Anti-Wrinkling Effect of 3,4,5-tri-O-caffeoylquinic Acid from the Roots of Nymphoides peltata through MAPK/AP-1, NF-κB, and Nrf2 Signaling in UVB-Irradiated HaCaT Cells

Nymphoides peltata has been widely used pharmacologically in traditional Chinese medicine to treat heat strangury and polyuria. The aim of this study was to isolate the bioactive components from N. peltata and evaluate their potential use as antioxidant and anti-wrinkle agents. Phytochemical investigation of the methanolic extract of N. peltata roots led to the isolation of 15 compounds (1–15), which were structurally determined as α-spinasterol (1), 3-O-β-D-glucopyranosyl-oleanolic acid 28-O-β-D-glucuronopyranoside (2), 4-hydroxybenzoic acid (3), protocatechuic acid (4), vanillic acid (5), p-coumaric acid (6), caffeic acid (7), ferulic acid (8), neochlorogenic acid (neo-CQA) (9), chlorogenic acid (CQA) (10), cryptochlorogenic acid (crypto-CQA) (11), isochlorogenic acid B (3,4-DCQA) (12), isochlorogenic acid A (3,5-DCQA) (13), isochlorogenic acid C (4,5-DCQA) (14), and 3,4,5-tri-O-caffeoylquinic acid (TCQA) (15). Of these 15 compounds, compound 2 was a new oleanane saponin, the chemical structure of which was characterized by 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data and high-resolution electrospray ionization mass spectrometry (HRESIMS), as well as chemical reaction. Biological evaluation of the isolated compounds revealed that 3,4,5-tri-O-caffeoylquinic acid (TCQA) significantly improved Nrf2 levels in an Nrf2–ARE reporter HaCaT cell screening assay. TCQA was found to potently inhibit the Nrf2/HO-1 pathway and to possess strong anti-wrinkle activity by modulating the MAPK/NF-κB/AP-1 signaling pathway and thus inhibiting MMP-1 synthesis in HaCaT cells exposed to UVB. Our results suggest that TCQA isolated from N. peltata might be useful for developing effective antioxidant and anti-wrinkle agents.


Introduction
The skin is the largest body organ, and it protects internal organs from physical harm and chemical irritants, and plays an important role in maintaining skin homeostasis [1].However, complex interactions between multiple intrinsic and extrinsic factors cause skin aging [2].Photoaging due to continuous ultraviolet (UV) exposure is a major contributor to skin aging and accounts for more than 80% of facial aging [3].In particular, UVB irradiation can penetrate the epidermis and dermis and induce oxidative stress by generating reactive oxygen species (ROS) within cells and tissues, which can lead to skin inflammation, aging, and skin cancer [4].Nuclear factor erythroid-2-related factor 2 (Nrf2) is activated by oxidative stress and regulates antioxidant activity and cellular redox levels by neutralizing ROS and reactive electrophiles [5][6][7][8].In other words, when exposed to oxidative stress, Nrf2 dissociates from Keap1 and binds to antioxidant response element (ARE) to induce the expression of antioxidant-related enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and thereby inhibits ROS-induced oxidative damage and skin aging [9,10].
Photoaging is characterized by the activation of key transcription factors, including mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) [11].These factors promote collagen degradation in skin tissue by inducing matrix metalloproteinase-1 (MMP-1), which leads to an abnormal skin structure [12,13].MAPKs are phosphorylated and activated by ROS and oxidative stress, and they enter the nucleus and upregulate the transcription factors AP-1 and NF-κB [4,14].AP-1 increases the expression of MMPs, including MMP-1, MMP-3, and MMP-9, which degrade the extracellular matrix (ECM) and basement membrane components [15][16][17][18].In particular, MMP-1 is a collagen-degrading protease that promotes the degradation of procollagen type-I, a crucial constituent of ECM; thus, it contributes to skin aging and wrinkle formation [19,20].NF-κB is another transcription factor that increases MMP-1 levels in the dermis and is activated by UVB-generated ROS [21].Furthermore, activation of the NF-κB pathway results in the synthesis of proinflammatory enzymes and cytokines, leading to inflammation and tissue damage, enhanced MMP-1 production, and skin aging [22][23][24].Therefore, agents with antioxidant properties that promote Nrf2 activation and inhibit the MAPK/NF-κB/AP-1 signaling pathway are considered potential anti-photoaging and anti-wrinkle agents.For this reason, research is being actively conducted to identify phytochemicals with antioxidant, anti-aging, and anti-wrinkle effects [25,26].

Plant Material
N. peltata roots were collected at the Hantaek Botanical Garden Foundation (Yongin-si, Gyeonggi-do, Republic of Korea) and authenticated by Dr. Jung Hwa Kang.Voucher specimens (PNU-0040) were deposited at the Medicinal Herb Garden, Pusan National University.

Isolation of Compounds from N. peltata Extract
Dried roots of N. peltata (2.8 kg) were first ground to a powder, soaked in 100% methanol (MeOH), ultrasonicated for 90 min at room temperature twice, and immediately freeze-dried.The yield of MeOH extract obtained was 15.25% (427 g).This extract was then suspended in 2 L of H 2 O and sequentially extracted with n-hexane (Hex), ethyl acetate (EA), and n-butanol (n-BuOH) to obtain three fractions: NPH (80.3 g), NPE (24 g), and NPB (108 g), respectively.

Luciferase Reporter Gene Assay
HaCaT cells were seeded into 24-well plates, cultured for 24 h, and co-transfected with ARE-Luc reporter plasmid (300 ng/well) and internal control plasmid pRL-SV-40 (5 ng/well) using the FuGENE ® 4K Transfection Reagent (Fugent LLC, Madison, WI, USA).Twenty-four hours after transfection, cells were treated with the indicated concentrations of TCQA (15) for an additional 24 h.Luciferase activities of cell lysates were then measured using the Dual-Luciferase ® Reporter Assay System according to the manufacturer's instructions (Promega, Madison, WI, USA).To determine transfection efficiencies, luciferase activities were expressed as ratios of Renilla luciferase activity (SV40).

Real-Time Quantitative PCR (qPCR)
HaCaT cells were seeded at 2 × 10 5 cells/well in 12-well plates.Then, 24 h later, they were pretreated with the indicated concentrations of TCQA (15) for 4 h, washed with PBS, and treated with UVB (15 mJ/cm 2 ) and TCQA (15) for 24 h when total RNA was isolated.This was carried out using an RNeasy mini kit (Qiagen, Hilden, Germany) and reverse transcribed using a RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).Real-time PCR was conducted using QuantaStudio 6 pro (Thermo Fisher Scientific) using SYBR1 Green (Power SYBR Green PCR Master Mix; Applied Biosystems, Foster City, CA, USA).PCR settings were as follows: initial incubation for 2 min at 50 • C, denaturing for 10 min at 95 • C, followed by 40 cycles of PCR (15 s at 95 • C and 60 s at 60 • C).The primer sets used were MMP-1 (accession no.DQ399597), forward 5 -GCC CAG ATG TGG AGT GCC TG-3 and reverse 5 -GTT TGC TCC CAG CGA GGG TT-3 ; and GAPDH (accession no.NM_001357943), forward 5 -ACA CCC ACT CCT CCA CCT TT-3 and reverse 5 -TGC TGT AGC CAA ATT CGT TG-3 .The qPCR data were analyzed using the QuantaStudio 6 pro System (Thermo Fisher Scientific).Transcript levels were normalized versus GAPDH.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism software v4.0 (Graph-Pad, La Jolla, CA, USA), and results are presented as the means ± SDs of 2 to 3 independent experiments.One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test for independent samples was used to determine the significances of intergroup differences, and statistical significance was accepted for p-values < 0.05: # p < 0.05, ## p < 0.01, and ### p < 0.001; * p < 0.05, ** p < 0.01, and *** p < 0.001.
Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 18 absolute configuration for both glucose and glucuronic acid as the D-form.The anomeric coupling constants (J = 7.5 Hz for glucose and J = 8.0 Hz for glucuronic acid) were indicative of the β-form, confirming the sugar units as β-D-glucose and β-D-glucuronic acid, respectively [41,42].Finally, the aglycone, derived from acid hydrolysis, was identified as oleanolic acid by comparison of the optical rotation and NMR data [41,42].Accordingly, the chemical structure of compound 2 was elucidated as 3-O-β-Dglucopyranosyl-oleanolic acid 28-O-β-D-glucuronopyranoside, and named peltatasaponin A.

Effects of TCQA on MMP-1 Expression in HaCaT Cells Exposed to UVB
The relative MMP-1 mRNA expression levels in HaCaT cells were measured by qRT-PCR.In comparison to the CON group, the NC group showed a two-fold increase in MMP-1 mRNA expression.However, the MMP-1 mRNA expression level after treatment with TCQA was significantly decreased by 1.7-fold at 5 µM, 1.5-fold at 10 µM, and 2fold at 20 µM, compared to the NC group (Figure 7A).After UVB treatment, MMP-1 concentration measured by ELISA increased to 9142 pg/mL and TCQA treatment after UVB exposure reduced MMP-1 concentrations to 8152 pg/mL at 5 µM, 7619 pg/mL at 10 µM, and 7700 pg/mL at 20 µM (Figure 7B).

Discussion
Photoaging is a significant extrinsic factor that causes skin aging, and the increase in ROS caused by UV exposure upregulates the MAPK/NF-κB/AP-1 pathway and Nrf2-ARE signaling pathway and causes skin aging and wrinkling [46,47].Recently, to prevent photoaging, natural products with antioxidant and anti-aging activities have been actively studied [25,26].N. peltata is a perennial aquatic plant in the family Menyanthaceae and has been used in Traditional Chinese Medicine to treat heat strangury and polyuria [27,48].Extracts of this plant have been reported to have anti-inflammatory and anti-tumor activities [30,48].Furthermore, we previously reported on the anti-atopic and antioxidant activities of the EtOH extract of N. peltata [31].In this study, we isolated and identified 15 phytochemicals, including a new oleanane-type triterpenoid saponin, 3-O-β-D-glucopyranosyl-oleanolic acid 28-O-β-D-glucuronopyranoside, in a methanolic extract of N. peltata roots and evaluated their protective effects on UVB-induced cellular injuries in HaCaT cells.Of these 15 compounds, α-spinasterol, ferulic acid, CQA, and TCQA markedly enhanced Nrf2-ARE luciferase activity; the antioxidant effects of α-spinasterol, ferulic acid, and CQA have been previously reported [43][44][45].Therefore, we conducted further experiments on TCQA.
UV exposure causes oxidative stress and accelerates skin aging [49], and activation of the Nrf2 signaling pathway inhibits oxidative stress by regulating various antioxidant enzymes, including HO-1 [50].These help to maintain skin homeostasis by inhibiting DNA damage, cell membrane destruction, and lipid damage [50].HO-1 regulates oxidative stress and immune responses [51], and the Nrf2 pathway has been demonstrated to suppress ROS increases in UVB-exposed HaCaT cells and, thus, to alleviate apoptosis and reduce skin damage [52,53].In the present study, TCQA exhibited strong antioxidant activity by upregulating Nrf2 and HO-1 expressions and reducing UVB-induced ROS levels in HaCaT cells.Furthermore, UVB exposure and oxidative stress induce the activation of NF-κB [54], which is involved in cellular senescence and apoptosis by causing protein damage [55].They also induce the nuclear translocation of p65, which stimulates various inflammatory cells and the expressions of various inflammatory cytokines, like TNF-α and IFN-γ [56,57].In addition, inflammation mediated by TNF-α enhances ROS production and increases NF-κB activation and the expressions of other cytokines [58].In addition, activated NF-κB has been demonstrated to be involved in the synthesis of proteins that inhibit collagen production in dermal fibroblasts and HaCaT cells [59], disrupting skin homeostasis and inflammatory cycles, and causing rapid skin aging [60].We also observed that TCQA suppressed NF-κB, p65, and IκBα transcript levels in UVB-and TNF-α/IFN-γ-induced HaCaT cells, which indicated that TCQA inhibits oxidative stress and inflammatory pathways.
Caffeoylquinic acid (CQA) derivatives are secondary plant metabolites, and many studies have reported their strong antioxidant and anti-inflammatory effects [61,62].These bioactivities have been attributed to the presence of an ortho-hydroxy group and esterification groups on caffeic acid and quinic acid moieties [63,64].According to the research on the different bioactivities of dicaffeoylquinic acid (DCQA) isomers, DCQAs (4,5-or 3,4-) with caffeoyl groups substituted at C-4 exhibit stronger physiological activities than 3,5-DCQA [65][66][67], which suggests the importance of the presence of the cis-caffeoyl group in DCQA [68].Nevertheless, TCQA has been reported to have more potent pharmacological activities than DCQA.3, 4, 5-TCQA has a stronger antiradical ability than DCQA [69], induces ATP synthesis [70], and effectively inhibits proinflammatory substances such as TNF-α [71].This enhanced bioactivity has been attributed to increasing the steric hindrance of TCQA due to the presence of three caffeoyl groups [69,72].Our in vitro results obtained using Nrf2-ARE reporter HaCaT cells confirmed that TCQA better inhibits Nrf2 activation compared with several CQAs and DCQAs.
MAPKs are composed of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase [73].They are involved in a variety of cellular activities, including cell proliferation, differentiation, and death [74].UV-induced oxidative stress increases ROS levels and promotes the phosphorylation of ERK, JNK, and p38, thereby activating the MAPK signaling pathway [75,76].In this study, TCQA significantly decreased the protein expressions of p-ERK, p-JNK, and p38 in UVB-irradiated HaCaT cells.Thus, our results suggest that TCQA inhibits UVB-induced MAPK activation.AP-1 is a transcription factor composed of c-Jun and c-Fos and is mediated by an upstream MAPK signaling pathway consisting of ERK, JNK, and p38 [77].In other words, the expression of c-Fos is induced by ERK activation, and the expression of c-Jun is induced by JNK and p38 [78,79].Furthermore, c-Fos and c-Jun combine to form either a heterodimer (Fos-Jun) or a homodimer (Jun-Jun), which regulate the transcriptions of numerous genes, including AP-1 [80].Moreover, the activation of AP-1 transcription can induce ceramide production, leading to the overproduction of proinflammatory cytokines [81].
In the present study, TCQA significantly and dose-dependently reduced the protein expressions of phosphorylated c-Fos and c-Jun in UVB-irradiated HaCaT cells and significantly inhibited the dose-dependent upregulation of AP-1 induced by PMA exposure.The ROS-induced MAPK, AP-1, and NF-κB signaling pathways activate MMPs, which leads to MMP secretion from keratinocytes and fibroblasts, and accelerates protein and collagen degradation [82].In particular, MMP-1 is a key marker of skin photoaging and causes collagen breakdown in the dermis [83], which contributes to the aging responses of dermal cells, such as dermal layer atrophy, skin wrinkling, and loss of elasticity [84].TCQA inhibited the UVB-induced activation of MMP-1 at all concentrations examined.Taken together, we conclude that TCQA protects against photoaging and skin wrinkling by modulating MAPK/NF-κB/AP-1 signaling, reducing MMP-1 activity, and activating Nrf2 in UVB-damaged HaCaT cells.

Conclusions
Phytochemical investigation of the MeOH extract of N. peltata roots led to the isolation of 15 compounds (1-15).Among these compounds was a new oleanane-type triterpenoid saponin, 3-O-β-D-glucopyranosyl-oleanolic acid 28-O-β-D-glucuronopyranoside (peltatasaponin A), the chemical structure of which was characterized by 1D and 2D NMR spectroscopic data and HRESIMS, as well as chemical reaction.Among the isolates, TCQA effectively inhibited ROS production by inducing the expression of Nrf2/HO-1 antioxidant enzyme in HaCaT cells.In addition, treatment with TCQA significantly inhibited MMP-1 expression by suppressing MAPK, AP-1, and NF-kB pathways and their respective subunits in HaCaT cells exposed to UVB.Thus, our study supports the potential use of TCQA as an antioxidant and anti-wrinkle treatment.

Figure 7 .
Figure 7. Effect of TCQA on MMP-1 expression.(A) MMP-1 mRNA expression was analyzed in HaCaT cells exposed to UVB by qRT-PCR and (B) the concentration of MMP-1 was assessed using