Heat Treatment Improves UV Photoprotective Effects of Licorice in Human Dermal Fibroblasts

External stimulation of the skin by ultraviolet B (UVB) radiation induces oxidative stress or inflammation, causing skin aging and skin cancer. Glycyrrhiza uralensis (licorice) has been used as a medicinal plant for its antioxidant, anti-inflammatory, antiviral, antimicrobial, anticarcinogenic, and hepatoprotective properties. The present study analyzed the effects of thermal processing on the bioactivities of licorice. Heat-treated licorice (HL) extracts had better antioxidant and antiinflammatory activities than non-treated licorice (NL) extract. HL extracts also had higher total phenol contents than NL extract. In particular, contents of isoliquiritigenin, an antioxidant and anti-inflammatory substance of licorice, increased in proportion to the skin-protection effects of HL extracts. Heat treatment increased the contents of phenolic compounds such as isoliquiritigenin in licorice extract, which improved the UV photoprotective effect of licorice in human dermal fibroblasts.


Introduction
Ultraviolet (UV) radiation is subdivided into several main types based on wavelength, including ultraviolet A (UVA), ultraviolet B (UVB), and ultraviolet C (UVC) (100-400 nm). UVC and UVB radiation are completely or mostly absorbed, respectively, by the ozone layer in the atmosphere [1]. As the ozone layer is being depleted due to environmental pollution, UVB is increasingly reaching Earth's surface [2]. Exposure to UVB leads to DNA damage, pigmentation, and inflammatory diseases, and excessive exposure can lead to skin cancer [3,4]. Exposure to UVB also indirectly induces oxidative DNA damage through the generation of reactive oxygen species (ROS) [5]. Inflammation-related pathways in the skin, such as the nuclear factor kappa B (NF-κB) pathway, are activated by ROS [6]. Activated NF-κB in the cytoplasm migrates into the nucleus, activating inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines such as interleukin (IL)-6 and IL-1β [7].
Thermal processes are commonly used for preservation and are known to alter the physicochemical and biological properties of plants [22]. During heat treatment, antioxidant activity increases as bioactive substances are synthesized from insoluble components of plants [23]. However, excessive heat can reduce the antioxidant capacity and bioactivity via the loss of nutrients, phenolic compounds, and carotenoids [24]. The appropriate temperature and time for heat treatment can lead to beneficial changes in the components of plants [25]. Previous studies have reported that thermal processes increase phenolic compounds [26] and improve antimutagenic activity and antimicrobial effects [27]. The aim of this study was to confirm the effects of thermal processes on the UV photoprotective activity of licorice in dermal fibroblasts and determine the components related to the activity.

Sample Preparation
The licorice (NL) used in this study was cultivated in Jecheon, Korea, in 2018, and dried at 60 • C for 20 h. Dried licorice (5 g) was placed in a glass container with 1 mL distilled water and treated in an autoclave (Jisico, Seoul, Korea) for 1 h at 120 • C or 130 • C (HL-1 and HL-2, respectively). NL and HL were extracted using 70% ethanol (sample:solvent, 1:50, v:v) for 24 h at room temperature (RT). After filtration, all extracts were evaporated in vacuo, freeze-dried, and stored at −80 • C. All extracts were dissolved in DMSO (Sigma, St. Louis, MO, USA) for use in each experiment.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Activity
DPPH scavenging activity was measured according to the Mishra et al. method, with some modifications [28]. DPPH solution in 95% ethanol was prepared. Next, 50 µL of sample and 200 µL of DPPH solution were mixed, covered with aluminum foil, and incubated for 30 min at RT. Absorbance was measured using a microplate reader (BioTek, Winooski, VT, USA) at 515 nm. The results were calculated according to the following equation, with each value as the half-maximal inhibitory concentration (IC 50 ): where A Treatment = absorbance value of DPPH with sample treatment; A Blank1 = absorbance value of ethanol with DMSO; A Blank2 = absorbance value of ethanol with sample treatment; A Control = absorbance value of DPPH with DMSO.

ABTS + Scavenging Activity
ABTS + scavenging activity was measured as described by Lee et al., with some modification [29]. ABTS + solutions in distilled water were prepared and incubated for 24 h. Next, 20 µL of sample and 180 µL of ABTS + solution were mixed, covered with aluminum foil, and incubated for 30 min at RT. Absorbance was measured using the microplate reader at 732 nm. The results were calculated according to the following equation, with each value as the IC 50 : where A Treatment = absorbance value of ABTS + with sample treatment; A Blank1 = absorbance value of water with DMSO; A Blank2 = absorbance value of water with sample treatment; A Control = absorbance value of ABTS + with DMSO.

Cell Culture and UVB Treatment
Human dermal fibroblasts (HDFs) (ATCC, Manassas, VA, USA) were grown in DMEM supplemented with 10% FBS and 1% p/s (Gibco, Grand Island, NY, USA) in a humidified 5% CO 2 atmosphere at 37 • C. The cells were washed with PBS (Gibco) and exposed to 100 mJ/cm 2 UVB irradiation using a Spectrolinker (XL-1000, Spectronics, Westbury, NY, USA) without the culture plate cover. Then, the UVB-exposed cells were immediately placed in a serum-free medium containing licorice extract for 24 h.

Cell Viability
The effects of HL extracts on the viability of UVB-exposed HDFs were investigated using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega, Madison, WI, USA). HDFs were seeded into 96-well culture plates at a density of 5 × 10 4 cells/well for 24 h. Cells were treated with licorice extracts at various concentrations (25-200 µg/mL) and incubated at 37 • C for 24 h. Then, MTS was added and the absorbance was measured using the microplate reader at 490 nm.

Intracellular ROS Generation
To measure the intracellular ROS levels in UVB-exposed HDFs, HDFs were seeded into 96-well plates at a density of 5 × 10 4 cell/mL for 24 h. Cells were then treated with licorice extracts (100 µg/mL) and UVB radiation (100 mJ/cm 2 ) for 24 h. The cells were treated with 20 µM of DCF-DA (Sigma) and incubated at 37 • C for 20 min. Intracellular ROS generation was measured using the microplate reader at excitation and emission wavelengths of 485 and 535 nm, respectively.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
HDFs were pretreated with NL and HL extracts (100 µg/mL) for 24 h with UVB irradiation (100 mJ/cm 2 ). Total RNA was extracted with TRIzol reagent (Ambion, Austin, Processes 2021, 9, 1040 4 of 12 TX, USA), and 1 µg was reverse transcribed into cDNA using the Reverse Transcriptase Premix Kit (Elpis Biotech, Daejeon, Korea) according to the manufacturer's instructions. RT-PCR was performed using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-, IL-6-, and IL-1β-specific primers (Bioneer, Daejeon, Korea). PCR to evaluate the expression of proinflammatory cytokines was performed using the Maxime PCR PreMix Kit (iNtRON Biotechnology, Seongnam, Korea), according to the manufacturer's instructions. Primers were designed using Primer-BLAST (NCBI, Bethesda, MD, USA). Table 1 shows the sequences of the primers used for RT-PCR. The PCR products were size fractionated by 1% agarose gel electrophoresis. After electrophoresis, the DNA bands were visualized using a UV transilluminator imaging system (Davinch-K, Seoul, Korea). Quantitative measurements were made using free ImageJ. Table 1. Forward and reverse primer sequences for RT-PCR.

Gene
Primer Sequence

Total Phenolic Content (TPC)
TPC was determined using the Folin-Ciocâlteu method [31]. Each extract (500 µL) was mixed with 1 N Folin-Ciocâlteu reagent (50 µL, Sigma) for 3 min, after which 20% sodium carbonate solution (100 µL) was added. After 1 h, the absorbance was measured at 725 nm using the microplate reader. TPC was calculated from the calibration curve using gallic acid (GA, Sigma), and the results were expressed as GA equivalents (y = 0.7615x + 0.0649).

Statistical Analysis
All experimental results are presented as the mean ± standard deviation (SD) of three independent measurements. Statistical analysis was performed using one-way analysis of variance, followed by Tukey's multiple comparison test using Prism 5.02 (GraphPad Software, San Diego, CA, USA), in addition to Duncan's multiple range test using IBM SPSS, ver. 22.0 for Windows. p-values < 0.05 were considered to indicate statistical significance.

Heat Treatment Improved the UV Photoprotective Effect of Licorice Extract by Enhancing Antioxidant Properties
MTS assay showed >100% HDF cell viability for extract treatments of up to 100 µg/mL; however, treatments of 200 µg/mL for both NL and HL extracts showed toxic effects on HDFs ( Figure 1). Therefore, subsequent experiments used extracts of 100 µg/mL. Exposure to UVB leads to accelerated cellular ROS levels [32]. ROS levels were 5.2-fold higher in the UVB control cells than in the non-treated control (p < 0.001) and 1.9-fold lower in the NL extract (100 µg/mL)-treated samples than in the UVB control (p < 0.001) (Figure 2). HL-1 and HL-2 extract (100 µg/mL) treatments showed better inhibitory activities (2.3-fold and 3.8-fold, respectively, vs. UVB control, p < 0.001) (Figure 2), indicating that heat treatment enhanced the antioxidant properties of licorice extract.

Heat Treatment Increased the Contents of Bioactive Compounds
Heat treatment causes an increase in antioxidant components [36]. In this study, the antioxidant and anti-inflammatory activities of the HL extracts increased with heat treatment. The total contents of phenolics, which are responsible for the antioxidant activities of plants, and isoliquiritigenin, a phenolic compound with antioxidant and anti-inflammatory activities, were measured in licorice extracts to determine whether the components related to these activities improved with heat treatment. The HL-1 and HL-2 extracts had higher TPC (12.1 ± 0.6 and 14.4 ± 0.7 mg GA/g extract, respectively) than NL extract (11.7 ± 0.7 mg GA/g extract) ( Table 3). HPLC analysis showed higher isoliquiritigenin contents in HL-1 and HL-2 extracts (2.82 ± 0.14 and 3.28 ± 0.18 mg/g extract, respectively) than in NL extract (2.40 ± 0.24) ( Figure 5). This indicates that heat treatment increased the phenolic contents of licorice extracts in proportion to the skinprotective effects of extracts. Therefore, phenolic compounds, especially isoliquiritigenin, likely play a crucial role in the UV photoprotective effects of licorice extracts.

Discussion
This study compared the protective effects of NL and HL extracts on UVB-exposed HDFs. HL-1 and HL-2 extracts showed better antioxidant activity than NL extracts in DPPH and ABTS+ radical scavenging assays ( Table 2). In addition, NL and HL extracts inhibited the expression of intracellular ROS, inflammation-related proteins, and proinflammatory cytokines in UVB-exposed HDFs. UV radiation directly induces photoaging and indirectly increases ROS production, thereby promoting inflammatory factors and causing skin cancer [4]. In addition, ROS promote the expression of p65, a subunit of NF-κB, in the cytoplasm. Phosphorylated p65 moves into the nucleus and acts as a transcription factor for COX-2, iNOS, and inflammatory cytokines and regulates inflammatory responses [37]. Intracellular ROS levels were effectively reduced in UVB-exposed HDFs treated with NL and HL extracts, most significantly by HL-2 extract (Figure 2). The expression of NF-κB p65 activated by ROS was also reduced by NL and HL extracts. HL-2 extract most strongly inhibited the activation of p65. By inhibiting ROS production and activation of NF-κB p65 by UVB, inflammation-related factors such as COX-2, iNOS, IL-6, and IL-1β were reduced. The HL-2 extract had the greatest inhibitory effect on the expression of inflammation-related factors (Figures 3 and 4). We found that the UV photoprotective effects of the NL and HL extracts were due to their antioxidant and anti-inflammatory activities. Phenolics exhibit various antioxidant and anti-inflammatory health effects [38]. During heat treatment, antioxidant capacity is improved through chemical reactions such as the Maillard reaction [39]. Melanoidin, a brown polymer produced by the reaction between sugar and amino acids in the Maillard reaction, has high antioxidant activity due to its high radical-scavenging activity and ability to chelate metals [40]. HL-2 extract had higher TPC than NL and HL-1 extracts (Table 3). Isoliquiritigenin contents were higher in HL extracts than in NL extract, and highest in HL-2 extract. These results indicate that time and temperature play important roles in the changes in phenolic composition during heat treatment [41]. Isoliquiritigenin inhibits ROS production and the NF-κB pathway [5]. Increased isoliquiritigenin in HL extracts may have increased antioxidant activity and inhibited ROS generation and activation of NF-κB p65 in UVB-exposed HDFs [15,42,43], thereby suppressing the expression of COX-2, iNOS, and cytokines. Therefore, heat treatment can improve the UV photoprotective effects of licorice extract on HDFs by increasing the contents of phenolics, such as isoliquiritigenin, in licorice ( Figure 6). HL-2 extract is a natural substance that may help to regulate inflammatory responses and act as a powerful antioxidant in dermal fibroblasts.
κB pathway [5]. Increased isoliquiritigenin in HL extracts may have increased antioxidant activity and inhibited ROS generation and activation of NF-κB p65 in UVB-exposed HDFs [15,42,43], thereby suppressing the expression of COX-2, iNOS, and cytokines. Therefore, heat treatment can improve the UV photoprotective effects of licorice extract on HDFs by increasing the contents of phenolics, such as isoliquiritigenin, in licorice ( Figure 6). HL-2 extract is a natural substance that may help to regulate inflammatory responses and act as a powerful antioxidant in dermal fibroblasts.

Conclusions
This study confirmed the effects of thermal processes on the UV photoprotective activity of licorice in dermal fibroblasts and determined the components related to this activity. The HL extracts had greater UV photoprotective effects, compared with the NL extract. The study showed that HL extracts can protect human dermal fibroblasts against UVB by suppressing intracellular ROS levels and the NF-κB signaling pathway, and heat treatment can be used to improve the UV photoprotective effects of licorice extract on dermal fibroblasts by increasing the contents of bioactive compounds. The HL-2 extract has antioxidant and anti-inflammatory effects that may be useful in cosmetics to protect dermal fibroblasts against UVB.

Conflicts of Interest:
The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; collection, analyses, or interpretation of the data; writing of the manuscript; or the decision to publish the results.