas Group B received the placebo. The lab\u0026rsquo;s technicians instructed to apply the assigned product twice daily, at least 12 hours apart, for 28 days. Training on product application was provided, with the first application supervised. Baseline measurements, including independent instrumental analysis and photographs, were conducted at the initial visit. Participants were reviewed at days 14 and 28, with pigmentation changes assessed using the melanin index (Mexameter MX 18) and skin luminance measured through L* and ITA\u0026deg; values (Skin-Colorimeter CL 400). Brown and UV spots resulting from photodamage were analyzed using VISIA 7th technology with RBX analysis \u003csup\u003e40\u003c/sup\u003e. Skin acceptability was previously tested with an occlusive patch test.\u003c/p\u003e\n\u003cp\u003e5.10. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eGraphPad Prism software version 9 (San Diego, CA, USA) was used for statistical analysis. t-test and ANOVA tests were used for comparison of two groups or multiple groups, respectively. The data were shown as mean percentage variation (%) \u0026plusmn; SEM. A p-value \u0026lt; 0.05 was considered statistically significant and was labelled with *; p-values \u0026lt; 0.01, 0.001, or 0.0001 were labelled with **, ***, or ****, respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAA: Annurca apple\u003c/p\u003e\n\u003cp\u003eAAO: Annurca apple oil\u003c/p\u003e\n\u003cp\u003eCREB: cAMP response element binding protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDCF: 2\u0026apos;,7\u0026apos;-dichlorofluorescin\u003c/p\u003e\n\u003cp\u003eDMEM: Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e\n\u003cp\u003eFLD: fluorescence detector\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGCLC: \u0026gamma;-glutamylcysteine ligase\u003c/p\u003e\n\u003cp\u003eHMOX-1: enzyme heme oxygenase\u003c/p\u003e\n\u003cp\u003eITA: Individual Typology Angle\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKA: kojic acid\u003c/p\u003e\n\u003cp\u003eMI: melanin index\u003c/p\u003e\n\u003cp\u003eMITF: microphthalmia-associated transcription factor\u003c/p\u003e\n\u003cp\u003eOD: optical density\u003c/p\u003e\n\u003cp\u003ePGI: Protected Geographical Indication\u003c/p\u003e\n\u003cp\u003eROS: reactive oxygen species\u003c/p\u003e\n\u003cp\u003eTYR: tyrosinase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTYRP: tyrosinase-related protein\u003c/p\u003e\n\u003cp\u003eUA: ursolic acid\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-MSH: alpha-melanocyte-stimulating hormone\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), and Ritamaria Di Lorenzo (R.D.L.);\u003c/p\u003e\n\u003cp\u003eData Curation: Ritamaria Di Lorenzo (R.D.L.), Maria Maisto (M.M.), and Giuseppe Ercolano (G.E.);\u003c/p\u003e\n\u003cp\u003eFormal Analysis: Lucia Ricci (L.R.), Vincenzo Piccolo (V.P.), Daniela Claudia Maresca (C.M.), Benedetta Romano (B.R.), and Adua Marzocchi (A.D.);\u003c/p\u003e\n\u003cp\u003eInvestigation: Ritamaria Di Lorenzo (R.D.L.), Lucia Ricci (L.R.), Vincenzo Piccolo (V.P.), Daniela Claudia Maresca (C.M.), Benedetta Romano (B.R.), and Adua Marzocchi (A.D.);\u003c/p\u003e\n\u003cp\u003eMethodology: Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), and Ritamaria Di Lorenzo (R.D.L.);\u003c/p\u003e\n\u003cp\u003eResources: Sonia Laneri (S.L.), Angela Ianaro (A.I.), and Giuseppe Ercolano (G.E.);\u003c/p\u003e\n\u003cp\u003eSupervision: Ritamaria Di Lorenzo (R.D.L.), Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), Sonia Laneri (S.L.), and Angela Ianaro (A.I.);\u003c/p\u003e\n\u003cp\u003eWriting – Original Draft Preparation: Ritamaria Di Lorenzo (R.D.L.), Maria Maisto (M.M.), and Giuseppe Ercolano (G.E.);\u003c/p\u003e\n\u003cp\u003eWriting – Review \u0026amp; Editing: Maria Maisto (M.M.), Giuseppe Ercolano (G.E.), and Ritamaria Di Lorenzo (R.D.L.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e The authors confirm that the data supporting the findings of this study are available within the article. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement:\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research received no external funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD\u0026rsquo;Mello, S. A. N., Finlay, G. J., Baguley, B. C. \u0026amp; Askarian-Amiri, M. E. 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Determination of free amino acids in isatidis radix by HILIC-UPLC-MS/MS. \u003cem\u003eBull Korean Chem Soc\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 197\u0026ndash;203 (2014).\u003c/li\u003e\n\u003cli\u003eZawodny, P., St\u0026oacute;j, E., Kulig, P., Skonieczna-żydecka, K. \u0026amp; Sieńko, J. VISIA Skin Analysis System as a Tool to Evaluate the Reduction of Pigmented Skin and Vascular Lesions Using the 532 Nm Laser. \u003cem\u003eClin Cosmet Investig Dermatol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 2187\u0026ndash;2195 (2022).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eDemographic characteristics of the study population.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 674px; height: 436.767px;\" width=\"674\" height=\"436.767\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e List of ingredients of AAO-based formulation (INCI name).\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"673\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIngredients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunctions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% w/w\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eGlyceryl-Stearate and PEG-100 Stearate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eEmulsifier\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eCetearyl Alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eConsistency factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eCetyl Ricinoleate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eEmollient/Consistency factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eCaprylic/Capric Triglycerides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eEmollient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e10.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eAqua\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eqs\u003csup\u003e1\u003c/sup\u003e to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eSodium Gluconate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eChelating agent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eCarbomer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eViscosizing agent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eAqua\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003ePhenoxyethanol (and) Ethylhexylglycerin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003ePreservative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eAAO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eActive ingredient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 248px;\"\u003e\n \u003cp\u003eNaOH 20% sol.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003epH modifier\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 150px;\"\u003e\n \u003cp\u003eQs to pH 5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e quantity sufficient.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ursolic acid, hyperpigmentation, melanogenesis, skin wellness","lastPublishedDoi":"10.21203/rs.3.rs-7028178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7028178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSkin-lightening molecules have gained widespread attention for their relevance in both cosmetic applications and the treatment of dermatological disorders. Hyperpigmentation and skin spots can be counteracted by suppressing oxidative stress, inhibiting tyrosinase activity, or regulating the expression of tyrosinase-related proteins (TYRP-1 and TYRP-2) and the microphthalmia-associated transcription factor (MITF). Among natural compounds, ursolic acid (UA), a triterpene primarily found in apple peel was investigated for its potential depigmenting properties. In this study, UA was extracted in oil form from Annurca apple (AAO) to formulate a standardized oleolite (784.40 ± 7.58 mg/mL) and evaluate its efficacy through in vitro and clinical models. AAO exhibited tyrosinase inhibitory activity and significantly reduced melanin content in A375 melanoma cells, accompanied by decreased expression of TYRP-1, TYRP-2, and MITF, as well as modulation of oxidative stress markers. These effects were confirmed in a randomized, double-blind, placebo-controlled clinical trial, in which a topical formulation containing 2.5% AAO significantly improved skin hyperpigmentation compared to placebo. Overall, the results highlight AAO as a promising natural agent for managing skin hyperpigmentation through multiple mechanisms, suggesting its potential utility in both cosmetic and dermatological settings.\u003c/p\u003e","manuscriptTitle":"Ursolic Acid-Based Oleolite for Hyperpigmentary Disorders: Molecular Mechanisms and Clinical Efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 15:51:22","doi":"10.21203/rs.3.rs-7028178/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-14T05:28:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T07:25:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40380089122614758289651646358669805726","date":"2025-08-11T06:08:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66638154900862071746118651023148400037","date":"2025-08-11T00:29:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T08:10:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232572079871305855619371903309837859242","date":"2025-07-24T22:46:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1331287295690100886427187539730494965","date":"2025-07-23T01:03:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-22T15:19:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-22T13:25:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-22T13:04:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-15T11:58:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-15T11:28:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf67c9a0-dfa1-4ad7-bb41-aecafc9754a0","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51958527,"name":"Biological sciences/Biochemistry"},{"id":51958528,"name":"Health sciences/Diseases"},{"id":51958529,"name":"Biological sciences/Drug discovery"},{"id":51958530,"name":"Health sciences/Health care"},{"id":51958531,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-12-15T16:00:53+00:00","versionOfRecord":{"articleIdentity":"rs-7028178","link":"https://doi.org/10.1038/s41598-025-29398-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-12 15:57:02","publishedOnDateReadable":"December 12th, 2025"},"versionCreatedAt":"2025-07-24 15:51:22","video":"","vorDoi":"10.1038/s41598-025-29398-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-29398-0","workflowStages":[]},"version":"v1","identity":"rs-7028178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7028178","identity":"rs-7028178","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}