JOURNAL OF MEDICINAL FOOD J Med Food 00 (0) 2014, 1–8 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.3088

FULL COMMUNICATION

Lithospermum erythrorhizon Extract Protects Keratinocytes and Fibroblasts Against Oxidative Stress Hee Geun Yoo,1,2 Bong Han Lee,1,2 Wooki Kim,1 Jong Suk Lee,3 Gun Hee Kim,4 Ock K. Chun,5 Sung I. Koo,1,5 and Dae-Ok Kim1,2 1

Department of Food Science and Biotechnology, Kyung Hee University, Yongin, Gyeonggi, Korea. 2 Skin Biotechnology Center, Kyung Hee University, Suwon, Gyeonggi, Korea. 3 Gyeonggi Biocenter, Gyeonggi Institute of Science and Technology Promotion, Suwon, Gyeonggi, Korea. 4 Department of Foods and Nutrition, Duksung Women’s University, Seoul, Korea. 5 Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut, USA. ABSTRACT Oxidative stress damages dermal and epidermal cells and degrades extracellular matrix proteins, such as collagen, ultimately leading to skin aging. The present study evaluated the potential protective effect of the aqueous methanolic extract obtained from Lithospermum erythrorhizon (LE) against oxidative stress, induced by H2O2 and ultraviolet (UV) irradiation, on human keratinocyte (HaCaT) and human dermal fibroblast–neonatal (HDF-n) cells. Exposure of cells to H2O2 or UVB irradiation markedly increased oxidative stress and reduced cell viability. However, pretreatment of cells with the LE extract not only increased cell viability (up to 84.5%), but also significantly decreased oxidative stress. Further, the LE extract downregulated the expression of matrix metalloproteinase-1, an endopeptidase that degrades extracellular matrix collagen. In contrast, treatment with the LE extract did not affect the expression of procollagen type 1 in HDF-n cells exposed to UVA irradiation. Thirteen phenolic compounds, including derivatives of shikonin and caffeic acid, were identified by ultrahigh-performance liquid chromatography–electrospray ionization–tandem mass spectrometry. These results suggest that LE-derived extracts may protect oxidative-stress-induced skin aging by inhibiting degradation of skin collagen, and that this protection may derive at least in part from the antioxidant phenolics present in these extracts. Further studies are warranted to determine the potential utility of LE-derived extracts in both therapeutic and cosmetic applications.

KEY WORDS:  caffeic acid derivative  matrix metalloproteinase-1  shikonin derivative  skin  ultraviolet irradiation  vitamin C equivalent antioxidant capacity

Skin aging can be influenced by both intrinsic and extrinsic factors that exert a synergistic effect on chronic degenerative changes throughout an individual’s life span.3 Intrinsic factors, such as genetic and biological predispositions, confer inevitable physiological changes with time. In contrast, extrinsic factors, such as UV radiation, pollution, and diet, can be controlled or modified.4 Among the extrinsic factors, photoaging induced by chronic exposure to UV radiation is a major cause of skin aging.5 Ultraviolet B (UVB, 280–315 nm) radiation penetrates the epidermis, and thus can be damaging to cellular macromolecules and constituents, such as DNA, proteins, and membranes. Ultraviolet A (UVA, 315–400 nm) radiation is also detrimental to cellular macromolecules. Exposure to UVA radiation has been shown to increase the generation of ROS through chemical interactions with intracellular chromophores.6,7 Overall, both UV radiation and ROS accelerate photoaging by inducing oxidative stress. Singlet oxygen and superoxide radical anion are enzymatically or spontaneously

INTRODUCTION

O

xidative stress is defined as an imbalance between the generation of reactive oxygen species (ROS) and antioxidant defense systems in the body, where the former is overwhelming the latter.1,2 ROS are produced during normal metabolism of oxygen and environmental stress such as ultraviolet (UV) in living organisms. ROS, such as superoxide radical anion (O2 - ), hydroxyl radical (OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2), are very reactive, resulting in damage to cellular macromolecules and functions. Oxidative stress is known to damage dermal and epidermal cells and degrade extracellular matrix proteins, such as collagen, ultimately leading to skin aging. Manuscript received 15 October 2013. Revision accepted 22 June 2014. Address correspondence to: Dae-Ok Kim, PhD, Department of Food Science and Biotechnology, Kyung Hee University, Yongin, Gyeonggi 446-701, Republic of Korea, E-mail: [email protected]

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YOO ET AL.

converted to hydrogen peroxide, which is further converted to hydroxyl radical through the Fenton reaction. Excessive intracellular ROS are highly detrimental to skin cells, including keratinocytes and fibroblasts, because of their reactivity with intracellular components.8,9 ROS also impact cell signaling by modulating the production of cytokines and growth factors and affecting mitogen-activated protein kinase (MAPK) pathways. In addition, chronic oxidative stress from a variety of sources of ROS upregulates nuclear factor kappa B (NF-kB) and activator protein 1 (AP-1) and downregulates transforming growth factor beta (TGF-b) expression, thereby inhibiting procollagen synthesis and activating the expression of matrix metalloproteinase (MMP), an endopeptidase that degrades extracellular matrix collagen.9,10 Lithospermum erythrorhizon (LE) is a plant native to East Asia, which has traditionally been used as a natural colorant for food and fabrics.11 In addition, LE has been used as a medicine to aid in healing or prevention of burns, inflammation, wounds,12 atopic dermatitis,13 and skin cancer.14 LE has also been demonstrated to have moisturizing effects on human skin.15 However, information is limited on whether LE has any protective effect against skin aging. Therefore, the aims of the present study were to investigate whether LEderived phenolics protect human keratinocytes and fibroblasts against oxidative stress caused by H2O2 or UV radiation, and to identify major phenolics present in LE-derived extracts using ultrahigh-performance liquid chromatography– electrospray ionization–tandem mass spectrometry (UHPLCESI-MS/MS). MATERIALS AND METHODS

Extraction of phenolics To establish optimal extraction conditions, phenolics were extracted from LE powder by homogenization and sonication in various ratios (20%, 40%, 60%, 80%, and absolute) of methanol/water (v/v) according to a modified version of the method described in our previous publication.16 LE powder (5 g) was homogenized in a 500-mL Erlenmeyer flask with a Polytron homogenizer (PT 10/35, Kinematica, KriensLuzern, Switzerland) at 15,000 rpm for 2 min in 100 mL of either aqueous methanol or absolute methanol. The flask, containing the homogenized sample, was immersed into the ultrasonic bath for 20 min under N2 purging. The resultant extracts were filtered through Whatman #2 filter paper (Whatman International Limited, Kent, England) using a chilled Bu¨chner funnel, and filter cakes remaining in the funnel were re-extracted twice by repeating the same procedure. The resultant filtrates were transferred to a 1-L, round-bottomed flask, and solvents were evaporated under reduced pressure using a rotary evaporator (Eyela, Tokyo, Japan) in a water bath at 37C. The concentrated extracts were dissolved in 50 mL of absolute methanol and brought up to final volumes of 100 mL with distilled deionized water (DDW). The final extracts were stored at - 20C until analysis. All experiments were conducted in triplicate. Determination of total phenolics Total phenolic contents of the LE phenolic extracts were measured colorimetrically using Folin-Ciocalteu’s phenol reagent.17,18 The contents of total phenolics were expressed as mg gallic acid equivalents (GAE)/100 g dry weight (DW) of LE.

Samples

Determination of total flavonoids

LE grown in Jecheon, Republic of Korea, was purchased from YakchoJangter ( Jecheon, Republic of Korea) and ground in a mill. The resultant powder was stored at - 20C prior to use.

Total flavonoid contents of the LE phenolic extracts were measured colorimetrically.18 The contents of total flavonoids were expressed as mg catechin equivalents (CE)/100 g DW of LE.

Chemicals

Determination of vitamin C equivalent antioxidant capacities

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Ascorbic acid, 2,2 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS), catechin, 20 ,70 dichlorofluorescein diacetate (DCFH-DA), 2,2-diphenyl1-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), Folin-Ciocalteu’s phenol reagent, gallic acid, H2O2, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2,20 -Azobis-(2-methylpropionamidine) dihydrochloride (AAPH) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dulbecco’s phosphate-buffered saline (DPBS), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Welgene, Inc. (Daegu, Republic of Korea). Fibroblast basal medium (FBM) and fibroblast growth kit were purchased from Lonza Cologne GmbH (Walkersville, MD, USA). All other reagents used were of analytical or HPLC grade.

The antioxidant capacities of the LE phenolic extracts were measured by two different methods, one detecting ABTS and one detecting DPPH radical-scavenging ability.18,19 Antioxidant capacities were expressed as mg vitamin C equivalents (VCE)/100 g DW of LE. Cell culture HaCaT cells, belonging to an immortalized human keratinocyte cell line, were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL of penicillin, and 100 mg/L of streptomycin. The HaCaT cells were kindly provided by Dr. Norbert E. Fusenig (German Cancer Research Center, Heidelberg, Germany). Human dermal fibroblast–neonatal (HDF-n) cells (American Type Culture Collection, Manassas, VA, USA) were cultured in complete FBM with FGM SingleQuot supplement containing insulin,

SKIN ANTIAGING EFFECT OF LITHOSPERMUM ERYTHRORHIZON

basic fibroblast growth factor, and 2% FBS. Both cell lines were maintained at 37C in a humidified incubator containing 5% CO2. Determination of cell viability To determine the nontoxic maximal concentration of the 80% (v/v) aqueous methanol LE extract, its cytotoxicity was determined with the MTT reduction assay.20 Cells were seeded in a 96-well plate at a density of 2 · 104 cells/well in 100 lL of FBS-containing culture medium for 24 h. Subsequently, the cells were treated with various concentrations of the LE phenolic extract, dissolved in serum-free culture medium, for 24 h. After removing the medium from each well, MTT reagent was added and cells were incubated for 2 h, during which time the plate was covered with aluminum foil. At 2 h, 50 lL of DMSO was added to dissolve the purple formazan resulting from MTT reduction. In this assay, a decrease in cell viability by more than 20% compared with control cells was considered to be cytotoxic. The LE phenolic extract showed no cytotoxicity in HaCaT cells up to concentrations of 120 mg/L (data not shown). However, at concentrations of 250, 500, and 1000 mg/L, cell viability decreased to 59.2%, 21.7%, and 4.6%, respectively, indicating cytotoxicity at these levels. This observation is consistent with an earlier report that demonstrates that the active fraction from the methanol extract of LE, containing lithospermic acid derivatives, was not cytotoxic at concentrations of 50–200 mg/L as determined by the MTT assay.21 However, in HDF-n cells, the LE phenolic extract was cytotoxic at much lower concentrations (data not shown). In our study, HDF-n cells were 100% viable in the presence of 12.5 mg/L LE phenolic extract, whereas cell viability in concentrations of 25, 50, and 100 mg/L LE phenolic extract decreased to 68.6%, 50.3%, and 29.0%, respectively. To determine whether the LE phenolic extract exerts a protective effect from oxidative stress, 500 lM of the oxidant H2O2 was added to each well after pretreatment with the extract at nontoxic levels. After 1 h, the absorbance was measured using a TECAN infinite M200 microplate reader (San Jose, CA, USA) at 570 nm (test wavelength) and 630 nm (reference wavelength). Cytotoxicity was expressed as the percentage of viable cells relative to control cells cultured without the LE phenolic extract. Measurement of oxidative stress in HaCaT cells Levels of oxidative stress were determined by fluorescence assays using DCFH-DA.18,20 This cell-permeable probe diffuses into cells, where it is deacetylated by cellular esterases. The product of this reaction is rapidly oxidized to generate a fluorescent product, DCF, upon reaction with intracellular radicals. In DCFH-DA assay, phenolic compounds can be taken up inside the cells or bind strongly at the cellular membrane, where they serve as antioxidants and neutralize ROS.22 HaCaT cells were seeded at a density of 2 · 104 cells/ well in 96-well plates and incubated for 24 h prior to pretreatment with serum-free culture medium, containing nontoxic concentrations of the LE phenolic extract, for an

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additional 24 h. After removing the supernatant, 50 lM of DCFH-DA in DPBS was added. At 30 min, oxidative stress was induced for 1 h with either 200 lM H2O2 or UVB (30 mJ/ cm2). The resultant fluorescence was measured using a TECAN infinite M200 microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The level of oxidative stress was expressed as the percentage fluorescence relative to control cells (set to 100%). Effect of the LE phenolic extract on expression of MMP-1 and procollagen type 1 in HDF-n cells exposed to UVA HDF-n cells were seeded in 96-well plates (2 · 104 cells/ well) and cultured in FBM supplemented with fibroblast growth kit–low serum for 42 h. Prior to UVA irradiation, cells were washed with DPBS and covered with a thin layer of DPBS. In parallel, nonirradiated cells were treated similarly. The cells were exposed to 5 J/cm2 of UVA light at 365 nm (Bio-Sun, Vilber Lourmat, Marne-la-Valle´e, France). After irradiation, DPBS was removed. Serum-free medium with or without the LE phenolic extract was added, and the cells were incubated for 24 h. The levels of MMP-1 in culture supernatants were measured using the Human Total MMP-1 DuoSet ELISA Development kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. Under the same cell culture conditions, the levels of procollagen type 1 in the supernatant were measured using the Procollagen Type 1 C-peptide EIA kit (Takara Korea Biomedical, Inc., Seoul, Republic of Korea) according to the manufacturer’s instructions. Identification of major phenolics using UHPLC-ESI-MS/MS Phenolics were analyzed by using a high-resolution LTQOrbitrap XL mass spectrometer connected to an Accela system (Thermo Fisher Scientific, Waltham, MA, USA). For polarity-based separation of phenolics, ACQUITY BEH C18 column (100 · 2.1 mm, 1.7 lm; Waters Corporation, Milford, MA, USA) was used. The flow rate and injection volumes were 0.3 mL/min and 2 lL, respectively. The solvent gradient conditions of binary mobile phases consisted of solvent A (DDW with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) were as follows: 96% A/4% B at 0 min, 70% A/30% B at 20 min, 30% A/70% B at 25 min, 5% A/95% B at 25.1 min, 5% A/95% B at 27 min, and 0% A/100% B at 30 min. Phenolics were detected at 350 nm. The MS conditions in negative ESI mode were optimized to the following: spray voltage, 4.0 kV; capillary voltage, 35 V; and temperature, 300C. Xcalibur software was used for data acquisition. Identifications of phenolic compounds were based on comparisons of retention times, UV spectra, masses, and MS/MS fragment ions with those of available standard compounds. Statistical analysis All experiments were performed in triplicate. Statistical analysis was performed using SAS software (version 8.2,

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YOO ET AL. Table 1. Levels of Total Phenolics, Total Flavonoids, and Antioxidant Capacities of the Lithospermum erythrorhizon Phenolic Extracts

Solvents 20% (v/v) Methanol 40% (v/v) Methanol 60% (v/v) Methanol 80% (v/v) Methanol Absolute methanol

Total phenolics (mg gallic acid equiv./100 g DW)

Total flavonoids (mg catechin equiv./100 g DW)

694.6 – 118.2d 754.1 – 65.1d 922.9 – 59.8c 1294.4 – 48.2a 1212.4 – 79.1b

166.7 – 35.8d 359.5 – 16.7c 809.0 – 96.6b 888.7 – 44.0a 763.0 – 55.5b

Antioxidant capacities (mg vitamin C equiv./100 g DW) ABTS

DPPH

866.2 – 43.4d 1154.5 – 47.1c 1183.6 – 94.8c 1527.9 – 74.0a 1461.1 – 56.7b

374.9 – 37.5d 568.6 – 44.6c 828.0 – 160.8b 1004.2 – 147.6a 943.2 – 171.3ab

All data are presented as means – standard deviations (n = 3). Lowercase letters in each column show significant differences as determined by Duncan’s multiplerange test (P < .05). DW, dry weight.

SAS Institute, Inc., Cary, NC, USA). One-way ANOVA was performed to determine the differences among means. Significance of differences (P < .05) was determined by Duncan’s multiple-range test. RESULTS Total phenolics, total flavonoids, and antioxidant capacities The levels of total phenolics, total flavonoids, and antioxidant capacities of the LE phenolic extracts obtained from the various concentrations of aqueous methanol (20%, 40%, 60%, 80%, and absolute) are shown in Table 1. The amounts of total phenolics varied depending on the methanol-water ratio, and ranged from 694.6 to 1294.4 mg GAE/100 g DW. The phenolic extract generated with 80% aqueous methanol yielded the highest amount of total phenolics. The amounts of total flavonoids ranged from 166.7 to 888.7 mg CE/100 g DW, with the 80% aqueous methanolic extract also yielding the highest amount of total flavonoids. The antioxidant capacities of the extracts were evaluated by performing ABTS and DPPH radical-scavenging assays. The antioxidant capacities of the extracts, as measured by the ABTS assay, ranged from 866.2 to 1527.9 mg VCE/100 g DW, whereas the antioxidant capacities determined by the DPPH assay ranged from 374.9 to 1004.2 mg VCE/100 g DW. Consistent with its total phenolic and flavonoid contents, the extract obtained with 80% methanol displayed the highest antioxidant capacities in both ABTS and DPPH assays. Considering its high levels of total phenolics, total flavonoids, and antioxidant capacities, the 80% methanol extract (heretofore referred to as ‘‘the LE phenolic extract’’) was selected for further analysis. Subsequent experiments were aimed to determine whether this LE phenolic extract protected skin from the damaging effects of oxidative stress, as induced by either H2O2 or UV irradiation. Protective effect of the LE phenolic extract against oxidative stress in HaCaT cells Upon exposure to 500 lM H2O2, the viability of HaCaT cells decreased to 62.7% compared with control cells (100%), whereas pretreatment with the LE phenolic extract (60 and 120 mg/L) prior to exposure to H2O2 significantly

increased cell viability compared with cells treated with H2O2 alone (Fig. 1). At 120 mg/L LE extract, cell viability was significantly increased compared with that at 60 mg/L. However, this protective effect was not observed at concentrations equal to or lower than 30 mg/L (Fig. 1). These results indicate that the LE phenolic extract attenuated oxidative stress and cell death at concentrations of 60 and 120 mg/L. Effect of the LE phenolic extract on intracellular oxidative stress in HaCaT cells The effect of LE phenolic extracts against intracellular oxidative stress in HaCaT cells exposed to H2O2 or UVB was also evaluated using the DCFH-DA assay (Fig. 2). Upon exposure to H2O2, oxidative stress in HaCaT cells increased to 162% compared with the level of oxidative stress in control cells (set to 100%, Fig. 2A). However,

FIG. 1. Effect of the Lithospermum erythrorhizon (LE) phenolic extract against oxidative stress in HaCaT cells as determined by the MTT assay. HaCaT cells were pretreated with various concentrations of the LE phenolic extract for 24 h, and then exposed to 500 lM H2O2 for 2 h. Lowercase letters above bars indicate significant differences as determined by Duncan’s multiple-range test (P < .05). Data are presented as means – standard deviations (bars) of three replicates.

SKIN ANTIAGING EFFECT OF LITHOSPERMUM ERYTHRORHIZON

FIG. 2. Effects of the Lithospermum erythrorhizon (LE) phenolic extract on oxidative stress induced by either H2O2 (A) or UVB (B) in HaCaT cells as determined by the DCFH-DA assay. HaCaT cells were pretreated with the LE phenolic extract for 24 h. Cell supernatants were removed, the DCFH-DA reagent was added, and, finally, H2O2 was added. Fluorescence was measured at 485 nm for excitation and 530 nm for emission. Lowercase letters above bars indicate significant differences as determined by Duncan’s multiple-range test (P < .05). Data are presented as means – standard deviations (bars) of three replicates.

pretreatment with the LE phenolic extract decreased this H2O2-induced oxidative stress, with a concentration of 120 mg/L being sufficient to completely rescue HaCaT cells from H2O2-induced oxidative stress (Fig. 2A). Likewise, UVB exposure increased intracellular oxidative stress to 137% the level of control cells, whereas pretreatment with phenolics at 40 mg/L decreased oxidative stress *30% compared with control cells (Fig. 2B). Effect of the LE phenolic extract on expression of MMP-1 and procollagen type 1 in HDF-n cells exposed to UVA We assessed the inhibitory effect of the LE phenolic extract on MMP-1 expression in HDF-n cells (Fig. 3A). The

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FIG. 3. Effects of the Lithospermum erythrorhizon (LE) phenolic extract on MMP-1 expression (A) and procollagen type 1 expression (B) in human dermal fibroblast–neonatal (HDF-n) cells. After 5 J/cm2 of UVA radiation at 365 nm, HDF-n cells were treated with the LE phenolic extract and incubated for 24 h. The expression of MMP-1 and procollagen type 1 was determined using the Human Total MMP1 DuoSet ELISA Development kit and the Procollagen Type 1 Cpeptide EIA kit, respectively. In parallel, nonirradiated control cells were treated with DPBS. Lowercase letters above bars indicate significant differences as determined by Duncan’s multiple-range test (P < .05). Data are presented as means – standard deviations of three replicates.

level of expression of MMP-1 following UVA exposure was 76% higher than that in nonirradiated controls. However, pretreatment of HDF-n cells with the LE phenolic extract downregulated MMP-1 expression in a dose-dependent manner. Also, we evaluated whether the phenolic extract of LE affected the expression of procollagen type 1 in HDF-n cells exposed to UVA (Fig. 3B). The expression of procollagen type 1 decreased by about 42% in control cells when exposed to UVA radiation. In our study, the LE phenolic extract did not significantly affect procollagen type 1 expression when used at concentrations of 0.1, 1.0, and 10 mg/L.

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YOO ET AL. Table 2. Identification of Main Phenolic Peaks in the Lithospermum erythrorhizon Phenolic Extract Using UHPLC-ESI-MS/MS in Negative Ion Mode

Peak No. 1 2 3 4 5 6 7 8 9 10 11 12 13

Retention time (min)

[M-H] - (m/z)

Formula

Error (ppm)

12.11 13.53 13.68 14.33 15.44 15.73 17.93 25.42 25.84 26.72 28.34 28.78 28.97

717.1457 537.1036 359.0770 537.1037 717.1455 537.1036 493.1135 355.1549 387.1447 329.1028 357.1342 369.1341 371.1500

C36H30O16 C27H22O12 C18H16O8 C27H22O12 C36H30O16 C27H22O12 C26H22O10 C21H24O5 C21H24O7 C18H18O6 C20H22O6 C21H22O6 C21H24O6

0.669 0.878 0.836 0.998 0.529 0.888 0.577 0.880 0.921 0.855 0.885 0.845 1.045

MS/MS ions (m/z) 519, 475, 493, 223, 197, 493,

365, 438, 179, 438, 519, 493, 438, 295, 313, 255, 269,

339 197 161 197 321 197 424 273 287 269 269 269 269

Identification Rabdosiina Lithospermic acid isomerb Rosmarinic acida Lithospermic acida Lithospermic acid Ba Lithospermic acid isomerb Salvianolic acid Aa Shikonofuran Ea b-Hydroxyisovalerylshikonina Acetylshikonina Isobutylshikonina b,b-Dimethylacrylshikonina Isovalerylshikonina

a

Identification was inferred from published literature. Identification was obtained by comparison with high-resolution MS and MS/MS data of lithospermic acid. UHPLC-ESI-MS/MS, ultrahigh-performance liquid chromatography–electrospray ionization–tandem mass spectrometry.

b

Identification of major phenolics using UHPLC-ESI-MS/MS analysis The LE phenolic extract was analyzed to identify bioactive phenolics using UHPLC-ESI-MS/MS (Table 2). We identified 13 phenolic compounds present in the LE phenolic extract (Fig. 4). Peaks 1, 3, 4, 5, 7, and 10 were identified as rabdosiin, rosmarinic acid, lithospermic acid, lithospermic acid B, salvianolic acid A, and acetylshikonin, respectively. Peaks 2, 4, and 6 were identified as isomers of lithospermic acid through comparisons of their product ions. Peaks 8, 9, 11, 12, and 13 were identified as shikonofuran E, b-hydroxyisovalerylshikonin, isobutylshikonin, b,b-dimethylacrylshikonin, and isovalerylshikonin, respectively.

FIG. 4. Ultrahigh-performance liquid chromatography chromatogram (350 nm) of the major phenolics present in the Lithospermum erythrorhizon phenolic extract. Through comparative study, thirteen compounds were identified (1, rabdosiin; 2, lithospermic acid isomer; 3, rosmarinic acid; 4, lithospermic acid; 5, lithospermic acid B; 6, lithospermic acid isomer; 7, salvianolic acid A; 8, shikonofuran E; 9, b-hydroxyisovalerylshikonin; 10, acetylshikonin; 11, isobutylshikonin; 12, b,b-dimethylacrylshikonin; 13, isovalerylshikonin).

DISCUSSION Phenolics, such as flavonoids and phenolic acids, are important aromatic secondary metabolites in plants. Many phenolic phytochemicals have been shown to have antioxidant properties and to protect cells against oxidative damage caused by ROS. Both endogenous and exogenous ROS can damage cell proteins, such as collagen, leading to photoaging in skin.9,23 Endogenous ROS are generated from phagocytosis, nucleotide degradation, cytochrome P450-mediated biotransformation, and the electron transport chain. These processes all generate superoxide radical anions (O2 - ), which can be converted to H2O2. On the other hand, UV radiation exogenously produces ROS like singlet oxygen (1O2) in the presence of photosensitizing chromophores, such as NADPH, riboflavin, cytochromes, and porphyrins. Using the DCFH-DA assay with HaCaT cells exposed to H2O2 or UVB, effects of LE phenolic extracts against cellular oxidative stress were revealed (Fig. 2). These findings suggest that the LE phenolic extract contains potent intracellular antioxidants that can protect cells against oxidative stress. In agreement with our findings here, a previous report demonstrated that the viability of normal human epidermal keratinocytes exposed to UVB (302 nm and 27 mJ/cm2) increased significantly when treated with LE phenolic extracts (2.5–7.5 mg/L).24 The high-resolution MS data and MS/MS spectral characteristics of the phenolics in the LE phenolic extract were compared with commercially obtained standards and published data.25–28 The data presented here suggest that LE phenolic extracts contain compounds possessing antioxidant properties (Table 1). Our mass spectrometry data as described later revealed derivatives of caffeic acid and shikonin present in our LE phenolic extract (Table 2). These data are consistent with previous reports that describe derivatives of caffeic acid21,29,30 and shikonin27,28,31 as major phenolic constituents of LE. Caffeic acid derivatives in LE

SKIN ANTIAGING EFFECT OF LITHOSPERMUM ERYTHRORHIZON

include rosmarinic acid, rabdosiin, salvianolic acid A, and lithospermic acid, whereas shikonin derivatives include shikonin, shikonofuran D, b-hydroxyisovalerylshikonin, and acetylshikonin. These caffeic acid derivatives are structurally similar to rosmarinic acid and other aromatic compounds.32 Interestingly, shikonin, b,b-dimethylacrylshikonin, and acetylshikonin have all been shown to exert a synergistic antioxidant effects with both vitamin E and citric acid.31 Further, the hexane extract of LE has been shown to contain acetylshikonin and shikonin and to inhibit ROS activity.33 Other previous studies have also shown that shikonin derivatives possess strong antioxidant activity, as measured by ABTS and DPPH radical-scavenging ability.34 Similarly, rabdosiin and rosmarinic acid have been shown to possess potent radical-scavenging activity for O2 - and OH; moreover, their scavenging activities are even stronger than that of vitamin C.30,35 Our data and findings from the aforementioned-cited studies suggest that these phenolic compounds, present in LE extracts, may be partly responsible for the reduced oxidative stress and improved viability of HaCaT cells upon exposure to H2O2 and UVA. In addition to their antioxidant properties, these phenolics exhibit various bioactivities, such as anti-HIV, antitumor, antihypertension, and anti-ischemic activities.36 Solar UV radiation is known to increase the synthesis of MMPs in human skin in vivo.37 The induction of MMP synthesis by UV radiation can promote skin aging, since MMPs degrade extracellular matrix constituents, such as collagen and elastin.38 Another damaging aspect of UV radiation is that it can trigger the release of ROS, such as H2O2 and OH, in fibroblasts.39 ROS are known to upregulate the expression of NF-kB and AP-1 and downregulate TGF-b expression, thereby leading to the activation of MMP expression and the inhibition of procollagen synthesis in their downstream.9,10 Oxidative stress generated by ROS has been shown to exacerbate mitochondrial stress, partially deplete mitochondrial DNA, cause loss of mitochondrial function, and ultimately result in the upregulation of MMP-1 expression.40 Specifically, MMP-1 is one of the interstitial collagenases that degrades collagen, thereby resulting in skin aging and wrinkles. In this regard, the inhibitory effect of the LE phenolic extract on MMP-1 expression in HDF-n cells was assessed (Fig. 3A). In our study, pretreatment of HDF-n cells with the LE phenolic extract downregulated MMP-1 expression in a dose-dependent manner. The LE phenolic extract (at a concentration of 10 mg/L) decreased the expression of MMP-1 by *33% (Fig. 3A). This result suggests that the phenolics present in the extract may act as antioxidants to attenuate UVA-induced oxidative stress, a major cause for the upregulation of MMP-1 in fibroblasts. Thus, it is possible that the combined antioxidant properties of LE phenolics may retard skin aging by inhibiting the degradation of extracellular matrix collagen in skin fibroblasts exposed to UVA. A substantial decrease in collagen synthesis has been reported in photoaged skin in vivo.41 Human dermis is predominantly composed of type I collagen, with lesser

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amounts of type III collagen; dermal fibroblasts synthesize type I procollagen precursor molecules that are later cleaved to mature collagen.42 The phenolic extract of LE did not affect the expression of procollagen type 1 in HDF-n cells exposed to UVA, demonstrating that it does not have a significant effect on procollagen type 1 expression (Fig. 3B). Taken together, the just-discussed findings indicate that the protective effect of the LE phenolic extract against skin damage is a result of its high antioxidant capacity, rather than an effect on collagen synthesis. In conclusions, the LE phenolic extract obtained with 80% aqueous methanol was shown to have the highest levels of total phenolics, total flavonoids, and antioxidant capacities. This LE phenolic extract protected human keratinocyte HaCaT cells from oxidative stress induced by H2O2 or UVB and inhibited the expression of MMP-1 in human fibroblast HDF-n cells exposed to UVA irradiation; however, it had no effect on procollagen type 1 expression. The results presented here suggest that the LE phenolic extract may protect against skin damage and aging by reducing cellular oxidative stress and downregulating the expression of MMP-1, a collagenase that degrades extracellular matrix collagen. Such protective effects may be attributable to the phenolics present in the LE phenolic extract, mainly, derivatives of caffeic acid and shikonin. Further studies are required to determine the bioactivities of individual LE phenolics and their synergistic effects in reducing oxidative stress and skin aging in vivo. The potential of LE phenolic extracts, and the phenolic components therein, for therapeutic and cosmetic applications remains to be evaluated.

ACKNOWLEDGMENT This study was conducted with the support of the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (No. 108037-03-2-CG000). AUTHOR DISCLOSURE STATEMENT The authors declare no conflicts of financial interest. REFERENCES 1. Betteridge DJ: What is oxidative stress? Metabolism 2000;49: 3–8. 2. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O: Oxidative stress and antioxidant defense. WAO J 2012;5:9–19. 3. Friedman O: Changes associated with the aging face. Facial Plast Surg Clin N Am 2005;13:371–380. 4. Bergfeld WF: The aging skin. Int J Fertil Menopausal Stud 1997;42:57–66. 5. Miyachi Y, Ishikawa O: Dermal connective tissue metabolism in photoageing. Australas J Dermatol 1998;39:19–23. 6. Tyrrell RM: Activation of mammalian gene expression by the UV component of sunlight—from models to reality. Bioessays 1995;18:139–148. 7. Routaboul C, Marguery MC, Garigue J, Bazex J: Influence of UVA pre-exposure on UVB-induced erythema. Photodermatol Photoimmunol Photomed 1999;15:52–58.

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