Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells

Abstract

Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

Introduction

Causes of skin aging include natural aging, stress, environment, and genetics, but the most harmful factor is sunlight (ultraviolet light, UV) and the resultant oxidative stress. With high levels of environmental pollutants and outdoor activities, UV exposure has increased along with skin photoaging.¹ UV radiation is classified as UVA (315–400 nm), UVB (280–315 nm), and UVC (200–280 nm) according to its wavelength. UVB, which comprises approximately 5% of the total UV radiation, penetrates the entire epidermis layer and the dermis of the human skin. It is mainly responsible for nonmelanoma and melanoma skin cancers.¹ Although less intense than UVB, UVA is much more prevalent during daylight since it can penetrate clouds, glass, and skin. UVA accounts for ∼95% of the UV radiation reaching Earth's surface and can pass through deeper skin layers, contributing to skin darkening, wrinkling, and aging.¹

UV exposure promotes substantial reactive oxygen species (ROS) production and consequent degradation of extracellular matrix (ECM) proteins (e.g., type I collagen, elastin, fibronectin, and proteoglycan).^1,2 Type 1 collagen, the major ECM component, is synthesized as procollagen type 1 (PC1) and provides the skin with strength and elasticity.^1
–3 Matrix metalloproteinase-1 (MMP-1) and elastase-1 degrade various ECM proteins, including elastin and collagen.^2,4 Since signs of skin photoaging, wrinkling, and flaccidity are associated with ROS-induced ECM degradation,^1

–4 it is important to elucidate the effects of natural compounds on skin aging-related factors (e.g., PC1, MMP-1, and elastase-1) in UV-irradiated skin cells.

Gastrodia elata Blume (GEB), an orchid plant devoid of chlorophyll, requires the symbiotic fungi, Mycena osmundicola and Armillaria mellea, for its germination and growth/maturation, respectively.^5,6 GEB has been used as an oriental medicinal remedy to treat neurodegenerative disorders (e.g., paralysis, vertigo, stroke, epilepsy, and dementia).^{6

–14} For example, GEB protected β-amyloid-induced neurotoxicity in drosophila and PC12 cells⁹ and MPP⁺-induced cytotoxicity in human dopaminergic SH-SY5Y cells.¹⁰ In addition, GEB exerted anticonvulsive effect on kainic acid-treated epilepsy in rats,¹¹ antiapoptotic effect in toxin-induced dopaminergic MN9D cells,¹² and anti-inflammatory effects in traumatic brain injury-treated rats,¹³ which were attributed to antioxidative,^13,14 antiplatelet, and antithrombotic activities¹⁵ by bioactive compounds. A variety of bioactive compounds have been isolated from GEB, the most important ones are gastrodin, vanillin, and 4-hydroxybenzaldehyde.^6

–13 Recent studies have identified ergothioneine (Ergo)^5,8 and diosgenin¹⁶ in GEB. Ergo is mainly found in mushrooms,^17,18 while diosgenin in wild yam.¹⁹ Both have been listed in the top 10 botanical ingredients present on antiaging creams in 2010 owing to their strong antioxidant activity,²⁰ suggesting that GEB may be used as a functional cosmetic as well as medicinal food.

Although there are reports on the composition of freeze-dried and steam-dried GEB,²¹ its chemical constituents,^8,14,22 and antioxidant,^13,14 antiplatelet, and antithrombotic¹⁵ effects, we believe that this is the first investigation of the protective effect of GEB against UV-induced skin photoaging. Since GEB has been used as a traditional remedy for skin problems and neurological disorders because of its bioactive compounds and antioxidant properties, we analyzed the bioactive compound contents and their activities, as well as the photoprotective effect on UVA-irradiated human skin cells. We demonstrated that GEB has the potential as a natural antiaging product by modulating the signs of skin photoaging through promotion of antioxidant activity and alteration of expression/activity of PC1, MMP-1, and elastase-1 in UVA-irradiated human dermal fibroblast cells (HDF).

Materials and Methods

Preparation of GEB extract

GEB rhizomes were obtained from the Hyewonchunma Farm (Chuncheon, Korea). The dried GEB (42°C for 16 h) was ground, and then, GEB powder (10 g) was extracted in 200 mL of deionized H₂O at 90°C for 3 h, followed by centrifugation at 3140 g (VS-24SMTI, Vision Scientific Co., Bucheon, Korea). The supernatant was filtered using filter paper with 6 μm pore size (No. 3; Whatman, Little Chalfont, England) and concentrated under vacuum using a rotary evaporator (Rotavapor R-100; BUCHI, Flawil, Switzerland). The frozen extract was evaporated to a freeze-dried powder for 72 h using a laboratory freeze dryer (Freezone plus 6; Labconco, Kansas City, MO, USA). The GEB extract powder was stored at −20°C before use.

Antioxidant levels and capacity of GEB

Total polyphenol and flavonoid content

The total GEB polyphenol and flavonoid contents were determined using the modified method of Folin and Denis²³ and Moreno et al.,²⁴ respectively. Absorbance of GEB (100 mg/mL d-H₂O) was measured at 750 nm for polyphenol and 450 nm for flavonoid using a spectrophotometer (Gen5.2; BioTek, Winooski, VT, USA). The polyphenol concentration was derived from a gallic acid (Sigma-Aldrich Co., St. Louis, MO, USA) standard curve and expressed as milligrams of gallic acid equivalents (GAE) per gram dry weight (DW). The flavonoid concentration was derived from a quercetin (Sigma) standard curve and expressed as milligrams of quercetin equivalents (QE) per gram DW.

Quantitative analysis of ergothioneine (Ergo)

The Ergo content of GEB was determined according to the modified method of Dubost et al. ¹⁷ In brief, analysis was performed using high-performance liquid chromatography (YL9100HPLC; Younglin, Anyang, South Korea) and separated on a C18 column (5 μm, 4.6 × 250 mm; Waters Corporation, Santry, Dublin, Ireland). The chromatographic separation was carried out using 50 mM sodium phosphate in 3% acetonitrile and 0.1% trimethylamine adjusted to a pH of 7.3 with a flow rate of 1 mL/min. An UV/VIS detector (Younglin) set to a wavelength of 254 nm was used. The injection volume was 10 μL with an internal standard of Ergo (Sigma). Ergo was quantified by comparing the peak area of the sample to peak areas obtained from authentic Ergo standard ranging from 0 to 50 μg/mL (R² = 0.9999). Ergo content of mushrooms was expressed as milligrams of Ergo per gram DW. The total running time was 25 min and four sets of GEB samples were analyzed in duplicate.

DPPH and ABTS radical scavenging capacity assay

The GEB (0, 5, and 10 μg/mL d-H₂O) antioxidant activity was determined on the basis of the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH; Sigma) and the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Sigma) free radicals, according to the modified method of Brand-Williams et al. ²⁵ and Re et al.,²⁶ respectively. A series of known concentrations (0–50 μg/mL) of gallic acid (Sigma) were used as a reference standard. Absorbance was measured at 515 nm for DPPH and at 734 nm for ABTS radical scavenging capacity, respectively.

Superoxide dismutase activity assay

GEB superoxide dismutase (SOD) activity was determined by the method of Marklund and Marklund.²⁷ Absorbance was measured at 420 nm and α-tocopherol (Sigma) was used as a reference standard.

Skin aging analysis in HDF

Cell culture

The HDF line (ATCC, Rockville, MD, USA) was cultured in Medium 106 (M106; Gibco, Grand Island, NY, USA) containing 2% low serum growth supplement and 1% penicillin–streptomycin (Gibco). In all experiments, confluent cells were treated with 0–2 mg/mL of GEB for 24 h, exposed to UVA irradiation (365 nm, 100 mJ/cm², 38 sec; UV crosslinker BLX-254, Vilber Lourmat, ECC, Marne, France), and incubated for 24 h according to the method of Jung et al. ²⁸

Cell viability assay

The confluent HDF were pretreated with 0–10 mg/mL of GEB for 24 h. After incubation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco, Solon, OH, USA) dye solution (5 mg/mL in PBS), absorbance of the formazan salts in the supernatant was measured at 570 nm.

Measurement of PC1 production and MMP-1

After pretreatment with 0–2 mg/mL GEB and UVA irradiation in HDF, PC1 and MMP-1 concentrations in the medium were determined using commercially available ELISA kits (PC1 C-Peptide ELA and Human Total MMP-1 kits; R&D System, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.

Measurement of elastase-1 activity

GEB elastase-1 activity was determined according to the modified method of Kraunsoe et al. ²⁹ The supernatant of lysated HDF was incubated with elastase-1 and its substrate (N-succinyl-Ala-Ala-Ala-p-nitroanilide; Sigma). Absorbance was measured at 410 nm and elastase-1 activity was expressed as units per milligram protein.

Analysis of real-time polymerase chain reaction

Isolation of total RNA was performed using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNA extraction kit (RNase Mini kit; Qiagen, Hilden, Germany). Total RNA (2 μg) was reverse-transcribed using random primers and reverse transcription master premix (ELPIS, Daejeon, Korea). Real-time polymerase chain reaction (real-time PCR) (LightCycler 96; Roche, Indianapolis, IN, USA) was performed using SYBR green supermix (Roche) and the primers (Bioneer, Daejeon, Korea) described in Table 1. The conditions for the PCR included initial denaturation for 3 min at 94°C, followed by 40 cycles of 10 sec at 94°C, 15 sec at 60°C, 30 sec at 72°C, and 5 min at 72°C for the final extension. The expression levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Table 1.

Gene Accession Numbers and Primers Used for Real-Time Polymerase Chain Reaction Amplification

Gene	Accession number	Primer sequences
PC1	XM_006721703.1	F: 5′-TTCTGTACGCAGGTGATTGG-3′
		B: 5′-CATGTTCAGCTTTGTGGACC-3′
MMP-1	NM_002421	F: 5′-TTGTGGCCAGAAAACAGAAA-3′
		B: 5′-TTCGGGGAGAAGTGATGTTC-3′
Elastase-1	NM_001971	F: 5′, -CTGGACACAAAGCTGGTCAC-3′
		B:5′-GAGATGGAGTTCGCTCTGGA-3′
GAPDH	NM_002046	F: 5′-AATGAAGGGGTCATTGATGG-3′
		B: 5′-AAGGTGAAGGTCGGAGTCAA-3′

PC1, procollagen type 1; MMP-1, matrix metalloproteinase-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Statistical analysis

Statistical analysis was performed using IBM SPSS software (Version 19, IBM-SPSS, Armonk, NY, USA). Student's t-test and one-way analysis of variance (ANOVA) followed by post hoc analysis with Tukey test were used to detect differences between experimental groups. At least three sets of experiments were performed with determinations made in duplicate for statistical analysis. A value of P < .05 was considered statistically significant.

Results

Total polyphenol, flavonoid, and Ergo content of GEB

The total polyphenol and flavonoid content of GEB was 21.8 mg GAE/g DW and 0.43 mg QE/g DW, respectively. GEB Ergo content was 0.41 mg/g DW (Table 2). The retention time of Ergo was around 4 min in the chromatog (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/jmf).

Table 2.

Contents of Total Polyphenol, Total Flavonoid, and Ergothioneine of Gastrodia elata Blume Extracts

	Total polyphenol (mg GAE/g DW)	Total flavonoid (mg QE/g DW)	Ergothioneine (mg/g DW)
GEB	21.8 ± 2.17	0.43 ± 0.95	0.41 ± 0.02

DW, dry weight; GEB, Gastrodia elata Blume; GAE, gallic acid equivalents; QE, quercetin equivalents.

DPPH and ABTS radical scavenging, and SOD activity of GEB

To determine the antioxidant effect of GEB, we measured DPPH and ABTS radical scavenging activity of GEB. The DPPH and ABTS radical scavenging activities at 5 and 10 mg/mL were 34.2% and 44% for DPPH and 31% and 44% for ABTS. DPPH and ABTS scavenging activities of gallic acid (a positive control) were 89.5% and 84%, respectively (Fig. 1A, B). The SOD activities of GEB at 10 and 25 mg/mL were 57% and 76%, respectively, while that of α-tocopherol (a positive control) was 91% (Fig. 1C). All markers showed higher antioxidant activities at higher dosages, suggesting that GEB displays a dose-dependent antioxidant activity.

FIG. 1.

Effect of GEB extract on antioxidant capacity. (A) DPPH radical scavenging capacity, (B) ABTS radical scavenging capacity, and (C) SOD of each bar represents the mean ± standard deviation (n = 3). *P < .01 5 versus 10 mg/mL of GEB for DPPH and ABTS radical scavenging capacity, 10 versus 25 mg/mL of GEB for SOD activity. α-TE, α-tocopherol as a positive control; GA, gallic acid as a positive control; GEB, Gastrodia elata Blume extract; SOD, superoxide dismutase.

Effects of GEB on cell viability in HDF

To examine its antiaging effects of GEB, we first evaluated GEB cytotoxicity by incubating HDF with various extract concentrations (0–10 mg/mL). Cell viability was not inhibited by low GEB concentrations (0.2–2 mg/mL), whereas higher concentrations (5–10 mg/mL) significantly induced toxicity (Fig. 2A). Therefore, a concentration of 0–2 mg/mL was used in further experiments.

FIG. 2.

Effect of GEB extract and UVA irradiation on cell viability in HDF. HDF were plated at 4 × 10⁴ cells/well and incubated in media containing various concentrations of GEB for 24 h, followed with/without exposure to UVA irradiation (365 nm, 100 mJ/cm², 38 sec) and incubated for another 24 h. Each bar represents the mean ± standard deviation (n = 3). Different letters mean significant difference according to ANOVA (P < .05). ANOVA, analysis of variance; HDF, human dermal fibroblast cells.

HDF were pretreated with 0–2 mg/mL GEB for 24 h and exposed to UVA irradiation for 38 sec, before a 24-h incubation. Cell viability in UVA-irradiated cells was 67% of control. However, GEB pretreatment restored cell viability to a level similar to control (Fig. 2B).

Effect of GEB on antiaging factors in HDF

To assess the antiaging activity of GEB, we measured PC1 and MMP-1 productions, and elastase-1 activity in UVA-irradiated HDF. The PC1 level in the control was 1.23 μg/mL and decreased by 17% upon UVA irradiation. However, PC1 production significantly increased after GEB treatment in a dose-dependent manner, and at 0.2–2 mg/mL was significantly higher than that of control and UVA-irradiated groups (Fig. 3A). The MMP-1 production level in the control was 27 μg/mL, increasing by 21% upon UVA irradiation. MMP-1 levels, however, decreased to 10 μg/mL upon GEB preincubation and were significantly lower than in control and UVA-irradiated groups (Fig. 3B). Elastase-1 activity in the control was 5.42 units/mg; it increased by 81% upon UVA irradiation but significantly decreased upon GEB application in a dose-dependent manner. The increased elastase-1 activity observed in UVA-irradiated HDF decreased to control levels after incubation with GEB (0.5–2 mg/mL) (Fig. 3C).

FIG. 3.

Effect of GEB on (A) PC1, (B) MMP-1 production, and (C) elastase-1 activity in UVA-irradiated HDF. HDF were treated with different concentrations (0–2 mg/mL) of GEB for 24 h, exposed to UVA irradiation (365 nm, 100 mJ/cm², 38 sec), and incubated for 24 h. Each bar represents the mean ± standard deviation (n = 3). Different letters mean significant difference according to ANOVA (P < .05). PC1, procollagen type 1; MMP-1, matrix metalloproteinase-1.

Effects of GEB on PC1, MMP-1, and elastase-1 messenger RNA levels

To determine whether GEB modulates skin aging-related factors at the molecular level, we examined PC1, MMP-1, and elastase-1 messenger RNA (mRNA) levels. PC1 mRNA significantly decreased by 60% in UVA-irradiated HDF compared to control. This was not affected by low GEB concentrations (0.2–0.5 mg/mL), but higher GEB dosage (1–2 mg/mL) had a tendency to restore PC1 mRNA levels in UVA-irradiated HDF (Fig. 4A). In comparison, while UVA irradiation markedly induced MMP-1 and elastase-1 mRNA levels, GEB (0.2–2 mg/mL) significantly decreased them (Fig. 4B, C).

FIG. 4.

Effect of GEB on mRNA levels of (A) PC1, (B) MMP-1, and (C) elastase-1 in UVA-irradiated HDF. HDF were treated with different concentrations (0–2 mg/mL) of GEB for 24 h, exposed to UVA irradiation (365 nm, 100 mJ/cm², 38 sec), and incubated for 24 h. Real-time polymerase chain reaction was performed using gene-specific primers for PC1, MMP-1, and elastase-1. Each bar represents the mean ± standard deviation (n = 3). Different letters mean significant difference according to ANOVA (P < .05). PC1, procollagen type 1; MMP-1, matrix metalloproteinase-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.

Discussion

Polyphenols and flavonoids play an important role in the antioxidant system by inhibiting free radical generation and preventing oxidative stress and cell injury. A recent study⁶ reported 64 GEB components using various chromatography techniques. There is cumulative evidence that phenolic compounds and their derivatives have pharmacological (antioxidant, anti-inflammatory, and anticonvulsant) properties.^6,7,14,15 In this study, the total polyphenol and flavonoid content of GEB was 21.8 and 0.43 mg/g DW, respectively (Table 2), lower than that found in fermented GEB.¹⁴ This difference is attributed to cultivation areas, drying conditions, different solvents, extraction methods, and fermentation.^5,8,14

Ergo is a natural water-soluble thiol amino acid,^17,18 newly found in GEB.⁵ At physiological pH, Ergo exists mainly in the thione ( = S) rather than the thiol (–SH) form.¹⁸ Its slow degradation, resistance to disulfide formation, and auto-oxidization make it more stable than glutathione.^17,18 It is not synthesized in humans but exists at different concentrations in blood and tissues, where it accumulates after consumption of mushrooms, meat, some beans,^5,17 and even from some topical skin treatments.³⁰ In our study, the Ergo content of GEB was 0.41 mg/g DW (Table 2). The Ergo content of GEB has been measured as 0.35–0.70 mg/g DW, depending on cultivation conditions, tuber weight, harvesting season, and tissue types.^5,8 The Ergo levels of GEB from three cultivation areas were 2.0–3.6 times higher than those of vanillin levels and 0.25–0.5 times lower than gastrodin.^5,8 Another bioactive component, 4-hydroxybenzyl alcohol, was 130–260 times lower than gastrodin in GEB.³¹ Since Ergo is quantitatively comparable to gastrodin, vanillin, and 4-hydroxybenzyl alcohol, it is considered one of the major compounds that contribute to antioxidant activity and pharmacological effects of GEB. In addition, Ergo was selected as one of the top 10 botanical ingredients in the 2010 antiaging creams.²⁰

Since the antioxidant properties of GEB and its total polyphenol, flavonoid, and Ergo contents are positively associated, we examined the GEB antioxidant potential by measuring DPPH and ABTS radical scavenging, and SOD activity. GEB showed a dose-dependent antioxidant activity. This was due to inhibition of ROS production and activation of antioxidant enzymes (e.g., catalase, glutathione peroxidase, and SOD) in PC12 cells.⁸ In SH-SY5Y cells, this was associated with caspase-3 cleavage, poly ADP ribose polymerase activation,¹⁰ and increased expression of heme oxygenase-1 through the p38 mitogen-activated protein kinase (MAPK)/nuclear factor erythroid 2 (Nrf2) signaling pathway.³² It has also hypothesized that the free hydroxyl groups linked to an aromatic ring in GEB phenolic compounds might, in part, be responsible for the antioxidant activity (i.e., donating electron and hydrogen, scavenging ROS, reducing metal ions, and inhibiting lipid peroxidation).^20,33 In addition, GEB fermentation boosts the radical scavenging capacity and antioxidant activity by increasing bioactive components (polyphenols, flavonoids, and Ergo) and their electron-donating ability.¹⁴ The positive control, 25 μg/mL GA, exerted a much higher antioxidant power than 5–10 mg/mL GEB. Among 20 natural and synthetic antioxidants, GA was reported to have the most efficient antioxidant activity by showing much higher antiradical efficiency and numbers of reduced DPPH radicals with slow kinetic behavior due to the presence of electron withdrawing groups (three hydroxyl groups and one carboxyl group).²⁵ In addition, the antioxidative value of efficient quantity (EQ) of vanillic acid and p-hydroxybenzoic acid (which are two of the main bioactive compounds of GEB) was >300 ppm, whereas EQ value of GA was 8 ppm to obtain an equivalent efficiency as an antioxidant,³⁴ suggesting that GA is one of the most powerful antioxidants.

Exposure to UV radiation is responsible for several skin disorders such as coarse wrinkles, sagging, roughness, mottled pigmentation, and eventually skin cancer.³ UVB radiation penetrates into the epidermis, while UVA penetrates into the epidermis and dermis, and causes ROS formation and accumulation, and consequent free radical damage.^1
–3 This event triggers degradation of ECM proteins, including type I collagen, elastin, fibronectin, and proteoglycan.^1,2 Type I collagen, the major structural ECM component, is synthesized as PC1 and is responsible for skin strength and elasticity.^1
–3 MMPs are considered key photoaging regulators as they are overexpressed in UV-exposed human fibroblasts. Particularly, MMP-1 degrades collagen types I, II, III, and gelatin, which are then further degraded by MMP-2 and MMP-9.^1,2,35 Elastase is able to degrade all major connective tissue matrix proteins, including elastin, collagen, keratin, and proteoglycan. UV exposure and consequent ROS production cause elastase secretion, activation, and therefore, elastic fiber degradation.^2,4 Hence, skin photoaging is associated with ROS generation, MMP-1 and elastase-1 upregulation, and dermal collagen degradation.

In this study, we found that UVA irradiation markedly decreased PC1 and increased MMP-1 and elastase-1 at the molecular level as well as at the secretory level, in HDF. In comparison, GEB significantly restored these effects in UVA-irradiated HDF (Figs. 3 and 4). In line with our results, several natural plant extracts showed similar effects on PC1, MMPs, elastase, and other mediators and comparable protection against ECM degradation and photoaging in UVA- and/or UVB-irradiated cells.^{4,28,35

–38} Based on these data, we confirmed that GEB has anti-photoaging therapy potential by regulating primary ECM components and providing structural support for the skin dermis.

At least three possible mechanisms have been proposed for UV irradiation-induced aging of human skin cells. (1) UV irradiation alters growth factor signaling.³⁹ For example, transforming growth factor β/Smad signaling and insulin-like growth factor I have been involved in triggering type I collagen synthesis.³⁹ (2) UV irradiation induces inflammatory cytokine production.^1,40

–43 Tumor necrosis factor α (TNF-α) impairs collagen synthesis through TNF-R55 activation in human skin,¹ while interleukin (IL)-1α stimulates macrophage migration inhibitory factor, which triggers MMP-1 expression via PKC, MAPK, and activator protein-1 (AP-1) in UV-irradiated dermal fibroblasts.^40,41 IL-1β induction by UV exposure⁴¹ and in human aging fibroblasts⁴² inhibits elastase activity and elastin synthesis. Moreover, IL-6 induced MMP-1 stimulation and collagen degradation.^1,43 (3) UV irradiation induces NF-κB activation through IκBα stabilization and IκB activation, which result in induction of inducible nitric oxide synthase and cyclooxygenase-2 in the skin.³ (4) MAPKs such as JNK, ERK, and p38, regulate AP-1.^2,32,33,37 AP-1 is a heterodimer transcription factor composed of c-Jun and c-Fos. UV irradiation potently induces c-Jun/c-Fos, hence activating AP-1 with consequent PC1 downregulation and MMP-1 upregulation.^28,37 These processes lead to ECM degradation in the dermis, connective tissue damage, and photoaging.

Although a relationship between GEB and skin fibroblasts has not been reported until now, the antioxidant and anti-inflammatory activities of gastrodin and p-hydrozybenzyl alcohol were associated with decreased nitric oxide production, inducible nitric oxide synthase, cyclooxygenase-2 expression, TNF-α and IL-1β production,⁴⁴ and with an induction of protein disulfide isomerase, 1-Cys peroxiredoxin, and Nrf2 gene expression in neuronal^43

–46 and inflammatory cells^17,47 and in rat models.^48,49 Therefore, it is suggested that GEB may modulate photoaging-related factors in UV-irradiated dermal fibroblasts through the modulation of ROS production and the relative GF signal transduction pathways, inflammatory cytokines, and NF-κB, and MAPK signaling.

UV irradiation modulates expression and/or activity of MMPs, elastase, and relevant factors in the dermal ECM, leading to the loss of collagen and elastin. Natural compounds may attenuate skin photoaging by regulating synthesis and degradation of collagen and elastin by MMPs and elastase. In this study, GEB ameliorated skin photoaging markers by promoting antioxidant activity and altering expression/activity of PC1, MMP-1, and elastase-1 in UVA-irradiated HDF, suggesting that GEB can be used as an antiaging ingredient for cosmeceutical applications. Further studies will be needed to elucidate the relationship between GEB and oxidative stress signal pathways in the skin photoaging process and characterize the underlying mechanisms in vivo.

Footnotes

Acknowledgments

We deeply appreciate Drs. Robert B. Beelman and Michael D. Kalaras in the Department of Food Science, the Pennsylvania State University, USA, and Dr. Eung-Jun Park in the Division of Forest Biotechnology, Korea Forest Research Institute, Korea, for technical advice. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2013R1A1A2012510).

Author Disclosure Statement

No competing financial interests exist.

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