The Antimelanogenic Activity of the Extract of Heukharang Lettuce ( Lactuca sativa L.) Leaf

Heukharang ( Lactuca sativa L.), the artifcially developed lettuce cultivar for enhancement of lactucin content, exhibits anti-oxidant activities and sleep-promoting efects. However, potential of Heukharang as a raw material for the skin-whitening agent has not been investigated yet. Tis study evaluated the efects of Heukharang extract (HHE) on α -melanocyte-stimulating hormone ( α -MSH)-induced melanogenesis in vitro and in vivo models. Our fndings revealed that HHE (25–100 μ g/mL) effectively inhibited α -MSH-induced melanin synthesis in both 2D and 3D cell culture environments without compromising the viability of the B16F10 murine melanoma cell line. Furthermore, HHE suppressed intracellular tyrosinase activity, along with both gene and protein expression of tyrosinase (TYR) and tyrosinase-related protein (TRP)-1 and TRP-2. HHE downregulated the mRNA level of microphthalmia-associated transcription factor ( MITF ) and the phosphorylation of cAMP response element-binding (CREB) protein. Te antimelanogenic activity of HHE (50 and 100 μ g/mL) was also confrmed using a zebrafsh embryo model, highlighting its efcacy in inhibiting α -MSH-induced melanogenesis through the downregulating CREB/MITF/TYR signaling pathways. Moreover, our study demonstrated that a corresponding amount of lactucin (5–20 μ M) to the HHE inhibited and regulated melanogenesis. Tese results collectively suggest that HHE, enriched with lactucin, holds promise as a potential skin-whitening agent for nutricosmetic industries.


Introduction
Te skin, constituting 16% of the total body weight, stands as the largest organ in the human body.Its vitality and aesthetics play a signifcant role in enhancing the overall quality of human life.Uniform skin color is one of the critical factors in determining skin health, but hyperpigmentation which is darkened patches or spots on the skin related to melanin synthesis can cause discomfort; thus, several researchers have tried to fnd out novel substances or agents inhibiting pigmentation of skin cells [1,2].Te color of the skin is determined by the synthesis and distribution of melanin, a pigment produced through the process of melanogenesis.Tis intricate biological mechanism involves epigenetic regulation, where melanin is synthesized within melanocytes and subsequently transported to keratinocytes.Te orchestration of multiple genes and signaling pathways is integral to the melanogenesis process [3].Tus, hyperpigmentation can be alleviated by controlling or regulating the process of melanin synthesis and transport in various ways, and an omnidirectional molecular investigation is required to determine whether specifc materials are efective for skin whitening or not [4,5].
On the other hand, lettuce (Lactuca sativa L.) enjoys widespread cultivation and global popularity, with signifcant consumption in regions such as China, the United States, and Western Europe.Its nutrition composition varied depending on the lettuce type, but all of them contain an abundance of moisture (∼95%) [18].Lettuce exhibits a diverse range of varieties distinguished by colors, sizes, and shapes.Within each type, further subdivisions into subtypes are possible, characterized by shared morphological and genetic similarities.However, the extensive genetic and morphological diversity of lettuce has led to the absence of a standardized classifcation system for this versatile plant.Instead, Mou [19] proposed a classifcation system comprising six primary lettuce types, categorized based on distinct characteristics such as had formation, leaf shape, size, stem type, and texture as follows: butterhead lettuce, crisphead lettuce, Latin lettuce, leaf or cutting lettuce, romaine or cos lettuce, and stem or stalk lettuce.A cultivar is a variety selected for desirable traits for cultivation, and several researchers have tried to select a pure line for their own purposes: higher yield, appearances [20], heat resistance [21], etc.
In Korea, Jang et al. [22] have collected and selected specifc cultivars from native seeds based on their traits and productivity from 2011 to 2015 to develop a new Korean lettuce variety which has excellent functional component contents and good texture.Tey successfully developed a specifc and novel type of lettuce named Heukharang, which was characterized by its more pungent, bitter taste, higher hardness, black-red leaf color, even distribution of anthocyanin color throughout the leaves, and substantially higher level of lactucin (C 15 H 16 O 5 , MW � 276.28 g/mol) exceeding those found in the typical lettuce, red skirt, by more than 100 times.Due to the improvement in lactucin content, several researchers have been interested in the potential use of Heukharang for developing food and/or health functional food products.Consequently, investigations into its physiological and/or biochemical efects have been conducted, encompassing areas such as antioxidant activities [22] and sleep-promoting efects [23].Lactucin, the target compound where the Korean researchers artifcially promoted its content in Heukharang, is a sesquiterpene lactone, and it has attracted attention owing to its multiple biological activities, including antimalarial [24], antihyperalgestic and anti-abiogenic [25], antiandrostenone metabolism [26], and anticancer [27].However, the efect of Heukharang and/or lactucin on skin cells has not been reported.Tis study examined whether Heukharang extract (HHE) and its bioactive compound lactucin would afect melanogenesis and explored the potential use of HHE as a skin-whitening agent in the nutricosmetic industry.Te expression of TYR, TRP-1, TRP-2, and cAMP/PKA/CREB/ MITF cascade was also investigated to elucidate how the HHE treatment on skin cells contributes to regulating melanogenesis, and the antimelanogenic efect of the HHE treatment was confrmed in vivo using a zebrafsh embryo model.

Melanin Content
Assay.B16F10 cells were seeded at a density of 2.4 × 10 5 cells per 60 mm dish) and treated with HHE (25-100 μg/mL) for 60 min prior to α-MSH treatment.Te cells were treated with 100 nM of α-MSH and incubated at 37 °C for 72 h.A three-dimensional melanoma cell culture system was conducted by the forced-foating method [28,29] with slight modifcations.1 × 10 4 cells were cultured in an ultra-low attachment (ULA) 96-well round plate (SPL, Seongnam, Republic of Korea) at 37 °C.Next day, the cells were cotreated by 100 nM α-MSH, HHE (25-100 μg/mL) or lactucin (5-20 μM).A ULA plate was incubated for 3 days at 37 °C.In both 2D and 3D cell cultures, 100 μL of the cultured medium was put on each well, and melanin contents were assayed at 490 nm absorbance.
2.6.Intracellular Tyrosinase Activity Assay.B16F10 cells were seeded at a density of 2.4 × 10 5 cells per 60 mm dish and incubated at 37 °C overnight.After pretreatment with HHE or lactucin for 1 h, the cells were treated with 100 nM α-MSH (37 °C for 72 h).Subsequently, the conditioned medium was discarded, and the cells were washed with PBS twice.Cell lysis was conducted using cell lysis bufer (Cell signaling Technology, Danvers, MA, USA), followed by centrifugation (15,000g, 10 min).Te supernatant was mixed with 10 mM L-DOPA and reacted (37 °C for 2 h).Quantitative protein concentrations were performed using the Pierce BCA protein assay kit (Termo Fisher, Waltham, MA, USA).Te absorbance of dopachrome was measured at 490 nm.

L-DOPA Staining Assay.
Te L-DOPA staining assay was conducted following the procedure outlined in prior studies [30,31], with certain modifcations.Te B16F10 cells were harvested with lysis bufer, and protein extracts were mixed with 10 mM Tris-HCl bufer (pH 7.0) including 1% sodium dodecyl sulfate (SDS), without β-mercaptoethanol or heating.8% SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed.Following electrophoresis, the gel was rinsed twice with 0.1 M sodium phosphate monobasic bufers (pH 6.8) containing 10 mM L-DOPA for 30 min each at room temperature (RT), followed by incubation in the dark at 37 °C for 1 h.TYR activity was confrmed with dark color bands on the gels.

Western Blotting.
Protein extracts obtained from 2.7 were used for western blotting.Protein samples were subjected to 5 min heat treatment (95 °C), followed by centrifugation (15,000g for 2 min at 4 °C), and subsequently separated by 8% SDS-PAGE.Te gels were transferred onto polyvinylidene difuoride membranes, and these membranes were blocked with Blotting-Grade Blocker (Bio-Rad, Hercules, CA, USA) for 1 h at RT. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies (diluted 1 : 1,000) in 1× Tris-Bufered Saline with 0.1% Tween ® 20 detergent (TBST).Following incubation with horseradish peroxidase-conjugated secondary antibodies (diluted 1 : 5,000) for 40 min at RT, the bands were detected using a chemiluminescence reader (LuminoGraph III Lite; ATTO, Tokyo, Japan) at the Biopolymer Research Center for Advanced Material (BRCAM, Seoul, Republic of Korea).Relative band intensity was calculated by the ImageJ software program (National Institutes of Health, Bethesda, MD, USA).
2.10.Maintenance of Zebrafsh.Adult zebrafsh were housed in a 3-liter acrylic tank for a period of 24 h.Te maintenance system was operated on a 14/10 h light/dark cycle, and the tank temperature was maintained at 28.5 °C.Zebrafsh were fed three times a day.Embryos were collected from natural spawning within 30 min by turning on the light, which induced sunrise.HHE (50 and 100 μg/mL) was treated zebrafsh embryos at 8 h after fertilization.Ten, the efects of the HHE treatment on the melanogenesis of zebrafsh were observed under the stereomicroscope.

Measurement of Melanin Contents in Zebrafsh Embryos.
Protein from zebrafsh embryos was extracted using zebrafsh embryos using PRO-PREP ™ protein extraction solution (Intron, Sungnam, Republic of Korea), followed by centrifugation for collecting the pellet.After exposing to 500 μL of 1 N NaOH (60 °C for 30 min), the melanin contents in the suspension were quantifed at 490 nm using a microplate reader (BioTeK, Santa Clara, CA, USA).

High-Performance Liquid Chromatography (HPLC)
Analysis.Contents of lactuin in HHE were quantifed using the HPLC system (Agilent Technologies, Santa Clara, CA, USA).Sample separation was used by Agilent Eclipse XDB-C18 (4.6 mm ID × 250 mm, 5 μm) at 0.8 mL/min of the fow rate.Te mobile phase was applied by solvent A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile), and the gradient program is summarized in Table S1.

HPLC-High-Resolution-MS/MS Analysis.
To identify lactucin in the HHE, LC-MS/MS analysis was performed by a method described in a previous study with some modifcations [32].For the HPLC analysis, an ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 50 mm) (Waters, Milford, MA, US) was used at 45 °C, and the column was equilibrated for 1 min.Te mobile phase was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) with 0.3 mL/min of the fow rate.Sample volume was used 10 μL to an injector, and the detection wavelength was 210 nm.HHE (200 mg/mL) and lactucin (1 μg/mL) were used for this experiment.An elution gradient program was as follows: 0-1 min, 2% B; 1-15 min, 20% B; 15-15.5 min, 90% B; 15.5-17.5 min, 90% B; and 17.5-18 min, 2% B. Mass spectrometry analysis was conducted by using an Orbitrap Exploris 120 mass spectrometer (Termo Fisher Scientifc, Hemel Hempstead, UK) with a heated electrospray ionization (H-ESI) interface (Termo Fisher Scientifc).Te mass spectrometric conditions were performed as follows: spray voltage, 2500 V in negative ion mode; collision gas (nitrogen); sheath gas, 50 arbitrary unit; auxiliary gas, 10 arbitrary unit; sweep gas, 1 arbitrary unit; ion transfer tube temperature, 325 °C; and vaporizer temperature, 350 °C.Te full-MS scan mode was detected at 120,000 resolution at m/z 100-1500 and RF Lens at 70%.Precursor ions were fragmented by higher energy collisional dissociation with a normalized collision energy of 15, 30, and 60% detected at 15,000 resolution.All data were analyzed using Freestyle software (Termo Fisher Scientifc).

Statistical Analysis.
All experimental data were presented as the mean ± standard deviation.Statistical analysis was conducted using Student's t-test with Microsoft Excel 2019 (Redmond, WA, USA).p < 0.05 was accepted to show a signifcant diference.

HHE Shows No Cytotoxicity and Suppresses
Melanogenesis.Before testing the efect of HHE on melanogenesis, a cell viability assay was conducted to assess the potential efect of HHE on B16F10 cells.HHE demonstrated no signifcant cytotoxicity up to a concentration of 400 μg/mL (Figure 1(a)), compared to that of 400 μg/mL of arbutin (positive control) that showed signifcant cytotoxicity (Figure S1(d)).When we measured extracellular melanin contents of the HHE (25-100 μg/mL)-treated cells, induction of melanogenesis by α-MSH (100 nM) was signifcantly decreased in both 2D (Figure 1(b)) and 3D melanoma cell culture systems (Figure 1(c)).In a specifc range of concentration (25-100 μg/mL), a dose dependency of the efect of HHE was clearly observed in a 2D culture (p < 0.05), and all HHE treatment groups reduced melanin contents to the similar level of the control group in a 3D culture.Te plateau of the efect was also observed in a higher concentration of HHE-treated groups (100-400 μg/mL) in a 2D culture (Figure S1(e)).

HHE Reduces Intracellular TYR Activity.
We applied L-DOPA, a substrate for the TYR reaction, to test the efect of HHE treatment on the intracellular TYR activity.Te pretreatment of HHE signifcantly suppressed intracellular TYR activity (Figure 2(a)).Te HHE pretreatment induced a decreased level of TYR activity, and intracellular melanogenesis induced by α-MSH was also signifcantly reduced as TYR activity decreases (Figure 2(b)).A dose dependency of the HHE treatment was clearly observed in both results.

HHE Inhibits and Regulates the Expression of Melanogenic
Enzymes.To validate the downregulatory efect of the HHE treatment on melanogenic enzymes, protein expressions of TYR, TRP-1, and TRP-2 in the HHE-treated B16F10 cells were compared by western blotting analysis (Figure 3(a)).Figure 3(b) shows that the HHE treatment signifcantly decreased gene expression of the key enzymes associated with melanogenesis (TYR, TRP-1, and TRP-2).To check the efect of the HHE treatment on the upstream regulatory action of the cAMP/PKA/CREB/ MITF cascade, changes in mRNA expression of MITF were confrmed (Figure 3(c)).We found that the HHE downregulated MITF expression.In addition, the 4 Journal of Food Biochemistry  phosphorylation level of CREB was confrmed (Figure 3(d)).Expression of phosphorylated CREB was decreased by HHE treatment, dose-dependently.

HHE Inhibits Melanogenesis in Zebrafsh. Figure 4(a)
shows that the HHE treatment inhibited the body pigmentation of zebrafsh with more than 90% of the survival and hatching rate (Figures S1(b) and S1(c), respectively).In addition, HHE treatment signifcantly reduced melanin contents in an embryo of zebrafsh in a dose-dependent manner (Figure 4(b)).

Lactucin Is an Active Constituent of the HHE on
Melanogenesis.HHE used in this study contained 0.23 mg of lactucin/g measured by HPLC analysis (Figures S2(a

Discussion
Heukharang which is well known for a sleep-promoting efect reported higher antioxidant activity compared with red skirt lettuce [23,33].Plant extracts with antioxidant activity could have potential of inhibitory efects on melanogenesis [34].Heukharang extracts used in this study showed antioxidant activities including 8.31 ± 0.61 mg gallic acid equivalent/g of phenolic contents, 18.81 ± 1.67 mg quercetin equivalent/g of favonoid contents, and increasing DPPH radical scavenging activity, dose-dependently (Figure S1(a)).Tis study represents the frst elucidation of the potential use of HHE as an antimelanogenic agent in the nutricosmetic industry.Te in situ test was performed by exposing the in vitro cell culture model to the HHE treatment and then α-MSH, inducer of melanogenesis [12,13] and/or substrate (L-DOPA) of key melanogenic enzyme action [35].Te skin-whitening efect of the HHE at an in vivo level was also confrmed using one of the animal alternative test methods, the zebrafsh model.
Indeed, pretreatment of the HHE only for 1 h greatly inhibited α-MSH-induced melanogenesis without afecting cell viability of the B16F10 cells, more than 90% of the cells remained viable even after 72 h of exposure to HHE at concentrations up to 400 μg/mL.Tis study employed the melanocyte aggregates as an alternative to the artifcial skin model, bridging the experimental gap between 2D cell culture systems and animal models [29].Notably, pretreatment with HHE (25-100 μg/mL) efectively inhibited α-MSH (100 nM)-induced melanin synthesis in both the 2D and 3D culture systems, as illustrated in Figures 1(b) and 1(c).In comparison of the experimental results reported in the previous study [36], which treated 50 μg/mL of arbutin, a well-known melanogenesis inhibitor in cosmetics and medicines, on 100 nM of α-MSH-stimulated B16F10 cells, the HHE applied in this study indicated a remarkable reduction efect on melanin synthesis (Figure S1(e)).
When intracellular responses of the cells to the HHE pretreatment were observed, increased conversion L-DOPA to L-DOPA chrome induced by α-MSH in eumelanogenesis was successfully suppressed.Te HHE decreased TYR  ) and also signifcantly reduced the production of dopachrome from L-DOPA, also called tyrosinase zymography.Protein expression of TYR is also signifcantly suppressed by the HHE treatment (Figure 3).TYR activity is crucial to melanogenesis [13]; thus, inhibition of enzymatic activity and/or expression of TYR by the HHE treatment exert a signifcant infuence on the overall pathway of melanogenesis.In addition, the pretreatment of HHE exhibited a dose-dependent impact on the relative expressions of TRP-1 and TRP-2.Notably, our result showed that changes in TRP-1 expression were more noticeable than in TRP-2 (Figure 3(a)).In an eumelanogenesis pathway, TRP-1 oxidizes 5,6-dihydroxyindole-2carboxylic acid (DHICA) to a carboxylated indolequinone, a precursor that is ultimately transformed into eumelanin.On the other hand, TRP-2 catalyzes the rearrangement of dopachrome to DHICA [37].It is assumed that pretreatment of HHE inhibits melanin synthesis by suppressing TYR itself and preventing reorganization L-DOPA chrome and oxidizing DHICA to an eumelanin precursor.Te process of melanin synthesis is very complex and involves a number of molecular biological factors [13,14].Tis study considered expressions of p-CREB, CREB-1, and MITF to examine the efect of the HHE on the B16F10 cells in a cAMP/PKA/CREB/MITF cascade [14].Te results of this study show that the HHE treatment downregulates p-CREB and mRNA of MITF.Te role of MITF as a master regulator in melanocyte development been extensively studied and established [8,38].α-MSH induces the production of cAMP, and the increase in cAMP levels triggers the phosphorylation of the CREB transcription factor, subsequently facilitating MITF activation.Tis cascade of events positively regulates the transcription TYR, TRP-1, and TRP-2 [39][40][41][42].Tus, this study shows that treatment of the HHE negatively regulates melanogenesis by suppressing p-CREB and MITF, which are transcription factors of key melanogenic enzymes TYR, TRP-1, and TRP-2.
Zebrafsh is a valuable model system to evaluate antimelanogenesis activity because it has similar genetic structures and organs to human beings [43].To confrm the HHE treatment successfully control melanin synthesis without safety issue at an in vivo level, this study treated zebrafsh with HHE (50 and 100 μg/mL).Melanogenesis in zebrafsh is successfully controlled, and more than 90% of zebrafsh survived and hatched.Tus, reviewing HHE as a skin-whitening agent in the nutricosmetic industry is reasonable.

Conclusions
Tis is the frst report elucidating antimelanogenic activity of Heukharang and/or lactucin in vitro and in vivo.We suggest the mode of action of the HHE treatment on eumelanogenesis (Figure 6) based on scientifc data demonstrating that pretreatment HHE on the skin inhibits melanin synthesis by regulating the CREB/MITF signaling pathway.In addition, lactucin, a well-known bioactive constituent in Heukharang lettuce, is one of the major active compounds in the HHE on antimelanogenesis.Terefore, HHE could be quite useful as a skin-whitening agent, preventing the abnormal accumulation of melanin in the cosmetic industry's melanogenesis process.

Data Availability
Te string data that support the fndings of this study are included within the article and the supplementary information.Also, the integer data used to support the fndings of this study are available from the corresponding author upon request.

Figure 1 :
Figure 1: Efects of Heukharang (HHE) on cytotoxicity and melanin synthesis in B16F10 cells.(a) Cell viability of the HHE-treated B16F10 cells ( * p < 0.05 in comparison with the untreated group).(b) Extracellular melanin contents of the 2D cell culture system.(c) Extracellular melanin contents of the 3D cell culture system ( ### p < 0.001 versus the untreated group; * * * p < 0.001 in comparison with the α-MSH-treated group).