Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and prevents UVB-induced oxidative stress and DNA damage in a skin cell model

https://doi.org/10.1016/j.jdermsci.2016.08.004Get rights and content

Highlights

  • Lemon balm extract contains rosmarinic and salvianolic acids as the main polyphenols.

  • LBE protects skin cells against UVB-induced cytotoxicity and ROS generation.

  • LBE decreases UVB-induced DNA damage and the DNA damage response in keratinocytes.

  • Lemon balm extract promotes melanogenesis in melanoma cells.

  • LBE has the potential to protect human skin against UV-induced damage.

Abstract

Background

Solar ultraviolet (UV) radiation is one of the main causes of a variety of cutaneous disorders, including photoaging and skin cancer. Its UVB component (280–315 nm) leads to oxidative stress and causes inflammation, DNA damage, p53 induction and lipid and protein oxidation. Recently, an increase in the use of plant polyphenols with antioxidant and anti-inflammatory properties has emerged to protect human skin against the deleterious effects of sunlight.

Objective

This study evaluates the protective effects of lemon balm extract (LBE) (Melissa Officinalis, L) and its main phenolic compound rosmarinic acid (RA) against UVB-induced damage in human keratinocytes.

Methods

The LBE composition was determined by HPLC analysis coupled to photodiode array detector and ion trap mass spectrometry with electrospray ionization (HPLC-DAD-ESI-IT-MS/MS). Cell survival, ROS generation and DNA damage were determined upon UVB irradiation in the presence of LBE. The melanogenic capacity of LBE was also determined.

Results

RA and salvianolic acid derivatives were the major compounds, but caffeic acid and luteolin glucuronide were also found in LBE. LBE and RA significantly increased the survival of human keratinocytes upon UVB radiation, but LBE showed a stronger effect. LBE significantly decreased UVB-induced intracellular ROS production. Moreover, LBE reduced UV-induced DNA damage and the DNA damage response (DDR), which were measured as DNA strand breaks in the comet assay and histone H2AX activation, respectively. Finally, LBE promoted melanogenesis in the cell model.

Conclusions

These results suggest that LBE may be considered as a candidate for the development of oral/topical photoprotective ingredients against UVB-induced skin damage.

Introduction

The skin is the largest organ of the human body, participates in sensitivity and temperature maintenance and offers protection from chemicals, microorganisms and UV radiation [1]. An excessive UV exposition can lead to several skin pathological disorders, including erythema, immunosuppression, edema, sunburn, hyperplasia, hyperpigmentation, premature aging and skin cancer [2]. UV radiation is divided into long wave UVA (315–400 nm, 90% of UV radiation), medium wave UVB (280–315 nm, 5% of UV radiation), and short wave UVC (200–280 nm). UVB is one thousand times more capable of causing sunburn than UVA and is considered the most damaging and genotoxic [3].

Melanin is the main skin protective barrier that acts by absorbing and scattering UV radiation. Nevertheless, other intracellular molecules are targeted (DNA, RNA, lipids and proteins). The direct effect of UVB on DNA leads to the formation of cyclobutane pyrimidine dimers (CPDs) and to a lesser extent pyrimidine (6–4) pyrimidone (6-4PPs) photoproducts. When these alterations are not well repaired, the resulting substitution/transition mutations (cytosine-thymine) in the epidermal cells can lead to the development of skin cancer [4], [5].

UVB, together with UVA, generates superoxide (O2radical dot), either directly or through enzyme activation [6], [7]. This is the most promptly generated oxygen radical species (ROS) and is rapidly derived into H2O2, which forms OH upon the Fenton reaction [4]. UVB induced radical dotOH is postulated to be responsible for the formation of DNA single-strand breaks (SSBs) and also for lipid peroxidation through the generation of lipoperoxy radicals (ROO), malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). Additionally, UVB-induced ROS interact with numerous cellular targets and receptors that regulate crucial pathways related to inflammation, cell survival, cell growth and differentiation in human keratinocytes: the NF-κB, the AP-1 transcription factor, the mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2) pathways [8], [9], [10]. Most of these effects lead to extracellular matrix degeneration by proteases activation and skin photoaging [1].

Plant polyphenols possess strong free radical scavenging properties and have exhibited the capacity to modulate multiple cellular pathways [11]. Recently, their potential skin photoprotective effects have gained considerable attention [12]. The UVB protective effects of botanical extracts, such as Punica granatum [13], citrus and rosemary [14], green tea polyphenols [15] and pure compounds, such as resveratrol [16], genistein [17], [18] and hydroxytyrosol [19] has been reported. Recently our group has demonstrated the synergistic protective effect of rosemary and citrus polyphenolic extracts both in vitro and in vivo [14].

Lemon balm (Melissa officinalis, L.) is a representative of the Lamiaceae family, native to Europe but with a worldwide distribution. This herb is used not only for ornamental purposes but also for medicine and cosmetics. It is commonly used for insomnia and anxiety [20], herpes [21], and indigestion [22], as an antioxidant [23] and as an antimicrobial agent [24]. Lemon balm extraction may lead to the essential oil and the lemon balm polyphenolic extract (LBE), which is enriched in phenylpropanoid derivatives and flavonoids. The major phenolic compound found in the polyphenolic extract is rosmarinic acid (RA), which is an ester of caffeic acid, and 3,4-dihydroxyphenyllactic acid [25] (Fig. 1, see insert). The antioxidant activity of LBE has been previously characterized in both in vitro and in vivo models [26], [27], [28], [29].

In the present study, the UVB protective effects of LBE and its major polyphenol RA were explored and compared in human keratinocytes. The potential of LBE to protect human keratinocytes from UVB-induced oxidative stress and to alleviate DNA damage was studied. The protective effect through melanogenesis activation was also studied in a cellular model.

Section snippets

Materials and LBE

Human keratinocytes (the spontaneously immortalized cell line HaCaT) were obtained from Cell lines Service GmbH, CLS (Eppelheim, Germany). Dulbeccós modified Eaglés medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco/Thermo Fisher Scientific (Waltham, MA, USA). RA (96%) and the rest of the reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). LBE standardized containing 18.0 ± 0.3% RA (w/w) was kindly provided by NUTRAFUR, SA – Frutarom Group

Characterization of LBE by HPLC–MS/MS

The major components of LBE were identified by HPLC-DAD-ESI-IT-MS/MS as described in the Methods section. The main polyphenolic compounds were identified using a library of phenolic compounds by comparing their retention times, UV spectra and MS/MS data with those of commercial standards or reports in the literature. Fig. 1 shows the chromatogram obtained at 280 nm for LBE. Thirteen major phenolic compounds were identified in LBE as detailed in Table 1 and were assigned numbers 1–13 according to

Discussion

In this study, the capacity of a lemon balm extract to prevent signs of cellular damage induced by UVB was explored. The major compound in LBE was RA (18% w/w), but other phenylpropanoid derivatives were also present (caffeic acid, salvianolic acid, yunnaneic acid, sagerinic acid and lithospermic acid; Supplementary Fig. S3). Additionally, small amounts of the flavone luteolin-3′-O-glucuronide were found. Hence, most of the effects observed must be due to RA and related polyphenols. Our results

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

This investigation was supported by project AGL2015-67995-C3-1-R and the Torres-Quevedo (PTQ-14-07243) fellowship to E. Barrajón-Catalán from the Spanish Ministry of Science and Innovation, grants PROMETEO/2012/007 and 2016/006 and VALi+D fellowships (ACIF/2010/162 and ACIF/2013/064) from Generalitat Valenciana (GV) and CIBER (CB12/03/30038, Fisiopatología de la Obesidad y la Nutrición, CIBERobn, Instituto de Salud Carlos III). We thank NUTRAFUR, SL for providing us with the raw materials. We

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    1

    These authors have equally contributed to this research and are listed in random order.

    2

    Frutarom Group.

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