Wound Healing-Promoting and Melanogenesis-Inhibiting Activities of Angelica polymorpha Maxim. Flower Absolute In Vitro and Its Chemical Composition

Angelica polymorpha Maxim. (APM) is used in traditional medicine to treat chronic gastritis, rheumatic pain, and duodenal bulbar ulcers. However, it is not known whether APM has epidermis-associated biological activities. Here, we investigated the effects of APM flower absolute (APMFAb) on responses associated with skin wound healing and whitening using epidermal cells. APMFAb was obtained by solvent extraction and its composition was analyzed by GC/MS. Water-soluble tetrazolium salt, 5-bromo-2′-deoxyuridine incorporation, Boyden chamber, sprouting, and enzyme-linked immunosorbent assays and immunoblotting were used to examine the effects of APMFAb on HaCaT keratinocytes and B16BL6 melanoma cells. APMFAb contained five compounds and induced keratinocyte migration, proliferation, and type IV collagen synthesis. APMFAb also induced the phosphorylations of ERK1/2, JNK, p38 mitogen-activated protein kinase, and AKT in keratinocytes. In addition, APMFAb decreased serum-induced B16BL6 cell proliferation and inhibited tyrosinase expression, melanin contents, and microphthalmia-associated transcription factor expression in α-melanocyte-stimulating hormone-stimulated B16BL6 cells. These findings demonstrate that APMFAb has beneficial effects on skin wound healing by promoting the proliferation, migration, and collagen synthesis of keratinocytes and on skin whitening by inhibiting melanin synthesis in melanoma cells. Therefore, we suggest that APMFAb has potential use as a wound healing and skin whitening agent.


Introduction
Skin wound healing is a complex process and comprised of inflammation, proliferation, and remodeling phases [1]. The skin healing process involves interactions between multiple factors such as different cell types in skin, growth factors, cytokines, and extracellular matrix (ECM) [1,2]. The occurrence of a skin wound causes keratinocytes, which make up most of the epidermis, to proliferate and migrate to the damaged area to facilitate re-epithelialization [1]. The migratory and proliferative activities of keratinocytes are activated by epidermal growth factor (EGF) and other growth factors. Collagen, a key ECM protein, can be synthesized by keratinocytes or fibroblasts and is a component of skin basement membrane [3]. Collagen is involved in all phases of wound healing and plays important roles during the proliferative and remodeling phases of wound healing [1]. The cleavage of collagen is closely associated with the activation of keratinocyte migration

Effects of APMFAb on the Migratory and Proliferative Activities of HaCaT Cells
To determine whether APMFAb affects skin wound healing, we investigated its effects on keratinocyte migration and proliferation, which are known to play important roles in the healing process [12]. Initially, we evaluated the effect of APMFAb (0.1-250 µg/mL) on HaCaT cell viability using a water-soluble tetrazolium salt (WST) assay. Treatment with APMFAb (0.1-150 µg/mL) had no cytotoxic effect on HaCaT cells, but rather accelerated cell viability significantly from 1 µg/mL to 100 µg/mL as compared with untreated controls. However, at 250 µg/mL APMFAb reduced HaCaT cell viabilty (Figure 1a). Cell migration was assessed using a Boyden chamber assay on HaCaT cells exposed to APMFAb (0.1-150 µg/mL), and APMFAb was found to increase migration significantly at 50 µg/mL (122.33 ± 6.33% of untreated control) (Figure 1b), indicating that APMFAb can stimulate the migration in HaCaT cells. We also investigated the effect of APMFAb (0.1-150 µg/mL) on the proliferation of HaCaT cells using a BrdU incorporation assay. The results obtained showed APMFAb at 1-50 µg/mL significantly increased HaCaT cell proliferation and increased it maximally at 10 µg/mL (159.62 ± 7.75% of untreated control) (Figure 1c), which showed that APMFAb can promote HaCaT cell proliferation.
Molecules 2021, 26, x FOR PEER REVIEW 3 of 12 (0.1-150 μg/mL) on the proliferation of HaCaT cells using a BrdU incorporation assay. The results obtained showed APMFAb at 1-50 μg/mL significantly increased HaCaT cell proliferation and increased it maximally at 10 μg/mL (159.62 ± 7.75% of untreated control) (Figure 1c), which showed that APMFAb can promote HaCaT cell proliferation. Cell migration. HaCaT cells were exposed APMFAb (0.1-150 μg/mL) for 210 min. Migration levels were determined using a Boyden chamber assay as described in Materials and Methods (upper panels). Representative images of the results. Black spots indicate migrated cells. Scale bar = 100 μm.
(lower panels) Graphical representation of the results. Recombinant human epidermal growth factor (EGF: 1 ng/mL) was used as the positive control. Results are presented as mean percentages ± SEMs of non-treated controls (Con) (n = 4). * p < 0.05 vs. non-treated cells. (c) Cell proliferation.
HaCaT cells were incubated with APMFAb (0.1-150 μg/mL) for 48 h, and proliferations were determined using a BrdU incorporation assay as described in Materials and Methods. Recombinant human epidermal growth factor (EGF: 50 ng/mL) was used as the positive control. Results are presented as mean percentages ± SEMs of non-treated controls (Con) (n = 5). * p < 0.05 vs. non-treated cells.

Effect of APMFAb on Keratinocyte Sprout Outgrowth
To simultaneously evaluate the proliferative and migratory effects of APMFAb on keratinocytes, we used a collagen sprout assay. APMFAb (1-100 μg/mL) significantly elevated HaCaT cell sprouting at 10 and 50 μg/mL, peaking at 50 μg/mL (326.24 ± 14.99% versus untreated control) ( Figure 2). This assay is commonly used to confirm keratinocyte proliferation and migration results in vitro [12]. (b) Cell migration. HaCaT cells were exposed APMFAb (0.1-150 µg/mL) for 210 min. Migration levels were determined using a Boyden chamber assay as described in Materials and Methods (upper panels). Representative images of the results. Black spots indicate migrated cells. Scale bar = 100 µm. (lower panels) Graphical representation of the results. Recombinant human epidermal growth factor (EGF: 1 ng/mL) was used as the positive control. Results are presented as mean percentages ± SEMs of non-treated controls (Con) (n = 4). * p < 0.05 vs. non-treated cells. (c) Cell proliferation. HaCaT cells were incubated with APMFAb (0.1-150 µg/mL) for 48 h, and proliferations were determined using a BrdU incorporation assay as described in Materials and Methods. Recombinant human epidermal growth factor (EGF: 50 ng/mL) was used as the positive control. Results are presented as mean percentages ± SEMs of non-treated controls (Con) (n = 5). * p < 0.05 vs. non-treated cells.

Effect of APMFAb on Keratinocyte Sprout Outgrowth
To simultaneously evaluate the proliferative and migratory effects of APMFAb on keratinocytes, we used a collagen sprout assay. APMFAb (1-100 µg/mL) significantly elevated HaCaT cell sprouting at 10 and 50 µg/mL, peaking at 50 µg/mL (326.24 ± 14.99% versus untreated control) ( Figure 2). This assay is commonly used to confirm keratinocyte proliferation and migration results in vitro [12].

Effect of APMFAb on the Activations of Kinases in HaCaT Cells
To investigate the association between signaling molecules and the migration and proliferation of HaCaT cells exposed to APMFAb (0.1-150 µg/mL), we performed western blotting. APMFAb significantly elevated the activations of extracellular signal-regulated kinase1/2 (ERK1/2) from 10 µg/mL to 100 µg/mL (Figure 3a

Effect of APMFAb on the Activations of Kinases in HaCaT Cells
To investigate the association between signaling molecules and the migration and proliferation of HaCaT cells exposed to APMFAb (0.1-150 μg/mL), we performed western blotting. APMFAb significantly elevated the activations of extracellular signal-regulated kinase1/2 (ERK1/2) from 10 μg/mL to 100 μg/mL (Figure 3a

Effect of APMFAb on the Activations of Kinases in HaCaT Cells
To investigate the association between signaling molecules and the migration and proliferation of HaCaT cells exposed to APMFAb (0.1-150 μg/mL), we performed western blotting. APMFAb significantly elevated the activations of extracellular signal-regulated kinase1/2 (ERK1/2) from 10 μg/mL to 100 μg/mL (Figure 3a   MAPKs regulate many different cellular responses besides proliferation and migration, and ERK1/2, JNK, and p38 MAPK are major members of the MAPK family [13,14]. It has been reported that MAPKs and AKT importantly signal the migration and proliferation of keratinocytes [14]. Increased activation of ERK1/2 is known to promote keratinocyte migration and proliferation [14], and reductions in ERK1/2 activation have the opposite effect [15]. Increased p38 MAPK activation also promotes keratinocyte proliferation and migration [16], whereas though JNK participates in keratinocyte migration and proliferation, its effects are consistent. For example, JNK activation has been reported to promote, and on the other hand, not to be involved in keratinocyte migration and proliferation [14,17], which implies that JNK signaling does not play a critical role in migratory and proliferative activities in keratinocytes. As mentioned above, we found that APMFAb increased the phosphorylation of ERK1/2, p38 MAPK, and JNK in HaCaT cells, which suggests activations of these MAPKs are associated with APMFAb-induced migration and proliferation in HaCaT cells. We also showed that APMFAb increased AKT activation in HaCaT cells, and AKT activation has been reported to promote HaCaT cell migration and proliferation [14]. These observations indicate APMFAb might induce keratinocyte migratory and/or proliferative activities by activating the AKT and/or MAPK signaling pathways.

Effect of APMFAb on Collagen Synthesis by HaCaT Cells
To examine the effect of APMFAb on collagen synthesis by keratinocytes, we performed sandwich enzyme-linked immunosorbent assay (ELISA) using conditioned medium, which was obtained by culturing HaCaT cells in the presence of APMFAb. Treatment of HaCaT cells with APMFAb from 1 µg/mL to 100 µg/mL did not affect type I collagen synthesis (Figure 4a) but significantly elevated type IV collagen synthesis at 100 µg/mL (202.87 ± 14.79% versus untreated control, Figure 4b).
MAPKs regulate many different cellular responses besides proliferation and migration, and ERK1/2, JNK, and p38 MAPK are major members of the MAPK family [13,14]. It has been reported that MAPKs and AKT importantly signal the migration and proliferation of keratinocytes [14]. Increased activation of ERK1/2 is known to promote keratinocyte migration and proliferation [14], and reductions in ERK1/2 activation have the opposite effect [15]. Increased p38 MAPK activation also promotes keratinocyte proliferation and migration [16], whereas though JNK participates in keratinocyte migration and proliferation, its effects are consistent. For example, JNK activation has been reported to promote, and on the other hand, not to be involved in keratinocyte migration and proliferation [14,17], which implies that JNK signaling does not play a critical role in migratory and proliferative activities in keratinocytes. As mentioned above, we found that APMFAb increased the phosphorylation of ERK1/2, p38 MAPK, and JNK in HaCaT cells, which suggests activations of these MAPKs are associated with APMFAb-induced migration and proliferation in HaCaT cells. We also showed that APMFAb increased AKT activation in HaCaT cells, and AKT activation has been reported to promote HaCaT cell migration and proliferation [14]. These observations indicate APMFAb might induce keratinocyte migratory and/or proliferative activities by activating the AKT and/or MAPK signaling pathways.

Effect of APMFAb on Collagen Synthesis by HaCaT Cells
To examine the effect of APMFAb on collagen synthesis by keratinocytes, we performed sandwich enzyme-linked immunosorbent assay (ELISA) using conditioned medium, which was obtained by culturing HaCaT cells in the presence of APMFAb. Treatment of HaCaT cells with APMFAb from 1 μg/mL to 100 μg/mL did not affect type I collagen synthesis (Figure 4a) but significantly elevated type IV collagen synthesis at 100 μg/mL (202.87 ± 14.79% versus untreated control, Figure 4b).
(a) (b) Figure 4. Effects of Angelica polymorpha Maxim. flower absolute on the syntheses of type I and IV collagens. HaCaT cells were incubated in the absence or presence of APMFAb (1-100 μg/mL) for 48 h. Conditioned media were subjected to sandwich ELISA using anti-type I (n = 3; a) or anti-type IV collagen antibody (n = 3; b). Collagen levels in conditioned media are expressed as percentages of those in nontreated controls (Con). Results are presented as means ± SEMs. * p < 0.05 vs. non-treated cells.
Collagen synthesis is required for all skin healing processes but is especially important during the proliferation and remodeling phases [1,18]. Types I and IV collagen were found to increase keratinocyte migratory activity in vitro [18], and these two collagens are associated with skin recovery [19] and skin membrane formation [18,20], respectively. Furthermore, both collagens are produced and secreted by keratinocytes or fibro- Collagen synthesis is required for all skin healing processes but is especially important during the proliferation and remodeling phases [1,18]. Types I and IV collagen were found to increase keratinocyte migratory activity in vitro [18], and these two collagens are associated with skin recovery [19] and skin membrane formation [18,20], respectively. Furthermore, both collagens are produced and secreted by keratinocytes or fibroblasts exposed to various plant extracts [12]. Therefore, our findings indicate that APMFAb facilitates skin wound healing by enhancing type IV collagen production in keratinocytes.
MFAb facilitates skin wound healing by enhancing type IV collagen production in keratinocytes.

Chemical Composition of APMFAb
Gas chromatography/mass spectrometry (GC/MS) analysis showed that APMFAb contained 5 compounds ( Figure 6 and Table 1), viz., isopimpinellin (34.95%), bergapten (30.74%), nonadencane (24.50%), aromadendrene (8.70%), and methoxsalen (1.12%) ( Table 1). It has been reported that methoxsalen, in combination with long wavelength UVA inhibits HaCaT cell migration [24]. On the other hand, isopimpinellin or bergapten-stimulated melanogenesis in melanoma cells [25,26]. Accordingly, it might be expected that APMFAb would have a negative effect on skin wound healing or whitening response. However, other compounds are not reported about their skin wound healing-or melanogenesislinked bioactivities on keratinocytes and melanocytes. Angelica dahurica extract increased human keratinocyte proliferation [10] and Angelica tenuissima root extract inhibited melanin production in melanoma cells [11]. In the present study, we found that APMFAb promoted skin wound healing and whitening response. These observations indicate that APMFAb may contain components that promoted these responses. Further studies are required to identify the main bioactive components in APMFAb responsible for its wound healing and/or whitening-related responses.
Melanin biosynthesis is influenced by the survival, proliferation, and differentiation of melanocytes [21] and is regulated by tyrosinase, TRP-1, and TRP-2 [22], the expressions of which were reported to be reduced by inhibiting MITF [23], implying that MITF can regulate tyrosinase, TRP-1, and TRP-2-induced effects. Furthermore, it has been shown that inhibitions of the expression of these proteins reduced melanin production in melanocytes [22], and in another study, melanin biosynthesis was attenuated by inhibiting melanocyte proliferation [23]. Similarly, we observed APMFAb inhibited the expressions of these proteins and proliferation and melanin production in B16BL6 melanoma cells. Therefore, our findings indicate APMFAb inhibits melanin biosynthesis in melanocytes.

Chemical Composition of APMFAb
Gas chromatography/mass spectrometry (GC/MS) analysis showed that APMFAb contained 5 compounds ( Figure 6 and Table 1), viz., isopimpinellin (34.95%), bergapten (30.74%), nonadencane (24.50%), aromadendrene (8.70%), and methoxsalen (1.12%) ( Table  1). It has been reported that methoxsalen, in combination with long wavelength UVA inhibits HaCaT cell migration [24]. On the other hand, isopimpinellin or bergapten-stimulated melanogenesis in melanoma cells [25,26]. Accordingly, it might be expected that AP-MFAb would have a negative effect on skin wound healing or whitening response. However, other compounds are not reported about their skin wound healing-or melanogenesis-linked bioactivities on keratinocytes and melanocytes. Angelica dahurica extract increased human keratinocyte proliferation [10] and Angelica tenuissima root extract inhibited melanin production in melanoma cells [11]. In the present study, we found that AP-MFAb promoted skin wound healing and whitening response. These observations indicate that APMFAb may contain components that promoted these responses. Further studies are required to identify the main bioactive components in APMFAb responsible for its wound healing and/or whitening-related responses.

Materials
Trypsin-ethylenediamine tetra-acetic acid (EDTA), FBS, and penicillin/streptomycin (P/S) were purchased from Gibco BRL (Gaithersburg, MD,  Absolute was obtained by solvent extraction, as previously described [12]. In brief, APM flowers (5.521 kg) were completely immersed in hexane at room temperature (RT) for 1 h. Extracts were collected, and the hexane was removed by rotary evaporation at 25 • C under vacuum to give a dark yellow waxy residue (concrete). This residue was then mixed with ethanol (99.5%), left at −20 • C for 12 h, filtered through a sintered funnel, and then the ethanol was removed by evaporation at 35 • C to leave a light-yellow anhydrous wax (APMFAb; 1.43 g, yield 0.026%, w/w). APMFAb was stored at −80 • C until required.

Identification of Compounds in APMFAb
Components in APMFAb were identified by GC/MS at the Korean Basic Science Institute (KBSI, Seoul, Korea). GC/MS analysis was executed using an Agilent 7890BGC/7010QQQ MS instrument (Palo Alto, CA, USA) and a DB5-MS capillary column (30 m × 0.25 mm, film thickness 0.25 µm) [12]. Helium was used as the carrier gas and its flow rate was 1 mL/min. Injector port, ion source, and interface temperatures were 290, 230, and 290 • C, respectively. The GC oven was programmed as follows; 40 • C for 3 min, 40 to 230 • C at 2 • C/min, 230 to 300 • C at 5 • C/min, and maintained at 300 • C for 15 min. The split ratio was 1:10. Masses were scanned from m/z 50 to 800. Retention indices (RIs) were determined using Kovats method using C 7 -C 40 n-alkanes as standards. Compounds were identified by comparing their RI values with Kovats indices [27] and matching their MS fragmentation patterns with those in the Wiley7NIST0.5L Mass Spectral library and mass spectrum catalogs. GC/MS data were reanalyzed as described by Adams [28].

Cell Culture
HaCaT cells (a human immortalized keratinocyte cell line) were obtained from the National Institute of Korean Medicine Development (NIKOM, Gyeongsan, South Korea) and B16BL6 cells (a murine melanoma cell line) were from the Korean Cell Line Bank (KCLB, Seoul, South Korea). Cells were cultured in DMEM with 1% (v/v) P/S and 10% (v/v) FBS in a humidified 5% CO 2 atmosphere at 37 • C. For experiments, cells were cultured until 70-80% confluent.

Western Blotting
Cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer; Cell Signaling), and lysates were centrifuged at 17,000× g for 15 min at 4 • C. Protein concentrations in supernatants were determined using DC protein assay reagents (Bio-Rad Laboratories, Hercules, CA, USA). Proteins (80-120 µg/lane) were subjected to 10% (w/v) SDS-PAGE and transferred to polyvinylidene fluoride membranes at 4 • C. Membranes were blocked in 3% (w/v) skim milk at RT for 2 h, washed with PBS containing 0.05% (v/v) Tween-20, incubated with primary antibodies (1: 1000-10,000 dilution), and then with secondary antibody (conjugated horseradish peroxidase) at RT for 1 h. Bands were visualized using a chemiluminescence substrate and detected using a chemiluminescence imaging system (LuminoGraph, ATTO, Tokyo, Japan).

Melanin Content Assay
Melanin contents were analyzed as described previously [29]. Briefly, B16BL6 melanoma cells (5 × 10 5 cells per well) were incubated in DMEM containing different concentrations of APMFAb with or without 200 nM α-MSH for 48 h at 37 • C in 60-mm dishes. After washing with PBS, cells were lysed using lysis buffer (0.1 M sodium phosphate buffer [pH 6.8] containing 0.2 mM phenylmethylsulfonyl fluoride and 1% [v/v] Triton X-100 and then centrifuged at 10,000× g for 15 min. Cell pellets were dissolved in 150 µL of 1 N NaOH containing 10% (v/v) DMSO, incubated at 80 • C for 1 h, and pipetted to solubilize the melanin. Absorbances were measured at 405 nm using an ELISA reader (Synergy 2, Bio-Tek Instruments, Winooski, VT, USA).

Statistical Analysis
Data values are presented as means ± standard errors of means (SEMs). The Student's t-test is used to analyze the significances of differences between pairs of groups. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test was conducted for multiple comparisons. The analysis was conducted using GraphPad Prism (version 5.0; GraphPad Software, Inc., San Diego, CA, USA), and p values < 0.05 were considered significant.

Conclusions
In the present study, we found APMFAb contained five compounds and that it stimulated HaCaT cell proliferation, migration, and sprout outgrowth, and increased the activations of AKT, ERK1/2, p38 MAPK, and JNK, and increased type IV collagen synthesis in HaCaT cells. In addition, in B16BL6 cells exposed to α-MSH, APMFAb reduced serum-induced proliferation and attenuated MITF expression, tyrosinase expression, and melanin production. These observations suggest APMFAb induces wound healing-linked migration, proliferation, and collagen synthesis in keratinocytes and promotes whiteningassociated responses in melanocytes. Our findings suggest that APMFAb provides a novel basis for the development of natural skin wound healing and whitening agents.