Laricitrin 3-Rutinoside from Ginkgo biloba Fruits Prevents Damage in TNF-α-Stimulated Normal Human Dermal Fibroblasts

Human skin comprises the epidermis and dermis, which perform interactive functional activities with each other in order to maintain the skin’s tensile strength. In particular, the dermal layer is crucial for skin protection. However, skin aging destroys collagen and elastin fibers, causing wrinkles, pigments, and sagging. Skin aging-related factors, such as tumor necrosis factor-α (TNF-α), promote the generation of intercellular reactive oxygen species (ROS). These are known to stimulate the hypersecretion of matrix metalloproteinase-1 (MMP-1), which degrades collagen and inhibits collagen synthesis. In this study, as part of our ongoing discovery of natural products, we investigated potential natural products derived from ginkgo fruit (Ginkgo biloba fruit) with protective effects against TNF-α-induced skin aging. Phytochemical investigation of the MeOH extract of G. biloba fruits, aided by liquid chromatography–mass spectrometry, led to the isolation of 14 compounds (1–14) from the n-butanol-soluble fraction. These were structurally determined to be: (E)-coniferin (1), syringin (2), 4-hydroxybenzoic acid 4-O-β-D-glucopyranoside (3), vanillic acid 4-O-β-D-glucopyranoside (4), glucosyringic acid (5), (E)-ferulic acid 4-O-β-D-glucoside (6), (E)-sinapic acid 4-O-β-D-glucopyranoside (7), ginkgotoxin-5-glucoside (8), ginkgopanoside (9), (Z)-4-coumaric acid 4-O-β-D-glucopyranoside (10), (1′R,2′S,5′R,8′S,2′Z,4′E)-dihydrophaseic acid 3’-O-β-D-glucopyranoside (11), eucomic acid (12), rutin (13), and laricitrin 3-rutinoside (L3R) (14). Biological evaluation of the isolated compounds for their effects on intracellular ROS generation showed that, of these 14 compounds, L3R (14) inhibited TNF-α-stimulated ROS generation (p < 0.001 at 100 μM). Inhibition of ROS generation by L3R led to the suppression of MMP-1 secretion and protection against collagen degradation. The inhibitory effect of L3R was mediated by the inhibition of extracellular signal regulated kinase (ERK) phosphorylation. Furthermore, L3R diminished the secretion of pro-inflammatory cytokines interleukin 6 (IL-6) and interleukin 8 (IL-8). Based on these experimental results, L3R is a potential bioactive natural product that can be used to protect against skin damage, including aging, in cosmetics and pharmaceuticals.


Introduction
The human skin is a primary barrier network matrix spanning the entire body, functioning to protect interior organs [1]. Skin is comprised of the epidermis and dermis layers, new bioactive natural compounds from the extract of G. biloba fruits and investigate their potential for pharmacological application.

General Experimental Procedure
Optical rotation was measured using a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA). Infrared spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer (Bruker, Karlsruhe, Germany). UV spectra were acquired using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were recorded using a Bruker AVANCE III HD 850 NMR spectrometer with a 5 mm TCI CryoProbe operated at 850 MHz ( 1 H) and 212.5 MHz ( 13 C). The chemical shifts were presented in ppm (δ) for 1 H and 13 C NMR analyses. Preparative and semi-preparative high-pressure liquid chromatography (HPLC) was performed using a Waters 1525 Binary HPLC pump with a Waters 996 photodiode array detector (Waters Corporation, Milford, MA, USA) and Agilent Eclipse C18 column (250 × 21.2 mm, 5 µm; flow rate: 5 mL/min; Agilent Technologies), as well as a Phenomenex Luna Phenyl-hexyl 100 Å column (250 × 10 mm, 5 µm; flow rate: 2 mL/min; Phenomenex, Torrance, CA, USA). LC/MS analysis was performed using an Agilent 1200 Series HPLC system equipped with a diode array detector, a 6130 Series Electrospray Ionizing mass spectrometer for electrospray ionization high-resolution mass spectrometry (ESI-HRMS), and an analytical Kinetex C18 100 Å column (100 × 2.1 mm, 5 µm; flow rate: 0.3 mL/min; Phenomenex). All ESI-HRMS data were obtained using an Agilent G6545B quadrupole time-of-flight mass spectrometer (Agilent Technologies). Silica gel 60 (230-400 mesh; Merck, Darmstadt, Germany) was used for the column chromatography. Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan) was used for the open-column chromatography. Thin-layer chromatography was performed using pre-coated silica gel F254 plates and RP-C18 F254s plates (Merck), and the spots were detected under UV light or by heating after spraying with anisaldehyde-sulfuric acid.

Plant Material
Whole fruits of G. biloba were collected on the campus of Sungkyunkwan University, Suwon, Korea, in October 2019. The plant was identified by one of the authors (K.H.K.). A voucher specimen (GBF-2019-10) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Cell Culture and Treatment Preparation
NHDFs (juvenile foreskin, cryopreserved, 5 × 10 5 cells) were obtained from PromoCell (Sickingenstr, Heidelberg, Germany) and stored in a nitrogen tank. NHDFs were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning, Manassas, VA, USA, pH 7.4) containing 10% fetal bovine serum (FBS; Atlas, Fort Collins, CO, USA) and 100 U/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA) in a humidified atmosphere containing 5% CO 2 at 37 • C in a cell incubator (Thermo Scientific, Waltham, MA, USA). The isolated compounds were dissolved in dimethyl sulfoxide (Biosesang, Seong-Nam, Korea) to prepare 10 mM stock solutions. The isolated compounds were diluted in DMEM without FBS at concentrations of 25, 50, and 100 µM. The concentration of the TNF-α stock solution was 20 µg/mL, and the final treatment concentration was 20 ng/mL. In all experiments, the cells were seeded onto a cell culture plate and starved in DMEM without FBS overnight. The cells were then co-treated with TNF-α and the isolated compounds.

Cell Viability
NHDFs were seeded in 96-well plates with a clear bottom (5 × 10 3 cells/well) and incubated for 24 h. The NHDF were then starved in DMEM without FBS for 24 h. The supernatant was removed and replaced with a serum-free medium. The isolated compounds were exposed to specified concentrations and incubated for 24 h in a cell incubator. Next, 100 µL EZ-Cytox solution (Dogen, Seoul, Korea) was added to each well and incubated for 1 h. The absorbance values were measured using a plate reader (SPARK 10 M; Tecan) at 450 nm.

Intercellular ROS Generation Assay
NHDFs were plated in black 96-well plates with a flat bottom (1 × 10 4 cells/well) and incubated for 24 h in a cell incubator. To arrest the cell cycle, cells were stored in DMEM without FBS for 24 h. After this process, the NHDFs were treated with 25, 50, and 100 µM of the isolated compounds for 1 h and co-treated with specific concentrations of TNF-α (20 ng/mL) and DCFDA (10 µM) (Sigma-Aldrich, St. Louis, MO, USA; CAT No. 35845-1G) for 15 min. After aspiration of the supernatant, phosphate-buffered saline (PBS, pH 7.4) (Welgene, Gyeongsangbuk, Korea) was added to each well for washing. The level of intercellular ROS generation was measured by DCFDA treatment using a microplate reader (SPARK 10 M) at wavelengths of 485 and 535 nm. For fluorescence imaging, NHDFs were seeded in 48-well clear plate (1 × 10 4 cells/well) and incubated for 24 h in a cell incubator. Subsequently, the next process was performed as described above. DCFDA-stained images were immediately observed using a fluorescence microscope (Olympus, Tokyo, Japan).

Enzyme-Linked Immunosorbent Assay (ELISA)
NHDFs were seeded in 48-well plates at a flat bottom (2 × 10 4 cells/well) and incubated for 24 h. The cells were then starved in a serum-free medium for 24 h. L3R was then added at the indicated concentrations for 1 h. Subsequently, cells were treated with 20 ng/mL TNF-α for 24 h. The secretion of MMP-1 and COLIA1 was measured using ELISA kits (R&D systems, Minneapolis, USA; MMP-1 kit, CAT NO. DY091B; COLIA1 kit, CAT NO. DY6220-05).

Western Blotting
NHDFs were seeded in 6-well plates at a flat bottom (3 × 10 5 cells/well) and incubated for 24 h. The NHDF were then starved in serum-free medium for 24 h. The cells were treated with L3R at the indicated concentration for 1 h. The cells were then treated with 20 ng/mL TNF-α for 15 min (for protein expression analysis of ERK, p-ERK, p38, p-p38, JNK, and p-JNK). Each well was then washed with PBS and lysed using radioimmunoprecipitation buffer (pH 8.0). After collection, the samples were centrifuged at 13,000 rpm for 30 min and harvested for western blot analysis. The protein concentrations of the harvested supernatants were detected using a BCA protein assay kit (Merck, Darmstadt, Germany). Standardized amounts of samples were loaded onto a 4-20% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membranes (PVDF) (Merck, Darmstadt, Germany). The membranes were blocked with 5% non-fat dry milk (BD Difco, CAT No. 232100) in Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 (TBS-T; Fisher Scientific, Fair Lawn, NJ, USA; CAT No. BP337-500) for 24 h. The membranes were incubated with primary antibodies (Cell Signaling Technology, Danvers, MA, USA) diluted in 1% BSA (ERK, p-ERK, JNK, p-JNK, p38, p-p38, and GAPDH) overnight at 4 • C and washed three times with TBS-T. The membranes were then incubated with secondary rabbit antibodies (Cell Signaling Technology) at room temperature for 2 h and washed with TBS-T thrice. Finally, the membranes were treated with SuperSignal Femto Substrate reagent kit (Thermo Scientific, Waltham, MA, USA). Protein bands were analyzed using the Fusion Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Statistical Analyses
All results are presented as mean ± standard error of the mean of at least two independent experiments. Statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference test. Statistical significance was set at p < 0.05.

Isolation and Structure Elucidation of Compounds
G. biloba fruits were extracted with 100% MeOH at room temperature and the resultant MeOH extract was successively partitioned with four representative organic solvents to obtain four main fractions: hexane, CH 2 Cl 2 , EtOAc, and n-BuOH-soluble fractions ( Figure 1). The analysis by LC-MS using the house-built UV library database revealed that the n-BuOHsoluble fraction was rich in phenolic compounds, including flavonoids. The column chromatographic procedures including semi-preparative HPLC separation led to the isolation of 14 compounds from the n-BuOH-soluble fraction ( Figure 1). The isolated compounds were structurally determined to be: (E)-coniferin (1) [37]; syringin (2)

Effect of Compounds 1-14 on NHDFs Viability
In this study, we evaluated the effect of the isolated compounds 1-14 against TNF-αinduced damage to NHDFs. Before evaluating this effect, the viability of NHDFs treated with the isolated compounds 1-14 was assessed. The NHDFs were treated with compounds 1-14 at specific concentrations to evaluate cell viability, and it was found that the cell viability did not decrease up to 100 µ M, except for compounds 1 and 7 ( Figure 3). On the other hand, the extract of G. biloba fruits did not decrease the cell viability up to 12.5 μg/mL ( Figure S29),

Effect of Compounds 1-14 on NHDFs Viability
In this study, we evaluated the effect of the isolated compounds 1-14 against TNF-αinduced damage to NHDFs. Before evaluating this effect, the viability of NHDFs treated with the isolated compounds 1-14 was assessed. The NHDFs were treated with compounds 1-14 at specific concentrations to evaluate cell viability, and it was found that the cell viability did not decrease up to 100 µM, except for compounds 1 and 7 ( Figure 3). On the other hand, the extract of G. biloba fruits did not decrease the cell viability up to 12.5 µg/mL ( Figure S29). The results are presented as the mean ± standard error of the mean (SEM) (n = 2). # p < 0.05, ## p < 0.01 , and ### p < 0.001 compared to the untreated group.

Effect of Compounds 1-14 on Intracellular ROS Generation in TNF-α-Stimulated NHDFs
The TNF-α receptor on the cell surface is activated by repetitive UV exposure of the skin [50]. As a result, increased TNF-α levels provoke inflammatory responses that cause oxidative stress, which then stimulates intracellular ROS generation in NHDFs [51]. Therefore, we assessed the effects of the isolated compounds 1-14 on intracellular ROS as the mean ± standard error of the mean (SEM) (n = 2). # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the untreated group.

Effect of L3R on ROS Generation in TNF-α-Treated NHDFs
To observe the effects of L3R on TNF-α-induced intercellular ROS generation, we conducted a DCFDA staining assay. As depicted in Figure 5, L3R exhibited an inhibitory effect on ROS generation in the TNF-α-treated groups. These findings further supported the results of L3R obtained from the intracellular ROS generation assay, as shown in Figure 4.

Effect of L3R on ROS Generation in TNF-α-Treated NHDFs
To observe the effects of L3R on TNF-α-induced intercellular ROS generation, we conducted a DCFDA staining assay. As depicted in Figure 5, L3R exhibited an inhibitory effect on ROS generation in the TNF-α-treated groups. These findings further supported the results of L3R obtained from the intracellular ROS generation assay, as shown in

Effect of L3R on ROS Generation in TNF-α-Treated NHDFs
To observe the effects of L3R on TNF-α-induced intercellular ROS generation, we conducted a DCFDA staining assay. As depicted in Figure 5, L3R exhibited an inhibitory effect on ROS generation in the TNF-α-treated groups. These findings further supported the results of L3R obtained from the intracellular ROS generation assay, as shown in Figure 4.

Effect of L3R on Pro-Inflammatory Cytokine Secretion in TNF-α-Treated HDFs
Pro-inflammatory cytokines cause chronic diseases, such as psoriasis, that destroy alpha hydroxy acids in the ECM and acclerate skin aging [55]. Therefore, we evaluated the effects of L3R on IL-6 and IL-8 secretion in TNF-α-treated NHDFs. Compared to the Figure 7. Effect of L3R on TNF-α-induced protein expression associated with inhibition of mitogenactivated protein kinases (MAPKs) phosphorylation in normal human dermal fibroblasts (NHDFs). (A) Cells were treated with 25, 50, and 100 µM L3R for 1 h and then exposed to 20 ng/mL TNF-α for 15 min. The phosphorylated bands were analyzed by immunoblotting for phospho-ERK, ERK, phospho-JNK, JNK, phospho -p38, p38, and GAPDH. (B) The graphs show the fold-increase in the phosphorylation of MAPKs compared to the untreated group. Data are presented as mean ± SEM (n = 3). # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the control group and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the TNF-α-treated group.

Discussion
The human skin is the primary barrier against harmful external factors such as bacterial fungi and chemical UV radiation [56]. The skin comprises two main functional layers: epidermal and dermal. The two layers interact with each other. The epidermis is a thin layer that easily recovers after injury and maintains moisture inside the body. However, it does not have a vascular layer, which should be supported by another assistant. The dermis is a thick layer of blood vessels, collagen, and elastin fibers [57]. It transports nutrients to the epidermis to maintain the moisture content [58]. Skin aging can be divided into two categories: endogenous and exogenous. Endogenous aging factors are generated within the human body, such as stress, genetic constitution of an individual person, and disease [59,60]. Exogenous factors, such as UV radiation, smoking, and pollution, are generated outside the body. Both aging factors decrease collagen and elastin fibers and fibroblasts [61]. In particular, UV radiation can directly destroy dermal collagen [62]. UV radiation induces various skin aging factors such as TNF-α. TNF-α causes inflammatory reactions in the skin that promote oxidative stress. An increase in oxidative stress is accompanied by ROS generation [63,64]. Excessive ROS generation promotes photoaging, which destroys the dermal layer and leads to epidermal damage. Consequently, skin aging occurs, and symptoms appear in human skin, such as wrinkles, pigments, and sagging [65,66].
As previously mentioned, inhibition of TNF-α-induced ROS is the key to protecting the skin against photoaging and skin disease. Therefore, we investigated potential natural products with protective effects against TNF-α-induced skin aging from G. biloba fruit. Phytochemical investigation of the MeOH extract of G. biloba fruits led to the isolation of

Discussion
The human skin is the primary barrier against harmful external factors such as bacterial fungi and chemical UV radiation [56]. The skin comprises two main functional layers: epidermal and dermal. The two layers interact with each other. The epidermis is a thin layer that easily recovers after injury and maintains moisture inside the body. However, it does not have a vascular layer, which should be supported by another assistant. The dermis is a thick layer of blood vessels, collagen, and elastin fibers [57]. It transports nutrients to the epidermis to maintain the moisture content [58]. Skin aging can be divided into two categories: endogenous and exogenous. Endogenous aging factors are generated within the human body, such as stress, genetic constitution of an individual person, and disease [59,60]. Exogenous factors, such as UV radiation, smoking, and pollution, are generated outside the body. Both aging factors decrease collagen and elastin fibers and fibroblasts [61]. In particular, UV radiation can directly destroy dermal collagen [62]. UV radiation induces various skin aging factors such as TNF-α. TNF-α causes inflammatory reactions in the skin that promote oxidative stress. An increase in oxidative stress is accompanied by ROS generation [63,64]. Excessive ROS generation promotes photoaging, which destroys the dermal layer and leads to epidermal damage. Consequently, skin aging occurs, and symptoms appear in human skin, such as wrinkles, pigments, and sagging [65,66].
As previously mentioned, inhibition of TNF-α-induced ROS is the key to protecting the skin against photoaging and skin disease. Therefore, we investigated potential natural products with protective effects against TNF-α-induced skin aging from G. biloba fruit. Phytochemical investigation of the MeOH extract of G. biloba fruits led to the isolation of compounds 1-14;  (12); rutin (13), and L3R (14). According to the literature survey, compounds 3-5 were isolated from G. biloba for the first time in this study. Among the isolated compounds, L3R (14) significantly inhibited intracellular ROS generation in TNF-α-treated NHDFs. However, the protective effects of L3R on TNF-α-induced NHDFs have not yet been reported. Thus, several assays should be conducted to identify the signaling pathway for the ROS-related downregulation of L3R in TNF-α-induced skin aging.
Furthermore, TNF-α-induced ROS generation is known to decrease collagen synthesis and increase the synthesis of collagenases, including MMPs [67]. TNF-α increased the expression of MMP-1, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9). Among these MMPs, MMP-1, which initiates collagen cleavage, is a crucial factor in skin aging [68]. In addition, an increase in collagenase expression is associated with type I collagenolysis [69]. Thus, we evaluated the inhibitory effect of L3R on MMP-1 secretion and destruction of type 1 collagen in TNF-α-exposed NHDFs. Figure 6 shows that L3R diminishes MMP-1 secretion and type 1 collagen degradation. Similar to our findings, previous studies have demonstrated that flavonoids derived from natural sources exhibit anti-skin aging activity. For instance, potentilloside A has been shown to inhibit ROS generation and MMP-1 secretion in TNF-α-induced HDFs [70]. Additionally, quercetin, myricetin, and kaempferol have been reported to inhibit MMP-1 expression and superoxide anion radicals in UV-induced human dermal fibroblasts [71]. Furthermore, 6-hydroxyflavones have been found to increase collagen type 1 mRNA expression in normal human skin fibroblasts [72].
TNF-α-induced ROS generation are known to upregulate skin aging-related molecular pathways such as MAPKs and NF-κB, while ROS trigger the activation of MAPKs phosphorylation, which in turn upregulates the transcriptional pathways NF-κB and AP-1 [73,74]. In a previous study, MAPKs were found to upregulate NF-kB and AP-1, which increased the expression of MMP-1 and pro-inflammatory cytokines such as IL-6, IL-8, and IL-1. Therefore, we investigated the inhibitory effect of L3R on MAPK phosphorylation in TNF-α-induced NHDFs. Figure 7 shows that L3R inhibits ERK phosphorylation in TNF-α-stimulated NHDFs. Similar to our results, several studies have demonstrated that flavonoids can effectively suppress the generation of ROS and inhibit MMP-1 production by targeting key signaling pathways such as MAPKs and NF-κB in human dermal fibroblasts and keratinocytes. For instance, apigenin and luteolin have been found to suppress UVAinduced matrix metalloproteinase-1 expression through MAPKs-and AP-1-dependent signaling in HaCaT cells [75]. Additionally, Skullcapflavone II has been shown to inhibit the degradation of type I collagen by suppressing MMP-1 transcription via ERK, JNK/AP-1, and NF-κB in TNF-α-induced human dermal fibroblasts [76].
As pro-inflammatory factors cause diverse inflammatory reactions that lead to chronic diseases [77], we investigated the effect of L3R on pro-inflammatory cytokine secretion in TNF-α-stimulated NHDFs. Figure 8 indicates that L3R has an inhibitory effect on proinflammatory factors in TNF-α-induced NHDFs. Flavonoids have been widely reported to exhibit suppressive effects on pro-inflammatory cytokines through the modulation of MAPKs/AP-1 or NF-κB signaling pathways in human skin cells. For instance, catechin has been shown to inhibit ROS generation and reduce levels of MMP-1 and pro-inflammatory cytokines. Furthermore, catechin has been found to increase the secretion of pro-collagen type 1 through MAPKs/AP-1 and NF-κB pathways in TNF-α-induced HDFs [78]. Similarly, fisetin has been demonstrated to ameliorate ROS generation, MMPs (MMP-1, 3,9), and pro-inflammatory cytokines through the inhibition of MAPKs and NF-κB signaling in UVB-induced human foreskin fibroblasts (HS68) [79]. The aforementioned studies provide support for the hypothesis that L3R can effectively ameliorate ROS generation and inhibit MMP-1 secretion through the suppression of ERK phosphorylation.
These results suggest that L3R is a potential protective agent against the TNF-αinduced skin aging process, and we have summarized the mechanism of the protective effect of L3R against TNF-α-induced NHDFs in Figure 9. In addition to the protective effect of L3R on TNF-α-induced skin aging, there are other potential effects associated with L3R. For instance, flavonoids have been found to inhibit fibrosis both in vitro and in vivo. Luteolin, quercetin, and myricetin have shown a dose-dependent inhibition of Smad2/3 phosphorylation in human dermal fibroblasts [80]. Additionally, quercetin has been shown to inhibit kidney fibrosis by reducing αSMA and fibronectin expression in mice with unilateral ureteral obstruction (UUO) [81]. Furthermore, rutin (quercetin 3-rutinoside) has demonstrated a decrease in α-SMA and collagen expression by inhibiting TGF-β/Smad signaling in BDL-induced lung tissue of Sprague-Dawley rats (SD rats) [82]. Therefore, L3R not only holds potential as a candidate for addressing skin aging but also shows potential application in other conditions, including fibrosis. ants 2023, 12, x FOR PEER REVIEW Luteolin, quercetin, and myricetin have shown a dose-dependent inhibition of Smad2/3 phosphorylation in human dermal fibroblasts [80]. Additionally, quercetin has been shown to inhibit kidney fibrosis by reducing αSMA and fibronectin expression in mice with unilateral ureteral obstruction (UUO) [81]. Furthermore, rutin (quercetin 3-rutinoside) has demonstrated a decrease in α-SMA and collagen expression by inhibiting TGFβ/Smad signaling in BDL-induced lung tissue of Sprague-Dawley rats (SD rats) [82]. Therefore, L3R not only holds potential as a candidate for addressing skin aging but also shows potential application in other conditions, including fibrosis.

Conclusions
In conclusion, in an ongoing study focused on the discovery of potential bioactive phytochemicals, we investigated protective natural products from ginkgo fruit (G. biloba fruit) against skin aging by TNF-α stimulation. Phytochemical examination of the MeOH extract of G. biloba fruit resulted in the isolation of 14 compounds (1-14) using column chromatography and HPLC purification aided by LC-MS analysis. Among the isolated compounds, L3R (14) was found to exhibit protective activity by inhibiting TNF-α-induced ROS generation in NHDFs. L3R also diminishes collagenase secretion and prevents collagen destruction by suppressing ERK phosphorylation. Furthermore, L3R inhibits pro-inflammatory cytokines, such as IL-6 and IL-8, which stimulate diverse inflammatory reactions associated with skin aging. Although further studies are needed, these results suggest that L3R is a potential protective agent against TNF-α-induced skin aging.

Conclusions
In conclusion, in an ongoing study focused on the discovery of potential bioactive phytochemicals, we investigated protective natural products from ginkgo fruit (G. biloba fruit) against skin aging by TNF-α stimulation. Phytochemical examination of the MeOH extract of G. biloba fruit resulted in the isolation of 14 compounds (1-14) using column chromatography and HPLC purification aided by LC-MS analysis. Among the isolated compounds, L3R (14) was found to exhibit protective activity by inhibiting TNF-α-induced ROS generation in NHDFs. L3R also diminishes collagenase secretion and prevents collagen destruction by suppressing ERK phosphorylation. Furthermore, L3R inhibits pro-inflammatory cytokines, such as IL-6 and IL-8, which stimulate diverse inflammatory reactions associated with skin aging. Although further studies are needed, these results suggest that L3R is a potential protective agent against TNF-α-induced skin aging.