Nonclinical evaluation of a Vitis vinifera extract towards a novel antiaging cosmetic ingredient

Skin aging is regulated by multiple physiological processes, such as oxidative stress. Natural products have been considered as a promising source of antioxidant compounds. As a result, few innovative products on the market based on natural products tackle additional underlying mechanisms of skin aging.


| INTRODUC TI ON
Aging is regulated by multiple physiological processes triggered, in great part, by oxidative stress.Increased reactive oxygen species (ROS) levels in the skin have long been related to aging, and ROS accumulation can be stimulated by intrinsic as well as extrinsic factors.
2][3] The antiaging market, led by the United States and Brazil, is one of the largest global cosmetics markets.In 2020 alone, this industry generated a total of US$ 58 billion in revenues.Different forecast analyses estimate that, by 2026, the global antiaging market will reach approximately US$ 88 billion, with an average growth of 7% per year. 4,5smetics products are developed using approaches to increase the concentration of active compounds in the formulations, modulate their delivery (i.e., via nanotechnology), improve sensory properties, or combine active compounds for a synergistic effect.
7][8] The development of novel chemical entities or active ingredients is challenging, as they should have an excellent safety profile with adequate efficacy and pharmaceutical properties.Nevertheless, innovation is imperative as new, superior therapeutic solutions have been long awaited in dermatology to tackle underlying mechanisms of skin aging that go beyond oxidative stress.In addition, the research strategy must be aligned not only with scientific readouts, but also must consider the global regulatory scenario.Since 2013, the use of animals to test the efficacy of cosmetic products has been banned and the data package must be obtained according to the (EC) No 1223/2009 regulation, focusing on alternative methods of testing. 9e use of natural ingredients represents an important strategy when launching new brands in the cosmetics industry and introducing novel therapeutic solutions.Natural products have been used extensively for different applications, including UV protection, antioxidant defense, DNA protection, emollience, and others. 10,11The constituents of different species of grapevine (Vitis spp.) have been investigated owing to the interest in their agricultural, enological, and biological properties. 12,13In particular, the inverse correlation between wine consumption and coronary disease incidence (the socalled French paradox) fostered considerable attention into the clinical implications of biologically active constituents in fruits of Vitis vinifera. 14Further, several biological activities have been reported for V. vinifera extracts in vitro and in vivo, including antibacterial effects in pomace, 15 steam, 16 and leaves 17 ; antiviral in leaves, 18 antiparasitic in leaves 19 ; and anti-inflammatory in fruits, 20 leaves, 21 and tendrils. 22lyphenolic compounds, such as flavonoids and their derivatives, represent the major active constituents present in V. vinifera. 23e to the complexity involved in understanding how two or more molecules can act synergistically, plant-derived extracts may represent more viable options than combination therapies to modulate multiple pharmacological targets, generating efficacy profiles far superior to that of small-molecule compounds acting on a single target.However, different extracts of the same plant species may possess distinct phytochemical fingerprints influenced by external factors, such as environmental or processing parameters.Thus, a precise control of agricultural, manufacturing, and quality variables is critical to ensure consistency in the biological response.
Herein, we investigated the biological properties of a novel V. vinifera extract (codified as ACH37 extract).We assessed its effects on ROS accumulation, inflammation, extracellular matrix (ECM) remodeling, and cellular senescence.Moreover, we observed ex vivo its protective effect against UV-induced histological and gene expression changes in reconstructed skin as well as permeation in human skin.The current findings underscore the preclinical efficacy of the ACH37 extract, suggesting its action through multiple mechanisms assessed both in vitro and ex vivo.These mechanisms are collectively responsible for the aging process and their significant modulation by the ACH37 extract makes it a promising candidate as an antiaging cosmetics ingredient.values were calculated using the nonlinear fitting of a curve built from at least five concentrations and are reported as mean (CI 95%).

| MATERIAL S AND ME THODS
The statistical analysis was done using the software GraphPad Prism 9.0 and specific details of the statistical tests applied in each assay are described in the next sections.The null hypothesis was rejected when the confidence level was >95% (p < 0.05).

| ACH37 extract
The ACH37 extract is a polyphenol-rich V. vinifera extract produced by Naturex®, part of Givaudan.Aché Laboratórios Farmacêuticos owns intellectual property related to the use of the extract in a cosmetic formulation (Patent Application WO2023010188A1).
V. vinifera fruit peels that would be discarded by wineries are used as used in the production process.The raw material is used as is, not being dried nor standardized regarding particle size.The fresh peels are submitted to percolation using water as solvent [ratio 1:10 (kg/L) peels: water].The pH is adjusted to seven if needed and the extraction takes places at 25°C for 2 h.After the first extraction, the extract is fractionated with 70% ethanol to remove undesirable metabolites [ratio 1:10 (L/L) aqueous extract: ethanol].The supernatant is concentrated using a falling film technique and then spray dried (intemperature of 270°C and out-temperature of 160°C) to obtain a fine powder (95% of the particles <420 μm).The major compounds present in the final extract are phenolic derivatives, such as gallic acid, malvidin, quercetin, anthocyanins, procyanidin, and others, which represent more than 70% (700 mg/g) of the ACH37 composition.Analytical details are described in the Appendix S1.

| Cytotoxicity assays
Prior to each cellular or tissue-based assays, the cytotoxicity of the ACH37 extract and other test substances was assessed using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) following the method described by Kumar et al (2018). 24The chosen test concentrations were deemed not cytotoxic (p > 0.05 in comparison to negative controls or less than 20% of reduction in metabolic activity) to those specific cell lines.The cytotoxicity data is available in Appendix S1.

| COX-2 expression in human fibroblasts (PCR)
Human fibroblasts (CCD-1059Sk cell line) were seeded in a 96-well plate (1 × 10 4 cells/well) and incubated at 37°C, 5% CO 2 , until adhesion.Then, the cells were treated in duplicate with the test items in different concentrations and incubated for 1 h.DMSO (1%) was used as negative control and dexamethasone (2 μg/mL) as positive control.After treatment, the cells were challenged with LPS (1 μg/ mL, obtained from E. coli) and incubated at 37°C, 5% CO 2 for 18 h.The cells were then collected with TRIzol® (Invitrogen) to obtain total RNA, following the procedure indicated by the supplier.The total RNA was quantified in spectrophotometer and approximately 30 ng were used for synthesizing complementary DNA (cDNA) using the enzyme Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) (Promega™).The cDNA was used to quantify the mRNA for COX-2 through a multiplex real-time PCR (StepOne Plus, Applied Biosystems) using a specific assay for human COX-2 (PTGS2, FAM-labeled™).The data were normalized employing the expression of GAPDH (VIC-labeled).The results of each treatment were analyzed applying one-way ANOVA followed by Dunnet test.

| Culture of full-thickness reconstructed skins (FTSK)
The experiment was conducted using an adaptation from Bernerd et al (2008) methodology. 25Five-day-old FTSK were placed in the assay medium and incubated in the dark for 24 h at 37°C, 5% CO 2 .
The medium was replaced each day for three more days.At day 5, FTSK were irradiated with UVA-6 J/cm 2 and UVB-310 mJ/cm 2 using a SOL500 Sun Simulator equipped with H1 filter (Dr.Hölne, AG).In experiments in which the FTSK were treated with a test substance, the medium was replaced with fresh medium containing no test substance prior to the irradiation.At the end of irradiation, the medium was replaced again to continue the treatment, and the FTSK were incubated for 24 h.The irradiation protocol was repeated each day for three more days.The analyses were performed at day 9. Nonirradiated and nontreated FTSK were used as controls.All experimental conditions were performed in triplicate.

| MMP-1 expression in FTSK (ELISA)
After the 9-day protocol described previously, the culture subnatants were collected and stored at −20°C for MMP-1 quantification.The ELISA quantification was done using the DuoSet Human Total MMP-1 kit (R&D systems) following the supplier's instructions.
The lowest and highest limits of detection were 6250 pg/mL and 400 000 pg/mL, respectively.The result was compared to the nontreated control T-student test.

| Telomerase activity
The experiment was conducted using an adaptation of Herbert et al (2006) methodology. 28Primary human fibroblasts cultivated in fibroblast medium (0.5 × 10 4 cells/well) were treated with different concentrations of the test substances (stock solution in DMSO 100%, final DMSO concentration of 0.5%) for 6, 12, and 24 h.The relative telomerase activity in whole cell lysates was measured by the Telomeric Repeat Amplification Protocol (TRAP), modified for real-time, quantitative PCR analysis (Q-TRAP).Ct values of the experimental conditions were normalized to the total protein concentration and compared to a standard curve (in HeLa cells, R 2 = 0.9745) to quantify the relative telomerase activity (RTA).All experimental conditions were performed in triplicate.The results of each treatment time were compared to nontreated controls using two-way ANOVA followed by Dunnet test.

| Ex vivo human skin absorption and intracellular antioxidant activity (DCFH-Da)
The experiment was conducted using an adaptation of Aggarwal et al (2019) and Aranda et al (2013) methodologies. 29,30The human skin fragments were collected from three healthy donors, phototype III, aged from 29 to 38 years old.The procedure was approved by the Ethics Committee CAAE (Protocol 82685618.9.0000.5514,Universidade São Francisco).The skin fragments were fractionated into pieces of approximately 1.5 cm 2 , placed in culture plates (Corning, USA) with DMEM culture medium containing 10% fetal bovine serum (FBS) and 0.1% gentamicine.To assess skin permeation and the ex vivo antioxidant activity, the skin fragments were treated with 15 mg of the test articles.After 48 h, the skin was submitted to 10 J/cm 2 of UV using an UVA Cube 400, SOL 500 H1 filter, and UV Meter device (Honle UV America Inc., MA, USA).Then, the skin fragments were embedded in Tissue-Tek® and, subsequently, the sections were washed and incubated with a solution of dichloro-dihydro-fluorescein diacetate (DCFH-DA).
The sections were analyzed under a microscope (OLYMPUS, JAP-BX53) using CellSens software (OLYMPUS).The fluorescence intensity parameter emitted by the DCFH-DA probe oxidation was evaluated using the ImageJ software (version 1.48, Arbitrary Units -A.U.).Each of the three experiments were done in triplicate.The results of each treatment were compared to nontreated controls using two-way ANOVA followed by Tukey multiple comparison test.

| Antioxidant screening assay (DPPH)
The experiment was conducted using an adaptation of Brand-Williams et al. (1995) and Chen et al. ( 2004) methodologies. 31,325 μL of the ACH37 extract in ethanol:H2O (1:1) and 75 μL of DPPH methanolic solution (final concentration of 60 μM = 24 μg/mL) were added to a 96-well plate.After 40 min in the dark, the absorbance of the samples was read in a UV-spectrophotometer at 517 nm.The ACH37 extract was evaluated in seven concentrations (1, 3, 10, 30, 100, 300, and 1000 μg/mL) to observe the dose-response relationship.From the results, the quantity of remaining DPPH was calculated using the following equation: The antioxidant potentials of ascorbic acid (vitamin C) and resveratrol were evaluated using the same methodology for comparative purposes.The antioxidant activity index (AAI) for a given compound correlates the DPPH concentration with IC 50 and is calculated using the following equation: The results were obtained from one experiment run in triplicate.Reduced cytochrome C is quantified by its absorption at 550 nm and its concentration is proportional to the concentration of the ion superoxide. 33e ACH37 extract and other test items (stock solution in The results were obtained from one experiment run in triplicate. The enzyme superoxide dismutase (10 U) was used as positive control and the absence of xanthine oxidase as blank control.The results of each treatment were analyzed using one-way ANOVA followed by Dunnet test.

| Elastase inhibition
The assay was run using a commercial kit with the recombinant enzyme neutrophil elastase (BioVision, Inc) reconstituted in a buffer solution.The methodology was indicated by the kit manufacturer and followed accordingly.At first, the activity of the enzyme was determined and compared to their activity after treatment with the inhibitor SPCK (30 μM) to validate the kit (data not shown).The en-

| The ACH37 extract has antioxidant properties in solution
We employed the α, α-diphenylβ-picrylhydrazyl (DPPH) assay to evaluate the antioxidant activity of the ACH37 extract in solution.
DPPH is a stable radical used to evaluate the radical scavenging ability of active ingredients. 34The extract was studied at concentrations ranging from 1 to 1000 μg/mL, and the IC 50 determined from the dose-response curve was 23.1 μg/mL (Figure 1A).The antioxidant activity index of 1.02 means that approximately 1 μg of extract is able to scavenge 1 μg of DPPH, which is suggestive of an excellent scavenging efficiency.The extract in vitro potency was comparable to the ones of vitamins C and E, and resveratrol, all of them commonly used in antiaging cosmetics formulations (Table 1).
In addition, we assessed whether the ACH37 extract could scav-  35 The therapeutic potential of formulations with SOD-like activity is currently explored for several pathologies, including cancer, inflammatory diseases, ischemia, and aging. 36In this work, SOD at 10 units/mL scavenged 61% of O 2 − when compared to the negative control.The ACH37 extract suppressed O 2 − as potently as SOD at the concentrations of 1 and 10 μg/mL.Vitamin C and resveratrol also exhibited SOD-like effects, as opposed to vitamin E (Figure 1B).

| The ACH37 extract downregulates COX-2, IL-8, and IL-1β expression
Inflammation is a major consequence of ROS accumulation within the skin.Moreover, inflammation is chronically active and linked to the remodeling of dermal and epidermal structures, determining the overall appearance of aged skin. 3,37In lipopolysaccharide (LPS)-activated human fibroblasts, we detected upregulation of cyclooxygenase-2 (COX-2), responsible for the synthesis of inflammatory mediators from arachidonic acid.Pretreatment of cells with the ACH37 extract in the low concentration range of 3-10 μg/mL suppressed COX-2 expression by twofold.At higher concentrations, COX-2 expression was completely suppressed (Figure 2A).

| The ACH37 extract inhibits the activity of elastase and matrix metalloproteases (MMP1, 3, and 12)
Loss of the elastic fiber network increases during aging and its reversal is a main target for antiaging therapies. 38,39Elastase is one of the enzymes implicated in this process, catalyzing the metabolism of the matrix protein elastin.The ACH37 extract inhibited the activity of recombinant elastase, displaying an IC 50 of 233.0 μg/mL (Figure 3A).
A major class of enzymes that participates in the metabolism of several ECM proteins are the matrix metalloproteases (MMPs).There are more than 20 different MMP isozymes, which are classified depending on their structural features and substrates. 40MMP overexpression can be induced via ROS dysregulation, which is commonly observed in aged skin.An ex vivo experiment in FTSK revealed that UV-A and UV-B exposure upregulates the expression and release of MMP-1 over twofold when compared to the levels in the nonexposed control (Figure 3B).This finding agrees with the findings of a previous study. 41To understand whether the ACH37 extract was able to halt the increase of MMP-1, we run an inhibition assay using the recombinant enzyme.MMP-1 is classified as a collagenase.The inhibitory effect of the extract was also investigated in MMP-3 (stromelysin) and MMP-12 (metalloelastase) to cover a range of structural diversity within the isozymes.Interestingly, we observed that the ACH37 extract inhibits the in vitro activity of MMP-1, 3, and 12, with IC 50 values ranging from 28.4 to 264.7 μg/mL (Figure 3C).The difference in potency suggests a degree of selectivity for specific isozymes in the superfamily.However, additional experiments are needed to fully comprehend what could be the impact of this selectivity.
As reference, we assessed vitamin C and E activities in the elastase and MMP inhibition assays.However, no inhibition was observed at concentrations up to 300 μg/mL.[44]

| The ACH37 extract prevents telomere shortening and promotes the activity of telomerase
Telomere shortening is one of the major events underlying cellular senescence.Telomerase is an enzyme responsible for the prevention of telomere shortening.However, in aging tissues, the activity of telomerase is not sufficient to halt or delay cellular senescence. 45eatment with the ACH37 extract prevented telomere shortening in human fibroblasts cultured under standard and oxidative stress conditions (10 μM H 2 O 2 ).We evaluated the median length, 20 th percentile length, and the percentage of telomeres shorter than 3 Kbp after treatment with the extract.In cells cultivated under standard conditions for 2 weeks, the ACH37 extract concentrations of 12.The treatment of human fibroblasts with the ACH37 extract in higher concentrations (50 μg/mL) was able to upregulate the relative telomerase activity for up to 24 h after treatment (Figure 4D).While the treatment of cells with vitamin C (50 μg/mL) also upregulated relative telomerase activity, this effect ceased after 6 h of treatment, indicating the need of successive dosing (Figure S1).Genes involved in ROS regulation (TXNDR1, FOXO3, HMOX1, MT1E, and MT1H) were highly upregulated in FTSK exposed to UV radiation.UV triggers ROS generation, and our observations were expected to represent the physiological attempt of counterbalancing oxidative stress in FTSK.We also confirmed that, in addition to LPS, UV also induces the expression of PTGS2 (COX-2), IL-1β, and IL-8 (Figure 2).Further, MMP-1 and MMP-3 were upregulated by UV and after treatment their expression returned to basal levels.

| The ACH37 extract protects against UV radiation-induced changes in reconstructed skin
Therefore, we suggest that the ACH37 extract can act in different molecular levels to maintain ECM cohesion through MMP, from gene expression to direct inhibition (Figure 3).Additional details on the affected genes are provided in the Appendix S1.
Treatment with the ACH37 extract reversed the above-described expression changes to an extent similar to that observed in nonexposed FTSK (p < 0.01), indicating that the extract might act as a protective agent against UV-induced ROS and their effects on the skin (Figure 5A,B).The effect was observed even at the lowest concentration evaluated (0.22 μg/mL).Different concentrations of the extract modulated expression to a different extent for most downregulated genes (p < 0.05).However, these results did not indicate a dose-response relationship, as no difference in the expression of upregulated genes were noted between groups receiving different concentrations of the ACH37 extract.Histological analysis revealed that repeated FTSK exposure to UV led to less pronounced morphological characteristics and the detachment of skin layers.Moreover, we observed keratinocytes with pycnotic nuclei in the epidermis, suggestive of UV irradiationinduced apoptosis in these cells (Figure 5C).Treatment with the ACH37 extract protected against some of these effects.More specifically, we observed a decrease in stratum corneum deformation and epidermis-dermis detachment.These observations further support the protective effects of the ACH37 extract noted on other phenotypic assays and gene expression changes.

| The ACH37 extracts absorbs UV radiation
Phenolic substances present in herbal preparations confers them the property to absorb UV light. 46Therefore, we assessed whether the effects seen in gene regulation in FTSK could have been caused by direct absorption of UV radiation by the ACH37 extract.To this end, we compared its UV absorption spectra with those of two common chemical sunscreens, namely avobenzone and octyl methoxycinnamate.
The area under the curve comparison revealed that the ACH37 extract absorbed the equivalent to 8.3% of UV-B radiation absorbed by octyl methoxycinnamate at the same concentration.The absorption of UV-A by the extract was negligible.This result demonstrates that part but not the totality of the effects might be explained by direct absorption of UV light in FTSK.In addition, it suggests that the ACH37 extract may contribute to increase the effect of commercial sunscreens when added to formulations, which would favor the overall antiaging efficacy.However, additional testing is necessary to confirm this assumption and understand the overall impact on solar protection efficiency.

| The ACH37 extract permeates human skin explants and promotes free radical scavenging ex vivo
In view of the efficacy profile of the ACH37 extract, we proceeded to attest its potential as a cosmetics ingredient in a more translational biological system.Specifically, we conducted a functional permeability evaluation in human skin explants.The skin explants were irradiated with UV-A, which represents the majority of UV radiation in sunlight and is known to increase ROS even in deeper skin layers. 47,48In this assay, the irradiation led to a threefold increase acts as an electron donor, neutralizing ROS that is generated via cellular metabolism or external factors.In addition, some phenolic compounds bind to iron ions, normally linked to ROS generation, as an alternative antioxidant mode of action. 51,52We demonstrated that the ACH37 extract scavenged DPPH and O 2 − in vitro with similar potency as observed for well-established small-molecule antioxidants.Other herbal derivatives have their antioxidant properties well described by the scientific literature. 53For instance, Pinus maritima bark extract, Quercus robus wood extract and Cinnamomum zeylanicum bark extract showed a free radical scavenging potential of 94.5%, 88.60%, and 84.43%, respectively, when assessed using the DPPH method- ology.An analysis of their chemical composition shows that the main class of metabolites present in the extract is polyphenolic compounds (361 mg/g in P. maritima, 397 mg/g; in Q. robus; and 309 mg/g in C. zeylanicum).In comparison to them, we highlight the higher polyphenolic content of the ACH37 extract (>700 mg/g).On the contrary, herbal extracts that have lower polyphenolic contents have minimal DPPH inhibitory activity.A good example of this is the Zinziger officinale root extract, which is composed by only 26 mg/g of polyphenols. 54inically, the efficacy of topical cosmetics products containing antioxidants has been extensively evaluated in end points related to skin aging.For instance, treatment with vitamin E could reduce wrinkling both in animal models and in clinical trials. 55Other studies have shown that resveratrol-containing cosmetics products improved skin firmness, elasticity, wrinkle depth, and other aging-related parameters. 56,57Vitamin C is one of the main antioxidants used in topical cosmetics products and is clinically efficacious, especially, in the treatment of photoaged skin. 58,59Different derivatives of vitamin C have been designed to improve stability, absorption, and efficacy.
Those have shown effects on different pillars of skin aging. 60The body of evidence suggest that in vitro antioxidant efficacy can be a good predictor of in vivo antiaging efficacy-which supports the application of such assays in the investigation of a new cosmetics ingredient candidate.
One of the consequences of ROS dysregulation in the skin is chronic inflammation, leading to an increase of circulating inflammatory mediators. 61,62The pro-inflammatory enzyme COX-2 is upregulated by UV radiation and is crucial in the development of nonmelanoma skin cancer, such as basal and squamous cell carcinomas. 63,64UV light and other factors affecting epidermal integrity contribute to the upregulation of IL-1β, IL-10, and TNFα, responsible for chemotaxis and other inflammatory events. 65,66Herein, the ACH37 extract reversed the LPS-and UV-induced upregulation of COX-2 in a dose-dependent manner.Moreover, IL-1β and IL-8 levels were dramatically reduced after UV irradiation in ACH37-treated cells.Taken together, the above-described findings emphasize the anti-inflammatory mechanism of the ACH37 extract.
During aging, the loss of the ECM proteins elastin and collagen contributes to skin atrophy and is associated with alterations in the mechanical properties of connective tissue. 38,39Elastin is cleaved by the serine protease elastase, whereas different isoforms of MMP degrade collagen.UV exposure and ROS contribute to the upregulation of elastase and MMPs.Inflammatory cytokines promote the activity of elastase, which in turn mediates the activation of proMMP-1. 67,68rther, ECM protein metabolism generates fragments that contribute to the establishment of an inflammatory microenvironment. 69e ACH37 extract directly inhibited the in vitro activity of recombinant elastases MMP-1, MMP-3, and MMP-12.Taken together with the aforementioned effects on ROS scavenging and inflammation, it is safe to argue that the ACH37 extract synergistically modulates ECM remodeling in direct and indirect manners.
In the present study, the ACH37 extract also delayed cellular senescence in human fibroblasts.One of triggers of senescence is the shortening of telomeres-DNA-protein structures located at the ends of chromosomes, whose function is to maintain the structural integrity of the genetic material.At each replication cycle, the length of telomeres is slightly reduced by the replication-related machinery. 70,713][74] Herein, the ACH37 extract increased the median telomere length and the 20th percentile length, while reducing the percentage of telomeres <3 Kbp in cells receiving up to 8 weeks of treatment.In cells cultivated under oxidative stress, the extract protective effects were apparent for up to 6 weeks of treatment.Considering the mechanism underlying telomere shortening, we suggest that the ACH37 extract may mediate its protective role via antioxidant and anti-inflammatory activities.It should also be noted that the relative telomerase activity in fibroblasts increased for up to 24 h after treatment with the ACH37 extract.Direct modulation of the enzyme activity is a possible contributor of the observed effects.Unfortunately, the lack of validated translational models hinders the exploration of the protective potential of the ACH37 extract in a more complex system.
Despite the widespread reports of the V. vinifera potential for antiaging purposes, only a few of its active ingredients have been extensively studied in terms of their efficacy.6][77] The interplay among UV, ROS, inflammation, and ECM degradation illustrates the complex molecular scenario of aging.The ACH37 extract acted on these multiple underlying mechanisms at both gene expression and phenotypic levels in direct and indirect manners.The current findings also highlight the protective role of the ACH37 extract against UVinduced damage.Further, appropriate skin permeation is an essential aspect for cosmetics ingredients. 78As observed in Figure 6, in the presence of reactive oxygen species, DCFH emits green fluorescence. 79The fluorescence in epidermis and dermis was significantly reduced when the cells were treated with the ACH37 extract (0.1%), showing that compounds present in the extract permeate the human skin, reach different cells layers, and promote the expected radical scavenging effect.Therefore, although not directly evaluated in this study, it is fair to suggest that other effects of the ACH37 extract seen in vitro could also translate to efficacy in different layers of human skin excerpts.

| CON CLUS ION
The nonclinical investigation presented in this work shows the action of the ACH37 extract in multiple targets, mechanisms, and biological models related to the skin aging process.This suggest that the ACH37 extract is a promising active ingredient, eligible for further studies to assess its efficacy on skin aging end points in a clinical trial.

2. 2 . 6 |
Telomere length determination The experiment was conducted using an adaptation of Canela et al (2007) and Kimura et al (2010) methodologies. 26,27Primary human fibroblasts (1.5 × 10 3 cells/well) cultivated in fibroblast medium (changed every 2-3 days) were treated with different concentrations of the test substances (stock solution in DMSO 100%, final DMSO concentration of 0.5%) for 2, 4, 6, and 8 weeks.At the end of the treatment period, cells were fixed with methanol: acetic acid (3:1) and treated with pepsin to digest the cytoplasm.The telomeres were hybridized with a fluorescent peptide nucleic acid probe (PNA, sequence Alexa488-OO-CCCTA ACC CTA ACC CTAA, Panagene) and the DNA stained with DAPI.The cells were incubated in mounting medium overnight at 4°C.Quantitative image acquisition and analysis are performed on a High Content Screening Opera System (Perkin Elmer), using the Acapella software (v 1.8, Perkin Elmer).Fifteen independent images were captured, using a 40 × 0.95 NA water immersion objective.UV and 488 nm excitation wavelengths are used to detect the DAPI and A488 signals, respectively.The fluorescence intensity of A488 was measured and exported to the Columbus software (v 2.8, Perkin Elmer).Telomere length distribution and median telomere length are calculated with a proprietary software (Life Length) using a standard curve (R 2 > 0.99).All experimental conditions were performed in quintuplicate.The results of each treatment time were compared to the respective nontreated controls using unpaired T-Student or Mann-Whitney U tests.

0. 1 M
PBS), 0.1 mL of xanthine oxidase (0.04 units/mL), 100 μM of xanthine (in 0.1 M PBS), and 1 mL of 100 UI/mL cytochrome C (in 0.1 M PBS) were mixed and the volume of the solutions was uniformized with phosphate buffer (0.1 M, pH 7.5).The solutions were incubated for 10 min at 25°C.The absorbance at 550 nm was read with a Hitachi U-2000 UV-VIS spectrophotometer.The percentage of scavenging activities (%) was calculated using the following equation:

2 . 4 |
zyme solution was added to a 96-well plate together with the inhibitor.The plate was incubated for 5 min at 37°C, and then 25 μL of the fluorescent substrate solution was added.Immediately after, the fluorescence was read in 400/505 nm (excitation/emission) and then, again, after 30 min.The fluorescence (RFU) values at both times were used to calculate the percentage of enzyme inhibition using the following equation:The same procedure was used to evaluate the percentage of inhibition caused by the ACH37 extract and other test items (stock solution in DMSO 10%, final DMSO concentration of 0.5%).The results were obtained from one experiment run in triplicate.DMSO 0.5% was used as negative control.The results of each treatment were compared to nontreated controls using one-way ANOVA followed by Dunnet test.2.3.4 | Metalloproteases inhibition (MMP-1, MMP-3, and MMP-12)The assays were run using commercial kits with the recombinant enzymes MMP-1, MMP-3 (BioVision, Inc), and MMP-12 (Abcam) reconstituted in a buffer solution.The methodology was indicated by the kits' manufacturer and followed accordingly.At first, the activity of each enzyme was determined and compared to their activity after treatment with the inhibitors GM6001 (at 10 nM for MMP-1 and 1.5 μM for MMP-3) and NNGH (at 1.3 μM for MMP-12) to validate the kits (data not shown).For MMP-1, 50 μL of the enzyme solution was added to a 96-well plate together with the inhibitor.The plate was incubated for 5 min at 37°C, and then 50 μL of the fluorescent substrate solution was added.Immediately after, the fluorescence was read in 490/520 nm (excitation/emission) and then, again, after 30 min.For MMP-3, 50 μL of the enzyme solution was added to a 96-well plate together with the inhibitor.The plate was incubated for 10 min at 37°C and then 10 μL of the fluorescent substrate solution was added.Immediately after, the fluorescence was read in 325/393 nm (excitation/emission) and then, again, after 30 min.For MMP-12, 20 μL of the enzyme solution was added to a 96-well plate together with the inhibitor.The plate was incubated for 50 min at 37°C and then 10 μL of the fluorescent substrate solution was added.Finally, the fluorescence was read in 328/420 nm (excitation/ emission) and then, again, after 10 min.The fluorescence (RFU) values at both times were used to calculate the percentage of enzyme inhibition using the following equation:The same procedure was used to evaluate the percentage of inhibition caused by the ACH37 extract and other test items (stock solution in DMSO 10%, final DMSO concentration of 0.5%).The results were obtained from one experiment run in triplicate.DMSO 0.5% was used as negative control.The results of each treatment were compared to nontreated controls using one-way ANOVA followed by Dunnet test.Other assays2.4.1 | UVA and UVB spectraThe absorbance spectrum of the ACH37 extract (0.01% in ethanol:water 1:1) in the wavelength range of 200-420 nm was obtained in a SpectraMax M5 spectrophotometer.The absorbance spectra of octyl-methoxycinnamate (0.001% in ethanol) and avobenzone (0.0002% in ethanol) in the same wavelength range Scavenging activity ( % ) = 1 − absorbance of samples at 550nm absorbance of blank at 550nm × 100 Elastase inhibition ( % ) = RFU 2 inhibitor − RFU 1 inhibitor RFU 2 blank − RFU 1 blank × 100 MMP inhibition ( % ) = RFU 2 inhibitor − RFU 1 inhibitor RFU 2 blank − RFU 1 blank × 100 were obtained to be used as reference for UVA (290-320 nm) and UVB (320-400) absorption, respectively.The absorbance curves were integrated, and the Lambert-Beer law was used to calculate the integrated absorbance of the ACH37 extract in concentrations equivalent to those of the comparators.From the results, it was possible to estimate how similar the UVA and UVB absorption of the ACH37 extract was in comparison to the ones of commercial sunscreens.

enge superoxide anions (O 2 −). O 2 −
is toxic to different cellular components.Superoxide dismutase (SOD) catalyzes O 2 − dismutation to hydrogen peroxide (H 2 O 2 ), which is subsequently converted to oxygen and water via catalase or glutathione peroxidase.Therefore, SOD maintains basal nontoxic O 2 − levels that are essential for cellular viability.
To further investigate the anti-inflammatory properties of the ACH37 extract, we measured the gene expression of the proinflammatory cytokines IL-8 and IL-1β in full-thickness reconstructed skin (FTSK) after exposure to UV-A and UV-B radiation, which are known to shift the ROS balance in skin and trigger inflammatory responses.UV exposure upregulated IL-8 and IL-1β expression by 10to 40-fold, respectively.Concomitant treatment with the ACH37 extract reduced their expression to nonirradiated control levels.The effect was observed even at submicrogram concentrations of 0.22-0.66μg/mL (Figure 2B).F I G U R E 1 The antioxidant activity of ACH37.(A) Dose-response curve of ACH37 antioxidant activity in solution.(B) The antioxidant activity of ACH37 in solution was similar to that of superoxide dismutase (10 U/mL).Bars represent the mean ± SEM of one experiment in triplicate.*p > 0.05 compared to the SOD group.*p > 0.05 compared to the basal group.(ANOVA/Dunnet).

5 and 3 .a
1 μg/mL led to significant changes in the evaluated parameters, indicating a protective effect exerted by the extract.Under oxidative stress conditions (10 μM H 2 O 2 ), the concentration of 12.5 μg/ mL led to an improvement in all parameters evaluated, in particular the reduction of the percentage of telomeres shorter than 3 Kbp (Figure 4A-C).Additional results at 4-, 6-, and 8-weeks are available in the Appendix S1.TA B L E 1 IC 50 and antioxidant activity indexes (AAI) of ACH37 extract, vitamins C and E, and resveratrol.P compared to the ACH37 extract group.F I G U R E 2 ACH37 suppressed inflammatory marker expression.(A) Relative mRNA expression of COX-2 in LPS-stimulated human fibroblasts.ACH37 treatment suppressed the inflammatory response.Bars represent the mean ± SEM of triplicates.Dexamethasone + LPS was used as a positive control (+).(B) Relative mRNA expression of cytokines IL-8 and IL-1β in FTSK exposed to UV-A/B radiation for 4 days.The experiment was done in duplicate, and the samples were pooled for PCR.Bars represent the mean ± SEM of the expression relative to three housekeeping genes (GAPDH, RPS28, and RPS9).*p < 0.001 compared to the control.

F I G U R E 4
Exposure to UV radiation-induced changes (FC >2) in the expression of twenty-eight genes in FTSK (Figure 5A,B).Genes related to ECM cohesion (COL1A1, COL3A1, COL4A1, FLG, KRT1, KRT10, DSC1, F I G U R E 3 ACH37 inhibits ECM metabolism-related enzymes.(A) Inhibitory effect of the ACH37 extract on elastase activity.(B) UVA-A and UV-B irradiation of FTSK upregulates the release of MMP-1 in the culture medium.The dashed line represents the limit of quantification for the assay.(C) Inhibitory effect of the ACH37 extract on MMP-1, 3, and 12 activities.Bars represent the mean ± SEM of one experiment in triplicate.IC 50 values are reported as the mean (CI 95%).Gray bars (−) in the graphs represent negative controls.*p < 0.05 compared to the control.Treatment with ACH37 for 2 weeks prevents telomere shortening in fibroblasts cultured under standard and oxidative stress conditions.(A) Median length.(B) 20 th percentile length.(C) Percentage of telomeres shorter than 3 Kbp in length.(D) Relative telomerase active (RTA).Bars represent the mean ± SEM of one experiment in quintuplicate.Gray bars in the graphs (−) represent untreated controls, *p < 0.05, ***p < 0.001, ****p < 0.0001 compared to the control.and DSG1) comprised the main class of genes downregulated by UV radiation.Genes related to DNA repair, apoptosis, and cell differentiation were downregulated (PCNA, CALML5, and CASP14) or upregulated (XPA and ERCC3).These changes in gene expression were associated with the upregulation of growth factors (EGF and FGF2) in UV-exposed cells, suggestive of a possible mechanism of UV-induced carcinogenicity.

F I G U R E 5
Treatment with ACH37 for 4 days protects FTSK against UV radiation-induced changes.(A) Downregulated genes.(B) Upregulated genes.Red color in the scales in (A) and (B) represents altered relative expression, either up or downregulated, normalized to three housekeeping genes (GAPDH, RPS28, and RPS9).All treatment concentrations protected against UV radiation-induced expression changes in comparison to the control (p < 0.01).(C) Histological analysis confirms that ACH37 protected FTSK from some deleterious effects after 4 days of UV exposition (HE staining, 40x objective lens).
in ROS compared to the nonirradiated control group (p < 0.0001), as measured by fluorescence intensity (DCFA-Da).The treatment of the skin with the ACH37 extract (0.1%) resulted in a steady decrease in fluorescence intensity, demonstrating it scavenged the UV-induced radicals.No statistical difference was seen between the scavenging efficacy of the ACH37 extract (0.1%) and a vitamin C containing cream (10%) used as a comparator.Figure6Cshows that the ACH37 extract permeated and scavenged the radicals present in inner skin layers.The results clearly demonstrate the ACH37 extract potential of permeating human skin and promoting radical scavenging effects-in 100-fold lower concentrations when compared to vitamin C. F I G U R E 6 ACH37 permeates human skin ex vivo and is capable of scavenging UV-induced free radicals.DCFH reacts with ROS emitting green fluorescence.Cell nucleus emits blue fluorescence (DAPI).(A) Human skin explants basal fluorescence (Control).(B) Human skin explants +10 J/cm 2 of UV (UV control).(C) Human skin explants +10 J/cm 2 UV + ACH37 (0.1%).(D) Human skin explants +10 J/cm 2 UV + Vitamin C (10%).Bars represent mean ± SEM of three experiments in triplicate.# p > 0.0001 compared to control group.*p < 0.0001 compared to UV-treated group.Multiple classes of biologically active secondary metabolites can be isolated from plants.These secondary metabolites harbor a range of protective functions against environmental stress factors. 49Further, their therapeutic potential has been explored by different cultures since ancient times. 50Natural extracts challenge the structure-and ligand-based paradigms commonly employed in drug discovery programs.Their unique chemical composition allows the engagement of multiple biological targets by different molecules contained in the active ingredient.This multitarget approach is essential to efficiently tackle the multifactorial nature of many pathologies.Herein, we demonstrated the ability of the polyphenol-rich V. vinifera ACH37 extract to modulate different aging-related molecular factors in nonclinical assays.The chemical structure of polyphenolic compounds present in the extract comprises fundamentally an aromatic ring bearing at least one hydroxyl group.This chemical moiety