Jasminum sambac Cell Extract as Antioxidant Booster against Skin Aging

Oxidative stress plays a major role in the skin aging process through the reactive oxygen species production and advanced glycation end products (AGEs) formation. Antioxidant ingredients are therefore needed in the skin care market and the use of molecules coming from plant cell cultures provide a unique opportunity. In this paper, the features of an hydroethanolic extract obtained by Jasminum sambac cells (JasHEx) were explored. The antioxidant and anti-AGE properties were investigated by a multidisciplinary approach combining mass spectrometric and bio-informatic in vitro and ex vivo experiments. JasHEx contains phenolic acid derivatives, lignans and triterpenes and it was found to reduce cytosolic reactive oxygen species production in keratinocytes exposed to exogenous stress. It also showed the ability to reduce AGE formation and to increase the collagen type I production in extracellular matrix. Data demonstrated that JasHEx antioxidant properties were related to its free radical scavenging and metal chelating activities and to the activation of the Nrf2/ARE pathway. This can well explain JasHEx anti-inflammatory activity related to the decrease in NO levels in LPS-stimulated macrophages. Thus, JasHEx can be considered a powerful antioxidant booster against oxidative stress-induced skin aging.


Introduction
Genetic intrinsic factors and extrinsic agents such as ultraviolet irradiation, infrared irradiation, xenobiotics and environmental pollutants cause the production of reactive oxygen species (ROS). They are generated by the activation of the mitochondrial respiratory chain, cytochrome p450 and NADPH oxidases [1]. ROS include free radicals (superoxide anion, hydroperoxyl radical, alkoxy radical and hydroxyl radical) and nonradical molecules (hydrogen peroxide, and singlet oxygen) [2]. When antioxidant systems are overwhelmed, ROS accumulate within cells and generate the so-called oxidative stress that is a main contributor to skin aging [3].
Indeed, oxidative stress causes both DNA damage and lipid and protein oxidation together with the triggering of the mitogen-activated protein kinases (MAPK) pathway (AKT, JNK, ERK and p38) [4]. This, in turn, promotes the expression of pro-inflammatory cytokines, growth factors and adhesive molecules through the stimulation of the transcription factors AP-1 and NF-κB. Moreover, MAPK pathway activation causes dermal matrix alterations by reducing collagen levels. In fact, it accelerates collagen breakdown by modulating the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors TIMPs and it reduces the synthesis of new collagen, by blocking the TGF-β type

Plant Tissue Cultures and Extract Preparation
A certified Arabian jasmine (Jasminum sambac) was obtained from a local nursery ("Ladre di Piante", Pistoia, Toscana Region, Italy). Jasminum sambac leaves were soaked in 70% ethanol (Sigma Aldrich, St. Louis, MO, USA) for 1 min and surface-sterilized with 1% (v/v) of commercial bleach supplemented with Tween 20 (Sigma Aldrich, St. Louis, MO, USA) for 8 min, followed by three rinses in sterile distilled water. Then, the leaves were excised into 0.5-1.0 cm pieces and cultured on full-strength MS medium [16] containing 3% (w/v) sucrose, 0.2 mg/L 2,4D and 8 g/L phyto-agar. The explants were monthly subcultured onto a fresh medium for three months. Once a yellow-green, friable and fast-growing callus was obtained, the plant cells were transferred to the liquid MS medium, supplemented with 3% (w/v) sucrose and 0.2 mg/L 2,4D. The suspension was stirred in a gyratory shaker at 110 rpm and 27 • C in dark climate room. The dark-grown cells were scaled up every week from small-scale to large-scale flasks, until liquid suspension cultures of about 177 g/L were reached. The preparation of Jasminum sambac cell culture hydro-ethanolic extract (JasHEx) was carried out by the addition of 2000 mL of a solution ethanol/water (90/10, v/v) to 500 g of cells. The mixture was homogenized 3 min at 1500 rpm and 6 min at 3800 rpm using a Grindomix GM300 knifemill (Retsch GmbH, Haan, Germany). The obtained suspension was stirred at 400 rpm for 2 h at 25 • C, avoiding light exposure. The suspension was then centrifuged at 6300 rpm for 10 min at 4 • C. The supernatant was removed, filtered and then concentrated under vacuum in a rotary evaporator (IKA RV8, IKA-Werke GmbH & Co., Staufen, Germany) set to 25 • C. Finally, the pH was brought to 7.0 with 10 N NaOH and then freeze-dried until gaining a fine powder.

UPLC-MS/MS Analysis for JasHEx Chemical Characterization
A biphasic butanol/water extraction was achieved. The butanolic fraction was desiccated and thawed in methanol (10 mg/mL) before the UPLC-MS/MS analysis, carried out on a Q-Exactive Classic Mass Spectrometer equipped with an UltiMate™ 3000 UPLC system (Thermo Scientific, Waltham, MA, USA). All the chromatographic runs were carried out as already described by Ceccacci et al. [17].

Global Natural Products Social Molecular Networking Analyses
For metabolite identification, Global Natural Products Social Molecular Networking (GNPS; https://gnps.ucsd.edu; Version 1.3.16-GNPS, UC San Diego on 14 November 2020) was employed [18]. All those MS and MSMS signals not assigned by GNPS were wisely examined and assigned accordingly to literature. Raw files were converted to mzXML format by MS Converter General User Interface software (ProteoWizard; Version 3; http:// proteowizard.sourceforge.net/project.shtml; Palo Alto, CA, USA, 94304 on 15 September 2020), before GNPS spectral library search. It was carried out using precursor ion mass tolerance of 0.025 Da, fragment ion mass tolerance of 0.02 Da, minimum matched peaks of 2 and score threshold of 0.7. The results were manually confirmed.

Skin cell Cultures and Explants
Immortalized Human Keratinocytes (HaCaT), bought from Addexbio Technologies (San Diego, CA, USA), were preserved in Dulbecco's Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) that was supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO, USA) in 95% air, 5% CO 2 , and humidified atmosphere at 37 • C. Human dermal fibroblasts (HDF) were preserved in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% of fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) in 95% air, 5% CO 2 , and humidified atmosphere at 37 • C. Skin explants, obtained from the skin of healthy female donors (aged 31 and 40) at the surgery center Villa Cinzia (Naples, Italy), were cultured in 24-transwell plates in DMEM/FBS plus antibiotics in air-liquid conditions at 37 • C in 5% CO 2 humidified air. All donors had given their written informed consent for the use of the skin tissues, according to the Declaration of Helsinki.

Cytosolic ROS Assay in H 2 O 2-Stressed HaCaT Cells
For this process, 1.8 × 10 4 HaCaT were seeded in 96-well plates and grown for 20 h. The cells were then treated for 2 h with different concentrations of JasHEx (0.0006%, 0.002% and 0.006% p/v) or with 500 µM ascorbic acid, used as the positive control. After that, they were washed in PBS (Phosphate-buffered saline) and incubated at 37 • C with 100 µL/well of a solution containing: 10 mM of Hepes, 1.3 mM CaCl 2 , 1 mM MgSO 4 , 5 mM of glucose and 5 µM CM-H2DCFDA (5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate, Invitrogen). After 45 min, a PBS wash was performed and the baseline fluorescence intensity of the cells was measured at 535 nm (excitation 485 nm), using the instrument EnVision (PerkinElmer, Waltham, MA, USA). Then, the oxidative stress was induced by adding 450 µM H 2 O 2 and the fluorescence of the samples measured after 30 min.

Enzyme-Linked Immunosorbent Assay (ELISA) for AGE Detection in Glyoxal Stressed Human Dermal Fibroblast (HDF) Cells
For this step, 1.5 × 10 4 HDF were seeded in 96-well plates and grown for 2 days. After washing with PBS, cells were fixed for 10 min with 100 µL of 4% formaldehyde in PBS. Subsequently, they were treated with JasHEx (0.0006% and 0.002% p/v) or the positive control of 1 µM Aminoguanidine in the presence of 0.5% glyoxal at 50 • C for 1 week. After incubation, they were processed for an enzyme-linked immunosorbent assay (ELISA) using a specific antibody against AGE (abcam ab23722).

ImmunoHistoFluorescence Assay on Methyl-Glyoxal Stressed Skin Explants for Fibrillin-1 Detection
Skin explants were derived from two patients, 31 and 40 years old. From each skin biopsy, three punches were generated for each treatment occurring at the air-liquid interface. The first day, the punches were treated with JasHEx (0.002% and 0.006% p/v) or the positive control (1 mM Aminoguanidine) and after 24 h, 500 µM methyl-glyoxal was added. The treatments were refreshed up each two days to total of seven days. At the end of the period, the punches were processed for histological analysis, fixed in 4% PFA, incubated in 15% sucrose, then in 30% sucrose and cryostored in OCT compound (Optimal cutting temperature) at −80 • C. Cryosection of 5 µm were obtained with the cryostat CM1520 Leica (Leica Biosystems, Buffalo, IL, USA). Slides with cryosections were hydrated for 30 min in PBS and placed in a "blocking" solution (6% BSA, 5% serum, 20 mM MgCl 2 , 0.2% Tween) for 1 h. Subsequently, they were incubated with the primary anti-Fibrillin 1 antibody (MA5-12770, Thermo Scientific, Waltham, MA, USA). for 16 h at 4 • C. The slides were washed with PBS for 30 min and then incubated with the secondary anti-rabbit Alexa-Fluor 546 antibody (A11035, Thermo Scientific, Waltham, MA, USA) for 1 h. The nuclei were stained with DAPI (4 , 6-5 diamidino-2-phenylindole) 1 g/mL in PBS for 10 min. The images were acquired with a fluorescence microscope and analyzed with the ImageJ software (Version 1.53a, National Institutes of Health, USA).

AlphaLISA Assay to Measure Procollagen Type I C-Peptide (PIP) Content
In this step, 8 × 10 3 HDF were seeded in a 96-well plate and treated for 24 h with JasHEx (0.0006%, 0.002% and 0.006% p/v) or with TGF-β (2.5 ng/mL). After treatment, the cells were processed according to the instructions of alpha LISA hPIP collagen kit provider (AL353HV, (PerkinElmer, Waltham, MA, USA)).

Nrf2 Luciferase-Based Transcription Activation Assay
Here, 6 × 10 3 HaCaT cells in 96-well plate were seeded and grown for 16 h. After that, they were subjected to a Nrf2 luciferase-based transcription activation assay, using the ARE reporter kit BPS Bioscience (San Diego, CA, USA, #60514). A transfection-ready ARE luciferase reporter vector (containing a firefly luciferase gene under the control of ARE responsive elements located upstream of a minimal promoter) together with an internal control (a constitutively expressing Renilla luciferase vector) were transiently co-transfected into HaCaT cells using X-TREME gene HP DNA transfection reagent (Roche, Basilea, Switzerland, #6366244001). After transduction for 24 h, cells were treated for 2 h with the extract (0.0006%, 0.002% and 0.006% p/v) or the positive control Resveratrol (50 µM). After that, they were subjected to the luciferase assay with the Dual-Glo Luciferase Assay System (Promega, Rome, Italy #E2920). Briefly, cells were incubated with firefly luciferase substrate for 10 min prior to measuring luminescence in a 96-well plate reader (Victor Nivo, Waltham, MA, USA). The ratio of luminescence from firefly and Renilla was calculated to normalize and compare Nrf2 transcriptional activity.

Analysis of the Expression of SOD-1(NM_000454.5) and OH-1(NM_002133.3) Genes in HaCaT Cells
For this process, 1.5 × 10 5 HaCaT cells per well were grown in 6-well plates for 16 h and incubated for 6 h with the extract (0.002% and 0.006% p/v) or 50 µM Resveratrol as the positive control. At the end of incubation, total RNA was extracted using the "GenElute™ Total RNA Purification" kit (from Sigma-Aldrich (Saint Louis, MI, USA) and treated with DNase I (Thermo Scientific, Waltham, MA, USA) at 37 • C for 30 min, to remove genomic DNA contaminant. 500 ng of total RNA was retro-transcribed using the enzyme Reverse transcriptase (Thermo Scientific, Waltham, MA, USA). Semi-quantitative RT-PCRs were conducted using the pair of universal primers 18S primer/competimer (Invitrogen-Thermo Scientific, Waltham, MA, USA) as internal standards. The PCR products were separated on 1.5% agarose gel, viewed using the iBright instrument (Invitrogen-Thermo Scientific, Waltham, MA, USA). The sequences of the primers used for amplification were the following: HsSOD1Fw: GAAAGTAATGGACCAGTGAAGG; HsSOD1Rv: ATTGGGCGATCCCAATTACACC; OH-1Fw GAACTTTCAGAAGGGTCAGG; OH-1Rv GCTCAATGTTGAGCAGGAA.

Nitric Oxide Assay in LPS-Stimulated RAW 264.7
NO concentration was determined in RAW 264.7 murine macrophages, seeded at a concentration of 1.5 × 10 5 cells/well in 96-well plates for 24 h, and pre-treated with the extract (0.0006%, 0.002% and 0.006% p/v) or with 10 µM TPCK (positive control) for 2 h, before the incubation with 2 µg/mL LPS for 18 h. The amount of NO, converted into nitrite, was calculated by adding Griess reagent (solution of N-(1-naphthyl)ethylenediamine and sulfanilic acid, Invitrogen-Thermo Scientific, Waltham, MA, USA) and, after 30 min, the absorbance was measured at 540 nm by the multiwell-plate reader (EnVision, PerkinElmer, Waltham, MA, USA).

Qualitative and Quantitative Analysis of Jasminum sambac Cell Culture Hydro-Ethanolic Extract (JasHEx)
UPLC-MS/MS analysis of JasHEx was performed and high-resolution spectrometric data were analyzed using Global Natural Products Social Molecular Networking (GNPS), a web-based mass spectrometry system that aids in the annotation of natural products (NPs) [18]. In particular, a GNPS spectral library was performed to achieve online dereplication. Chemical species not identified by GNPS were assigned accordingly to the literature. As shown in Figures 1 and 2 and Table 1, more than 50 compounds belonging to several classes of secondary metabolites, mainly polyphenols and terpenes, were identified. Indeed, JasHEx extract contains phenolic acid derivatives, lignans (secoisolariciresinol, nortrachelogenin and matairesinol) and triterpenoids (arjunolic acid, asiatic acid, maslinic acid, oleanolic acid and ursolic acid).

Qualitative and Quantitative Analysis of Jasminum sambac Cell Culture Hydro-Ethanolic Extract (JasHEx)
UPLC-MS/MS analysis of JasHEx was performed and high-resolution spectrometric data were analyzed using Global Natural Products Social Molecular Networking (GNPS), a web-based mass spectrometry system that aids in the annotation of natural products (NPs) [18]. In particular, a GNPS spectral library was performed to achieve online dereplication. Chemical species not identified by GNPS were assigned accordingly to the literature. As shown in Figure 1 and 2 and Table 1, more than 50 compounds belonging to several classes of secondary metabolites, mainly polyphenols and terpenes, were identified. Indeed, JasHEx extract contains phenolic acid derivatives, lignans (secoisolariciresinol, nortrachelogenin and matairesinol) and triterpenoids (arjunolic acid, asiatic acid, maslinic acid, oleanolic acid and ursolic acid).   Furthermore, a Feature-Based Molecular Networking (FBMN) job was also carried out. It is able to group related NPs within a network since structurally similar molecules share similar MS/MS fragmentation patterns [20]. The FBMN job allowed us to identify several chlorogenic acids, reported in Figure 2 and Table S1 [23].
The ions shown in Figure 2 and Table S1 and circled in orange were assigned to oxidation products of caffeic acid derivates, since they shared the same MS 2 peaks at m/z 177.02 and 133.03. In particular, species with parent ion at m/z 513. 11 [22].
Moreover, the extract contains feruloyl glycosides, circled in blue in Figure 2 (Table S1). In the MS2 spectrum they showed fragments at m/z 193.05 and 175.04, characteristic of the feruloyl moiety and/or at m/z 337.09, due to the loss of hexosyl moiety and one molecule of water and/or at m/z 295.08, 265.07 and 235.06, derived from by cross-ring cleavage of the remaining sugar residue [24]. In particular, the species at m/z 517. 16  Finally, the identified lignans and triterpenes were quantified. Quantification methods were validated as reported in Table 2. Nortrachelogenin and ursolic acid are the most abundant lignan and triterpene, respectively, present in JasHEx (Table 3).

Cytosolic ROS Detection in H 2 O 2-Stressed HaCaT Cells
Since the large majority of secondary metabolites identified in the extract such as chlorogenic acids, lignans and triterpenes possess well-known antioxidant activity [25][26][27], the JasHEx effect on cytosolic ROS was evaluated. Therefore, HaCaT cells were treated for 2 h with the extract (0.0006%, 0.002% and 0.006% p/v) or with the positive control ascorbic acid (500 µM), incubated with an indicator for ROS and then stressed with H 2 O 2 (450 µM). After oxidation, the indicator yields a fluorescent adduct. As shown in Figure 3A, the H 2 O 2 -induced stress increased cytosolic ROS formation by 50% and this was reduced by almost 30% both in case of ascorbic acid and JasHEx pretreatment.

AGE Detection in Glyoxal Treated HDF
Since AGE formation is dependent on oxidation reactions, JasHEx anti-glycation activity by an enzyme-linked immunosorbent assay (ELISA) was evaluated. It allowed us to detect, by a specific antibody, AGE products in human dermal fibroblasts (HDF), treated or not with the extract (0.0006% and 0.002% p/v), in the presence of 0.5% glyoxal at 50 °C for one week. Aminoguanidine (AG) 1 µM was used as the positive control. The treatment with 0.5% glyoxal stimulated the formation of AGE products by 90%, whereas the incubation with JasHEx at both concentrations was able to reduce it by 20% ( Figure  3B). HaCaT cells, (B) to inhibit AGE formation in glyoxal treated HDF and (C) to increment fibrillin-1 content in methylglyoxal stressed skin explants. Panel C has been obtained measuring the fluorescence intensity related to the photographs of skin sections (D) in which fibrillin-1 has been highlighted with a specific antibody labeled with fluorophore (green) and the nuclei (blue) have been stained with 4 ,6-diamidine-2-phenylindole (Dapi). Panel (E) shows the increase of collagen type I synthesis.

AGE Detection in Glyoxal Treated HDF
Since AGE formation is dependent on oxidation reactions, JasHEx anti-glycation activity by an enzyme-linked immunosorbent assay (ELISA) was evaluated. It allowed us to detect, by a specific antibody, AGE products in human dermal fibroblasts (HDF), treated or not with the extract (0.0006% and 0.002% p/v), in the presence of 0.5% glyoxal at 50 • C for one week. Aminoguanidine (AG) 1 µM was used as the positive control. The treatment with 0.5% glyoxal stimulated the formation of AGE products by 90%, whereas the incubation with JasHEx at both concentrations was able to reduce it by 20% ( Figure 3B).

Fibrillin-1 Detection in Methylglyoxal Stressed Skin Explants
On the basis of these results, to confirm the antiglycation effect in a physiological context, the JasHEx effect was tested on methylglyoxal stressed skin explants. Since fibrillin-1, an ECM protein essential for the dermal elastic network, is highly sensitive to glycation, we decided to use it as a biomarker. Indeed, the increase in fibrillin-1 glycation induced by methylglyoxal significantly alters its conformational structure and the modified protein is no longer recognized by the used antibody [28]. Therefore, skin explants were treated with JasHEx (0.002% and 0.006% p/v) before and after 500 µM methylglyoxal addition and the content of fibrillin-1 was detected by Immuno-Histo-Fluorescence assay. Aminoguanidine (AG) 1 mM was used as the positive control. As shown in Figure 3C and D, the addition of 500 µM methylglyoxal reduced fibrillin-1 levels by 30%. The incubation with the extract was able to protect fibrillin-1 from methylglyoxal-induced glycation. In particular, the treatment with JasHEx at the concentration of 0.002% p/v increased fibrillin-1 content by 35%, similar to the positive control.

Analysis of Collagen Type I Synthesis
To measure the JasHEx effect on the synthesis of collagen type I, further than its protective effect from glycation, Procollagen Type I C-peptide (PIP) was used as indicator. Indeed, the collagen type I is synthesized as procollagen that contains peptide sequences (propeptides) at both the amino-terminal and carboxy-terminal ends, essential for the winding of procollagen into triple helix. These propeptides are cleaved during secretion and the triple helix collagens polymerize into extracellular fibrils [29]. Therefore, the amount of released PIP stoichiometrically reflects the amount of synthesized collagen. Thus, HDF were treated for 24 h with JasHEx (0.0006%, 0.002% and 0.006% p/v) or with TGF-β (2.5 ng/mL), used as the positive control, and processed for an AlphaLISA assay to measure PIP levels. As shown in Figure 3E, the incubation with all extract concentrations significantly increased the content of PIP.

Analysis of Nrf2/ARE Pathway in HaCaT Cells
Due to the antioxidant and antiglycation activity of JasHEx, the effect of the extract on Nrf2/ARE (nuclear-related factor 2/antioxidant response element) pathway was investigated, since it is the most pivotal endogenous antioxidative system studied so far [30]. Therefore, an Nrf2 luciferase-based transcription activation assay on HaCaT cells, incubated for 2 h with the extract (0.0006%, 0.002% and 0.006% p/v) after transduction, was performed. The treatment with 0.006% p/v JasHEx increased luciferase activity linked to Nrf2 by 28% ( Figure 4A), similar to 50 µM resveratrol, used as the positive control.

Analysis of OH-1 and SOD-1 Gene Expression in HaCaT Cells
The effect of JasHEx in HaCaT cells on the expression of Nrf2 gene targets such as Superoxide dismutase 1 (SOD-1) and Heme oxygenase-1 (HO-1) was also tested. To do this, HaCaT cells were treated with JasHEx (0.002% and 0.006% p/v) for 6 h and then SOD-1 and OH-1 expression was analyzed by RT-PCR. The results, reported in Figure 4B,C, demonstrated that the extract, increased the expression of both genes, as the positive control resveratrol (50 µM). on Nrf2/ARE (nuclear-related factor 2/antioxidant response element) pathway was investigated, since it is the most pivotal endogenous antioxidative system studied so far [30]. Therefore, an Nrf2 luciferase-based transcription activation assay on HaCaT cells, incubated for 2 h with the extract (0.0006%, 0.002% and 0.006% p/v) after transduction, was performed. The treatment with 0.006% p/v JasHEx increased luciferase activity linked to Nrf2 by 28% ( Figure 4A), similar to 50 µM resveratrol, used as the positive control.

Analysis of OH-1 and SOD-1 Gene Expression in HaCaT Cells
The effect of JasHEx in HaCaT cells on the expression of Nrf2 gene targets such as Superoxide dismutase 1 (SOD-1) and Heme oxygenase-1 (HO-1) was also tested. To do this, HaCaT cells were treated with JasHEx (0.002% and 0.006% p/v) for 6 h and then SOD-1 and OH-1 expression was analyzed by RT-PCR. The results, reported in Figure 4B and

NO Determination in LPS-Stimulated RAW 264.7 Cells
Since Nrf2 also plays a role in counteracting NF-κB-driven inflammatory response and since inducible nitric oxide synthase (iNOS) is activated through the NF-κB pathway [31,32], JasHEx anti-inflammatory activity was evaluated performing a nitric oxide assay. RAW 264.7 cells were treated with the extract (0.0006%, 0.002% and 0.006% p/v) or with the positive control TPCK (10 µM) and then, were stressed with LPS (2 µg/mL). The amount of NO was revealed by adding Griess reagent and the absorbance was measured at 540 nm. As shown in Figure 4D, JasHEx reduced the levels of NO approximately by 30% at all tested concentrations.

Discussion
Here, an hydroethanolic extract derived from Jasminum sambac cell cultures (JasHEx) was studied. Its GNPS-aided mass spectrometry based chemical characterization revealed the presence of phenolic acid derivatives (mainly chlorogenic acids), lignans (secoisolariciresinol, nortrachelogenin and matairesinol) and triterpenes (arjunolic acid, asiatic acid, maslinic acid, oleanolic acid and ursolic acid). All of these secondary metabolites possess well-known antioxidant properties. Indeed, chlorogenic acids [25] and lignans [26,33,34], thanks to their phenolic moiety, have free radical scavenging and chain-breaking antioxidant activities: they donate hydrogen atoms to free radicals, giving rise to phenoxyl radicals stabilized by resonance, and thereby inhibiting the propagation of radical chain reactions and other biological oxidants. Moreover, they act as secondary antioxidants by binding metal ions (Fe(III) and Cu(II)) able to catalyze oxidative processes. Moreover, thanks to an accurate quantitative analysis, it emerged that JasHEx contains relevant amounts of arjunolic acid, asiatic acid, maslinic acid, oleanolic acid and ursolic acid: all these triterpenes can also act as good free radical scavengers, chain-breaking antioxidants or transition metal chelators [35][36][37][38][39].
On the basis of these results, the antioxidant activity of JasHEx was evaluated. It was able to reduce cytosolic ROS production in H 2 O 2-stressed keratinocytes. Furthermore, since the conversion step of Amadori products into AGEs is dependent on oxidation reactions [9], the antiglycation potential of the extract was tested. It was confirmed by both in vitro and ex vivo assays: JasHEx reduced AGE formation in glyoxal treated HDF and methylglyoxal stressed skin explants, an already used model to highlight the anti-glycation activity of natural substances [28].
In addition to this, JasHEx also showed an extracellular matrix booster effect increasing the production of collagen type I, that is highly sensitive to glycation [10][11][12] and whose levels are significantly reduced by oxidative stress [4].
In vitro assays proved that the antioxidant properties of JasHEx, such as those of chlorogenic acids [40] and triterpenoids [27], are not only related to free radical scavenging and metal chelating activities, but also to the enhancement of Nrf2/ARE pathway. This is the most pivotal endogenous antioxidative system studied so far: when cells are exposed to stressing conditions, Nrf2 dissociates from its cytoplasmic repressor kelch-like ECHassociated protein 1 (Keap1) and translocates to the nucleus where interacts with ARE, activating the transcription of its target genes; the expression of these genes involved in detoxification, NADH regeneration, glutathione (GSH) and thioredoxin (TXN)-based antioxidant system, lipid, eme and iron metabolism increases cell resistance to oxidative stress [41,42].
Moreover, the extract also showed an anti-inflammatory activity, decreasing the release of NO in LPS-stimulated macrophages. This effect is also related to the triggering of Nrf2/ARE pathway: Nrf2 upregulates the expression of HO-1, that, creating a more reducing environment, inhibits the activation of the pro-inflammatory transcription factor NF-κB [31].

Conclusions
On the basis of the chemical composition and the biological activity proved by in vitro and ex vivo experiments, JasHEx can be considered as a natural powerful antioxidant booster against oxidative stress-induced skin aging. Institutional Review Board Statement: Ethical review and approval were waived for this study, due to the experiment involving only cosmetic product testing.