Anti-Oxidative and Anti-Aging Effects of Ethanol Extract of the Officinal Breynia (Breynia vitis-idaea) In Vitro

The skin is the largest organ of the human body, and it is also the one most exposed to external environmental contaminants. The skin is the body’s first defense against harmful environmental stimuli, including ultraviolet B (UVB) rays and hazardous chemicals. Therefore, proper care of the skin is required to prevent skin-related diseases and age-related symptoms. In this study, we analyzed anti-aging and anti-oxidative effects of Breynia vitis-idaea ethanol extract (Bv-EE) in human keratinocytes and dermal fibroblasts. The Bv-EE had free radical scavenging activity and decreased the mRNA expression of MMPs and COX-2 in H2O2- or UVB-treated HaCaT cells. The Bv-EE also inhibited AP-1 transcriptional activity and phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and mitogen-activated protein kinase 14 (p38), which are major AP-1 activators upon H2O2 or UVB exposure. Furthermore, the promoter activity and mRNA expression of collagen type I (Col1A1) increased in HDF cells treated with Bv-EE, and Bv-EE recovered the collagen mRNA expression decreased by H2O2 or UVB exposure. These results suggest that Bv-EE has anti-oxidative effects by inhibiting the AP-1 signaling pathway, and shows anti-aging effects by upregulating collagen synthesis.


Introduction
The genus Breynia belongs to the family Phyllanthaceae, which is composed of 35 plant species distributed in tropical regions of the Pacific Islands, Australia, and Asia [1]. Many reports about the medicinal benefits of Breynia species have been published. Breynia nivosa possesses analgesic, anti-inflammatory, and antimicrobial properties [2]. Breynia retusa shows antioxidant and anti-diabetic activities, and Breynia distachia has shown hypoglycemic and anti-Alzheimer's activities [3,4]. Breynia vitis-idaea, commonly called Indian snowberry, is a large, evergreen shrub or treelet that grows up to 5 m in height [5]. These plants have ovate or elliptic leaves that are 1-3 cm long. The bark is yellowish grey, the flowers are small and greenish yellow or pink, and the fruits are dull red, purple, or white berries [6]. These plants are found in countries including Bangladesh, Cambodia, India, Malaya, Myanmar, Pakistan, Philippines, Sri Lanka, Thailand, and Vietnam, and have been used as a traditional herbal medicine for the treatment of wounds and chronic bronchitis, especially in China [7]. Recent studies have revealed that Breynia vitis-idaea has anti-hypoglycemic, anti-hypolipidemic, and anti-cancer activity [8,9]. Interestingly, Breynia vitis-idaea is known to contain 6-O-benzoyl arbutin, breynioside B, and 6-O-benzoyl-α-D-

Phytochemical Components of Bv-EE
The phytochemical composition of Bv-EE was analyzed by gas chromatography-mass spectrometry (GC-MS) ( Figure 1). The corrected percentage peak area was divided by the sum of the corrected area to obtain the total content of each compound in Bv-EE. The major compound was identified as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, also known as DDMP. This compound is produced in the intermediate stage of the Maillard reaction [25]. The contribution of DDMP to antioxidant activity has been well studied, and DDMP is known to exist in natural extracts and foods [26,27]. In particular, DDMP is known for its antioxidant effect and its ability to prevent and treat obesity or lipid-related metabolic diseases [28]. Other active components of Bv-EE are listed in Table 1. Nonetheless, confirming the identification of these components from Bv-EE will be continued with other extracts prepared with the same plant materials harvested according to different seasons, regions, and years to complete standardization of this extract for industrial application.

Bv-EE Exerts Reactive Oxygen Species Scavenging Activity
To determine whether Bv-EE has radical scavenging activity, 1-diphenyl-2-picryldydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assays were performed [29]. The Bv-EE scavenged DPPH radicals in a dose-dependent manner and showed significant scavenging activity at a concentration of 6.25 µg/mL (Figure 2a). In the ABTS assay (Figure 2b), Bv-EE exhibited ABTS radical scavenging activity, with IC 50 values of 10.8424 µg/mL and 5.36 µg/mL (Figure 2c,d). Ascorbic acid (300 µM) was used as a positive control (Figure 2a,b). The ferric reducing antioxidant power (FRAP) assay showed that Bv-EE can also reduce ferric acid dose-dependently ( Figure 2e). In addition, the cupric ion-reducing antioxidant capacity (CUPRAC) assay showed that Bv-EE reduced Cu ions in a dose-dependent manner. Trolox (3 mg/mL) was used as the positive control. Moreover, we determined the total phenolic content and total flavonoid content of Bv-EE to be 89.33 µg/mg and 73 µg/mg, respectively (Figure 2g,h). These results together show that Bv-EE is a potential antioxidant. showed that Bv-EE reduced Cu ions in a dose-dependent manner. Trolox (3 mg/mL) was used as the positive control. Moreover, we determined the total phenolic content and total flavonoid content of Bv-EE to be 89.33 μg/mg and 73 μg/mg, respectively (Figure 2g,h).
These results together show that Bv-EE is a potential antioxidant.

Bv-EE Prevents Cell Death Caused by UVB and H2O2 in HaCaT cells
Because Bv-EE showed ROS scavenging activity, we tested whether it has the same effect in human keratinocytes. First, we determined whether Bv-EE has cytotoxic effects on HaCaT cells. As shown in Figure 3a, Bv-EE did not show cytotoxicity and did not impact cell viability. Next, we induced cell death using UVB (30 mJ/cm 2 ) or H2O2 (200 μM) and found that the decreased cell number recovered with Bv-EE treatment (Figure 3b,c). In addition, to investigate whether Bv-EE can reduce ROS generation in keratinocytes, we stained HaCaT cells with 6-diamidino-2-phenylindole (DAPI) and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) after Bv-EE treatment. The ROS level increased upon treatment with H2O2; however, Bv-EE treatment reduced the ROS level in a dose-dependent manner. We also analyzed the ROS level in HaCaT cells using flow cytometry and similarly found that ROS generation decreased upon Bv-EE treatment ( Figure 3d). Thus, Bv-EE can reduce the ROS generation caused by UVB exposure or H2O2 treatment, suggesting that it has antioxidant and anti-aging effects.

Bv-EE Prevents Cell Death Caused by UVB and H 2 O 2 in HaCaT cells
Because Bv-EE showed ROS scavenging activity, we tested whether it has the same effect in human keratinocytes. First, we determined whether Bv-EE has cytotoxic effects on HaCaT cells. As shown in Figure 3a, Bv-EE did not show cytotoxicity and did not impact cell viability. Next, we induced cell death using UVB (30 mJ/cm 2 ) or H 2 O 2 (200 µM) and found that the decreased cell number recovered with Bv-EE treatment (Figure 3b,c). In addition, to investigate whether Bv-EE can reduce ROS generation in keratinocytes, we stained HaCaT cells with 6-diamidino-2-phenylindole (DAPI) and 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCFDA) after Bv-EE treatment. The ROS level increased upon treatment with H 2 O 2 ; however, Bv-EE treatment reduced the ROS level in a dose-dependent manner. We also analyzed the ROS level in HaCaT cells using flow cytometry and similarly found that ROS generation decreased upon Bv-EE treatment ( Figure 3d). Thus, Bv-EE can reduce the ROS generation caused by UVB exposure or H 2 O 2 treatment, suggesting that it has antioxidant and anti-aging effects.  All results are expressed as means ± standard deviations. ## p < 0.01 compared with the positive control group, and * p < 0.05, ** p < 0.01 compared with the normal group.

Bv-EE Prevents Cell Death Caused by UVB and H2O2 in HaCaT cells
Because Bv-EE showed ROS scavenging activity, we tested whether it has the same effect in human keratinocytes. First, we determined whether Bv-EE has cytotoxic effects on HaCaT cells. As shown in Figure 3a, Bv-EE did not show cytotoxicity and did not impact cell viability. Next, we induced cell death using UVB (30 mJ/cm 2 ) or H2O2 (200 μM) and found that the decreased cell number recovered with Bv-EE treatment (Figure 3b,c). In addition, to investigate whether Bv-EE can reduce ROS generation in keratinocytes, we stained HaCaT cells with 6-diamidino-2-phenylindole (DAPI) and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) after Bv-EE treatment. The ROS level increased upon treatment with H2O2; however, Bv-EE treatment reduced the ROS level in a dose-dependent manner. We also analyzed the ROS level in HaCaT cells using flow cytometry and similarly found that ROS generation decreased upon Bv-EE treatment ( Figure 3d). Thus, Bv-EE can reduce the ROS generation caused by UVB exposure or H2O2 treatment, suggesting that it has antioxidant and anti-aging effects.    The relative intensity of the ROS level was determined using ImageJ. All results are expressed as means ± standard deviations. ## p < 0.01 compared with the positive control group, and * p < 0.05, ** p < 0.01 compared with the normal group.

Bv-EE Inhibits AP-1 Promoter Activity and mRNA Expression of Aging Factors
Because Bv-EE was able to inhibit intracellular ROS generation and increase cell viability, we examined whether those effects involve AP-1, which is a main transcription The relative intensity of the ROS level was determined using ImageJ. All results are expressed as means ± standard deviations. ## p < 0.01 compared with the positive control group, and * p < 0.05, ** p < 0.01 compared with the normal group.

Bv-EE Inhibits AP-1 Promoter Activity and mRNA Expression of Aging Factors
Because Bv-EE was able to inhibit intracellular ROS generation and increase cell viability, we examined whether those effects involve AP-1, which is a main transcription factor that regulates ROS generation and cellular responses [30]. After confirming that Bv-EE has no cytotoxic effect on HEK293T cells (Figure 4a), we examined AP-1 promoter activity using a luciferase assay. As shown in Figure 4b, the AP-1 promoter activation level decreased significantly in a dose-dependent manner upon Bv-EE treatment in Tollinterleukin-1 receptor-domain-containing adapter-inducing interferon-β (TRIF)-induced HEK293T cells. Because Bv-EE can suppress AP-1 transcriptional activity, and AP-1 is a major regulator of the transcription of aging-and oxidative-related factors [31], we analyzed whether the mRNA levels of those related factors were regulated by Bv-EE. Interestingly, the mRNA expression of MMP-3 and MMP-9 was decreased by Bv-EE in H 2 O 2 -exposed HaCaT cells (Figure 4c). The COX-2 expression level was also decreased in the Bv-EEtreated groups in a dose-dependent manner (Figure 4d). In addition, we investigated the expression of those factors in UVB-treated HaCaT cells. The mRNA expression of MMP-3 and MMP-9 was reduced upon Bv-EE treatment (Figure 4e), and the COX-2 expression level was inhibited dose-dependently ( Figure 4f). Collectively, these data indicate that key factors related to aging and oxidation were inhibited by Bv-EE as a consequence of its inhibition of AP-1. factor that regulates ROS generation and cellular responses [30]. After confirming that Bv-EE has no cytotoxic effect on HEK293T cells (Figure 4a), we examined AP-1 promoter activity using a luciferase assay. As shown in Figure 4b, the AP-1 promoter activation level decreased significantly in a dose-dependent manner upon Bv-EE treatment in Toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β (TRIF)-induced HEK293T cells. Because Bv-EE can suppress AP-1 transcriptional activity, and AP-1 is a major regulator of the transcription of aging-and oxidative-related factors [31], we analyzed whether the mRNA levels of those related factors were regulated by Bv-EE. Interestingly, the mRNA expression of MMP-3 and MMP-9 was decreased by Bv-EE in H2O2exposed HaCaT cells (Figure 4c). The COX-2 expression level was also decreased in the Bv-EE-treated groups in a dose-dependent manner ( Figure 4d). In addition, we investigated the expression of those factors in UVB-treated HaCaT cells. The mRNA expression of MMP-3 and MMP-9 was reduced upon Bv-EE treatment (Figure 4e), and the COX-2 expression level was inhibited dose-dependently ( Figure 4f). Collectively, these data indicate that key factors related to aging and oxidation were inhibited by Bv-EE as a consequence of its inhibition of AP-1. (e) (f) The fold increase represents the ratio between the increase in mRNA expression in the Bv-EE treated groups and that in the normal group. GAPDH was used as an internal control. All results are expressed as means ± standard deviations. # p < 0.05, ## p < 0.01 compared with the positive control group, and * p < 0.05, ** p < 0.01 compared with the normal group.

Bv-EE Inhibits the AP-1 Signaling Pathway
We hypothesized that Bv-EE inhibited AP-1 transcription by inhibiting an upstream signaling pathway. Therefore, we studied whether phosphorylation of the kinases involved in the AP-1 signaling pathway would be reduced by Bv-EE in UVB-or H2O2treated HaCaT cells. First, we measured the phosphorylation levels of c-Jun and c-Fos, which are two main subunits of AP-1 [32]. Upon UVB irradiation, the expression of phosphorylated c-Jun and c-Fos was dramatically increased, and Bv-EE treatment decreased that expression in a dose-dependent manner (Figure 5a-c). We also examined the phosphorylation of AP-1 upstream kinases. Increased phosphorylation of the c-Jun N-terminal kinase (JNK) and p38 was decreased by Bv-EE treatment (Figure 5d-f). We then examined the same factors in cells subjected to H2O2 instead of UVB. We found downregulated phosphorylation of JNK, extracellular signal-regulated kinase (ERK), and p38, though the total form was unchanged (Figure 5g-i). These results indicate that Bv-EE can inhibit phosphorylation of the AP-1 signaling pathway to protect cells from UVB irradiation and oxidative stress.  The fold increase represents the ratio between the increase in mRNA expression in the Bv-EE treated groups and that in the normal group. GAPDH was used as an internal control. All results are expressed as means ± standard deviations. # p < 0.05, ## p < 0.01 compared with the positive control group, and ** p < 0.01 compared with the normal group.

Bv-EE Inhibits the AP-1 Signaling Pathway
We hypothesized that Bv-EE inhibited AP-1 transcription by inhibiting an upstream signaling pathway. Therefore, we studied whether phosphorylation of the kinases involved in the AP-1 signaling pathway would be reduced by Bv-EE in UVB-or H 2 O 2 -treated HaCaT cells. First, we measured the phosphorylation levels of c-Jun and c-Fos, which are two main subunits of AP-1 [32]. Upon UVB irradiation, the expression of phosphorylated c-Jun and c-Fos was dramatically increased, and Bv-EE treatment decreased that expression in a dose-dependent manner (Figure 5a-c). We also examined the phosphorylation of AP-1 upstream kinases. Increased phosphorylation of the c-Jun N-terminal kinase (JNK) and p38 was decreased by Bv-EE treatment (Figure 5d-f). We then examined the same factors in cells subjected to H 2 O 2 instead of UVB. We found downregulated phosphorylation of JNK, extracellular signal-regulated kinase (ERK), and p38, though the total form was unchanged (Figure 5g-i). These results indicate that Bv-EE can inhibit phosphorylation of the AP-1 signaling pathway to protect cells from UVB irradiation and oxidative stress. (e) (f)

Bv-EE Inhibits the AP-1 Signaling Pathway
We hypothesized that Bv-EE inhibited AP-1 transcription by inhibiting an upstream signaling pathway. Therefore, we studied whether phosphorylation of the kinases involved in the AP-1 signaling pathway would be reduced by Bv-EE in UVB-or H2O2treated HaCaT cells. First, we measured the phosphorylation levels of c-Jun and c-Fos, which are two main subunits of AP-1 [32]. Upon UVB irradiation, the expression of phosphorylated c-Jun and c-Fos was dramatically increased, and Bv-EE treatment decreased that expression in a dose-dependent manner (Figure 5a-c). We also examined the phosphorylation of AP-1 upstream kinases. Increased phosphorylation of the c-Jun N-terminal kinase (JNK) and p38 was decreased by Bv-EE treatment (Figure 5d-f). We then examined the same factors in cells subjected to H2O2 instead of UVB. We found downregulated phosphorylation of JNK, extracellular signal-regulated kinase (ERK), and p38, though the total form was unchanged (Figure 5g-i). These results indicate that Bv-EE can inhibit phosphorylation of the AP-1 signaling pathway to protect cells from UVB irradiation and oxidative stress.

Bv-EE Promotes Collagen Generation in Human Dermal Fibroblast (HDF) Cells
Aging and oxidative stress play important roles in wrinkle formation. Therefore, wrinkles are frequently considered an indicator of aging and oxidation [33,34]. Based on that concept, we tested whether Bv-EE can enhance the expression of wrinkle-related factors in human dermal fibroblast cells, which are responsible for collagen generation. After confirming that Bv-EE has no cytotoxic effect on HDF cells (Figure 6a), we examined whether Bv-EE can regulate Col1A1, a typical collagen-encoding gene [35]. As shown in Figure 6b, Bv-EE treatment dose-dependently upregulated Col1A1 promoter activity; retinol was used as a positive control. Correspondingly, mRNA expression of Col1A1 was also increased by Bv-EE treatment (Figure 6c). In addition, we tested whether Bv-EE can restore the Col1A1 expression decreased by exposure to UVB or H 2 O 2 . Interestingly, we found that the decreased mRNA expression of Col1A1 was recovered by Bv-EE treatment (Figure 6d,e). These results indicate that Bv-EE can promote collagen synthesis by upregulating its transcriptional activity. examined by Western blot. (d,g) The phosphorylation of ERK, JNK, and p38 was determined by Western blotting following Bv-EE treatment and exposure to UVB or H2O2. β-actin was used as an internal control (b,c), (e,f), (h,i) Band intensity was measured and quantified using ImageJ. All results are expressed as means ± standard deviations. ## p < 0.01 compared with the positive control group, and * p < 0.05, ** p < 0.01 compared with the normal group.

Bv-EE Promotes Collagen Generation in Human Dermal Fibroblast (HDF) Cells
Aging and oxidative stress play important roles in wrinkle formation. Therefore, wrinkles are frequently considered an indicator of aging and oxidation [33,34]. Based on that concept, we tested whether Bv-EE can enhance the expression of wrinkle-related factors in human dermal fibroblast cells, which are responsible for collagen generation. After confirming that Bv-EE has no cytotoxic effect on HDF cells (Figure 6a), we examined whether Bv-EE can regulate Col1A1, a typical collagen-encoding gene [35]. As shown in Figure 6b, Bv-EE treatment dose-dependently upregulated Col1A1 promoter activity; retinol was used as a positive control. Correspondingly, mRNA expression of Col1A1 was also increased by Bv-EE treatment (Figure 6c). In addition, we tested whether Bv-EE can restore the Col1A1 expression decreased by exposure to UVB or H2O2. Interestingly, we found that the decreased mRNA expression of Col1A1 was recovered by Bv-EE treatment (Figure 6d,e). These results indicate that Bv-EE can promote collagen synthesis by upregulating its transcriptional activity.

Discussion
Organic and nature-derived materials are attractive approaches to skin therapy due to their minimal toxicity and side effects [36]. This is an important factor in pharmaceutical and cosmetic formulations. In this study, we used UVB-and H2O2-induced damage in vitro to study the efficacy of a novel plant extract, Bv-EE, and demonstrated its protective effects in human keratinocytes and dermal fibroblasts. Importantly, Bv-EE showed no cytotoxicity to HaCaT or HDF cells, which are the cell lines most widely used for human skin research.
Oxidative stress is the largest cause of skin damage and is associated with skin aging [37]. Therefore, the use of antioxidants has become a leading approach for anti-aging therapy [38]. Several chemicals have been approved for application in the pharmaceutical and cosmetic industries [39]. In this work, we found that Bv-EE has ROS scavenging activity (Figure 2), indicating that it can directly scavenge generated ROS. More importantly, Bv-EE inhibits ROS generation in human keratinocytes (Figure 3). UVB and H2O2 treatment induce serious oxidation processes in cells and eventually lead to cell death. Bv-EE showed a remarkable ability to prevent cell death caused by ROS responses, indicating the potential of Bv-EE as a drug to treat skin diseases.
When COX-2 and MMPs are produced in response to UVB irradiation, they play an important role in inflammatory responses in skin cells. COX-2 mediates inflammatory processes in the skin, including inflammatory hyperalgesia and nociception [40][41][42]. In this work, we discovered that Bv-EE treatment significantly decreased COX-2 and MMP transcriptional activity and mRNA expression in UVB-and H2O2-damaged keratinocytes ( Figure 4). That finding clearly indicates that Bv-EE acts as an antioxidant, as well as an anti-aging agent, by recovering the damage induced by UVB irradiation and oxidative stress.
The AP-1 signaling pathway is prominently activated by external triggers, including UVB and oxidative stress. Therefore, its constitutive kinases are promising targets for disease therapies [43,44]. In this study, we focused on MAPK-related enzymes and confirmed that Bv-EE can inhibit the phosphorylation of JNK, ERK, and p38 in UVB-and H2O2-

Discussion
Organic and nature-derived materials are attractive approaches to skin therapy due to their minimal toxicity and side effects [36]. This is an important factor in pharmaceutical and cosmetic formulations. In this study, we used UVB-and H 2 O 2 -induced damage in vitro to study the efficacy of a novel plant extract, Bv-EE, and demonstrated its protective effects in human keratinocytes and dermal fibroblasts. Importantly, Bv-EE showed no cytotoxicity to HaCaT or HDF cells, which are the cell lines most widely used for human skin research.
Oxidative stress is the largest cause of skin damage and is associated with skin aging [37]. Therefore, the use of antioxidants has become a leading approach for anti-aging therapy [38]. Several chemicals have been approved for application in the pharmaceutical and cosmetic industries [39]. In this work, we found that Bv-EE has ROS scavenging activity (Figure 2), indicating that it can directly scavenge generated ROS. More importantly, Bv-EE inhibits ROS generation in human keratinocytes (Figure 3). UVB and H 2 O 2 treatment induce serious oxidation processes in cells and eventually lead to cell death. Bv-EE showed a remarkable ability to prevent cell death caused by ROS responses, indicating the potential of Bv-EE as a drug to treat skin diseases.
When COX-2 and MMPs are produced in response to UVB irradiation, they play an important role in inflammatory responses in skin cells. COX-2 mediates inflammatory processes in the skin, including inflammatory hyperalgesia and nociception [40][41][42]. In this work, we discovered that Bv-EE treatment significantly decreased COX-2 and MMP transcriptional activity and mRNA expression in UVB-and H 2 O 2 -damaged keratinocytes (Figure 4). That finding clearly indicates that Bv-EE acts as an antioxidant, as well as an anti-aging agent, by recovering the damage induced by UVB irradiation and oxidative stress.
The AP-1 signaling pathway is prominently activated by external triggers, including UVB and oxidative stress. Therefore, its constitutive kinases are promising targets for disease therapies [43,44]. In this study, we focused on MAPK-related enzymes and confirmed that Bv-EE can inhibit the phosphorylation of JNK, ERK, and p38 in UVB-and H 2 O 2 -damaged keratinocytes ( Figure 5). These findings suggest that the skin-protective characteristics of Bv-EE occur by regulating the AP-1 intracellular molecular signaling pathway.
One of the most typical symptoms and indicators of aging is wrinkles, which are caused by a decrease in ECM proteins such as collagen in fibroblast cells [45]. Therefore, the proper production of collagen is an important process for maintaining healthy skin [46]. In this work, we found that Bv-EE promotes collagen synthesis by increasing Col1A1 transcriptional activity and mRNA expression in human dermal fibroblasts ( Figure 6). More interestingly, Bv-EE can restore the collagen synthesis decreased by UVB irradiation and H 2 O 2 treatment ( Figure 6). These findings strongly suggest that Bv-EE can help to prevent skin wrinkles by promoting collagen synthesis.
Among all the skin-protecting agents currently used in the pharmaceutical and cosmetic industries, Bv-EE has shown its potential as a natural compound with anti-aging and antioxidant properties. This study provides novel insights about an organic and natural ingredient as a promising drug candidate because of its genetic and molecular regulation of potential targets.

Preparation of Breynia vitis-idaea
Breynia vitis-idaea was procured from the National Institute for Biological Resources (Incheon, Republic of Korea). The dried stems (1.3 kg) were extracted in 70% ethanol (3 × 18 L) at room temperature for 3 h using an ultrasonicator (Ultrasonic Cleaner UC-10, UC-20, 400 W) under dark conditions. After removing the macerate by filtration, the extracted solution was concentrated in vacuo at 40 • C using a rotary evaporator (IVT Co., Ltd., Daegu, Republic of Korea), and then freeze-dried for 48 h at −80 • C to make a completely dried powder (color of the extract: brown). The yield of this extraction was 13.97%.

GC-MS
The GC-MS analysis of dried Bv-EE (100 mg/mL in methanol) was carried out with an Agilent 8890 GC instrument (Santa Clara, CA, USA) equipped with an Agilent J&W DB-624 Ultra Insert GC column (60 m in length × 250 µM in diameter × 1.40 µM in thickness), and mass spectrometry was conducted with an Agilent 5977B MSD instrument (Santa Clara, CA, USA) equipped with a Series II triple-axis detector with a high energy dynode and long-life electron multiplier from the Cooperative Center for Research facilities of Sungkyunkwan University (Suwon, Republic of Korea), as previously reported [47,48]. Detailed conditions of the analysis are listed in Table 2. The spectrum of phytochemicals in the National Institute of Standards and Technology library was used to identify the unknown phytochemicals in Ca-EE, as reported previously [49,50].

Cell Culture
HaCaT cells (a human keratinocyte cell line) and HDF cells (a human fibroblast cell line) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin, and HEK293T cells (a human embryonic kidney cell line) were cultured in DMEM supplemented with 5% FBS and 1% penicillin-streptomycin. All cells were maintained in a 5% CO 2 incubator at 37 • C.

DPPH Radical Scavenging Activity Assay
The DPPH assay was performed to determine the radical scavenging capacity of the extract. We dissolved 300 µM DPPH in methanol and used ascorbic acid (100 µM) dissolved in PBS as the positive control. Different concentrations of Bv-EE were prepared, and the absorbance was measured at 517 nm and 37 • C for 15 min. Radical scavenging activity was calculated using the following equation, as reported previously [51,52].
where A 0 is the absorbance of DPPH, and A 1 is the absorbance of the extract.

ABTS Radical Scavenging Activity Assay
The ABTS assay was performed to determine the radical scavenging capacity of Bv-EE. First, 7.4 mM ABTS dissolved in PBS and 2.4 mM of potassium persulfate dissolved in PBS were mixed at a ratio of 1:1 and incubated overnight. Different concentrations of Bv-EE and ascorbic acid (100 µM) were prepared, and the absorbance was measured at 730 nm and 37 • C for 15 min. The percentage of scavenging was calculated using the same method as stated previously [53].

CUPRAC Assay
The CUPRAC assay was performed to determine the cupric reducing antioxidant capacity of Bv-EE. First, 100 mM CuCl 2 ·2H 2 O (copper (II) chloride solution) was dissolved in distilled or deionized water. NH 4 Ac (ammonium acetate) was dissolved in distilled or deionized water, and the pH was adjusted to 7.0. Neocuproine (Nc) solution (7.5 mM) was dissolved in pure ethanol. Next, the copper (II) chloride solution, ammonium acetate buffer, and Nc solution were mixed at a ratio of 1:1:1 to a final volume of 200 µL in e-tubes, and 200 µL of Bv-EE solution was added to each tube. Trolox (3 mg/mL) dissolved in pure ethanol was used as a positive control. After a 1 h incubation at room temperature, the mixed solution was transferred to a 96-well plate, and the absorbance was measured at 450 nm [54].

FRAP Assay
The FRAP assay was performed to determine the ferric reducing power of Bv-EE. First, 300 mM acetic acid buffer was prepared and mixed with anhydrous sodium acetate and glacial acetic acid (pH 3.6). Next, 10 mM TPTZ solution dissolved in distilled or deionized water and 20 mM FeCl 3 ·6H 2 O dissolved in distilled or deionized water were mixed with the FeCl 3 solution at a ratio of 10:1:1. Bv-EE solution was prepared at different concentrations and aliquoted to a 96-well plate at 100 µL per well. Then, 100 µL of FRAP working solution were added to each well and incubated for 15 min at 37 • C in the dark. Trolox was used as a positive control, and absorbance was measured at 593 nm [55].

Determination of Total Phenolic Content
At room temperature, 100 µL of Bv-EE (serially diluted five times in distilled or deionized water) and 100 µL of 10% Folin-Ciocâlteu reagent were mixed with 300 µL of distilled or deionized water and incubated for 5 min. Next, 500 µL of 8% sodium carbonate and 500 µL of distilled or deionized water were added to the tubes. After 30 min of incubation at room temperature in a dark room, the mixture was transferred to a 96-well plate, and the absorbance was measured at 765 nm. The calculation was performed using a standard curve obtained with gallic acid, and the total phenolic content is expressed as mg of gallic acid equivalent/g of extract.

Determination of Total Flavonoid Content
For this analysis, 100 µL of Bv-EE (serially diluted five times in distilled or deionized water) and 100 µL of aluminum chloride 2% reagent were mixed in a 1:1 ratio, as reported previously [56,57]. After a 1 h incubation at room temperature in a dark room, the mixture was transferred to a 96-well plate, and the absorbance was measured at 420 nm. The calculation was performed using a standard curve obtained with quercetin, and the total flavonoid content is expressed as mg of quercetin equivalent/g of extract.

ROS Generation Assay
The 2 ,7 -dichlorodihydrofluorescin diacetate (H2DCFDA) assay was used to detect intracellular ROS. HaCaT cells were pretreated with Bv-EE for 30 min and then treated with H 2 O 2 (200 µM) for 24 h. The cells were washed with PBS, stained with 10 µM H2DCFDA, and incubated for 20 min in the dark. The cells were then fixed in formaldehyde solution (100 µL/mL) for 10 min, washed with PBS two times, stained with DAPI (1 µL/mL), and incubated for 20 min in the dark. Photographs were captured using a Nikon Eclipse Ti (Nikon, Japan) fluorescence microscope.
For flow cytometry, HaCaT cells were treated with Bv-EE and exposed to H 2 O 2 , as stated above. The cells were harvested and resuspended in 300 µL of PBS. Then, 10 µM H2DCFDA was added to the tube and incubated for 20 min in the dark. The fluorescence was detected at 485/535 nm using a flow cytometer (Beckman Coulter, Brea, CA, USA), as described previously [58].

Cell Viability Assay
HaCaT and HEK293T cells were seeded at 3 × 10 5 cells/mL, and HDF cells were seeded at 1 × 10 5 cells/mL in 96-well plates and incubated for 24 h. Then, Bv-EE was added to all cells for 24 h. Next, 100 µL of the original media was removed, and 10 µL of MTT solution was added to each well and incubated for 4 h. To each well, 100 µL of MTT stopping solution was added and incubated overnight. The absorbance was measured at 570 nm using a multi-plate microreader, as previously reported [59,60].

H 2 O 2 Treatment and UVB Irradiation
HaCaT cells were plated in 6-well plates at 2 × 10 5 cells/mL and incubated for 24 h. The cells were pretreated with Bv-EE for 30 min, washed with cold PBS, and exposed to H 2 O 2 (200 µM) or UVB radiation (30 mJ/cm 2 ). After that, the cells were treated with Bv-EE at different concentrations for 24 h, as previously reported [61]. A BLX-312 (Vilber Lourmat, France) UVB lamp was used for UVB irradiation. Cell viability was calculated as follows: Cell viability (% of control) = A 1 /A 0 × 100 where A 1 refers to treated cells, and A 0 refers to normal untreated cells. HaCaT and HDF cells were exposed to UVB and H 2 O 2 as described above. Either Bv-EE or retinol (10 µg/mL) was added to the cells for 24 h. RNA was isolated using TRI reagent solution. cDNA was synthesized from total RNA (1 µg) using a cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). RT-PCR and real-time PCR were conducted as previously described [62,63]. The primer sequences used in this experiment are listed in Table 3.

Luciferase Reporter Gene Assay
HEK293T cells were seeded in a 24-well plate at 3 × 10 5 cells/mL. After 18 h of incubation, the HEK293T cells were co-transfected with luciferase-expressing genes (AP-1 and Col1A1) and the β-galactosidase gene using PEI. After 24 h of incubation, the cells were treated with Bv-EE or retinol for 24 h. Then, 300 µL of luciferase lysis buffer was added to each well, and the plate was frozen for 3 h at −70 • C. The luciferase assay was conducted using a luciferase assay system reported previously [64].

Preparation of Whole Cell Lysates and Western Blot Analysis
HaCaT cells were washed with cold PBS, collected using a cell scraper, and centrifuged at 12,000 rpm for 5 min at 4 • C. The cells were lysed for 15 min on ice in cell lysis buffer (20 mM Tris-HCl, pH 7.4; 2 mM EDTA; 2 mM ethyleneglycotetraacetic acid; 1 mM dithiothreitol; 50 mM β-glycerol phosphate; 0.1 mM sodium vanadate; 1.6 mM pervanadate; 1% Triton X-100; 10% glycerol; 10 µg/mL aprotinin; 10 µg/mL pepstatin; 1 µM benzamide; and 2 µM phenylmethylsulfonyl fluoride) and kept at −70 • C until use. The supernatant containing protein was collected and used for Western blotting. Protein concentrations were measured using the Bradford assay as described previously [65]. For that, 20 µg of protein from each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking the membranes with 3% BSA for 1 h at room temperature, we washed the membranes with tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) and 0.1% Tween-20 (TBST) three times at 10 min intervals. The membranes were incubated with primary antibodies (1:2500 dilution) overnight at 4 • C. Then, the membranes were washed with TBST three times for 10 min each and incubated with secondary antibody for 2 h at room temperature. After washing the membranes with TBST three times for 10 min each time, we detected their chemiluminescence using ECL reagent [66]. Relative band intensities were measured using ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).

Statistical Analyses
All data are presented as the mean ± standard deviation of at least three independent experiments. A Mann-Whitney test was used to compare statistical differences between experimental and control groups. p-values < 0.05 were considered statistically significant. All statistical analyses were conducted using SPSS (SPSS, Chicago, IL, USA).

Conclusions
Herein, we demonstrated the anti-aging and antioxidant capacity of Bv-EE by evaluating its inhibitory effect on ROS in human keratinocytes and dermal fibroblasts exposed to UVB irradiation and H 2 O 2 . Bv-EE was able to downregulate aging factors (COX-2 and MMPs). Correspondingly, the AP-1 (c-Jun/c-Fos) signaling activity was reduced upon Bv-EE treatment, as summarized in Figure 7. On the other hand, Bv-EE upregulated Col1A1 expression in human dermal fibroblasts. benzamide; and 2 μM phenylmethylsulfonyl fluoride) and kept at −70 °C until use. The supernatant containing protein was collected and used for Western blotting. Protein concentrations were measured using the Bradford assay as described previously [65]. For that, 20 μg of protein from each sample was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking the membranes with 3% BSA for 1 h at room temperature, we washed the membranes with tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) and 0.1% Tween-20 (TBST) three times at 10 min intervals. The membranes were incubated with primary antibodies (1:2500 dilution) overnight at 4 °C. Then, the membranes were washed with TBST three times for 10 min each and incubated with secondary antibody for 2 h at room temperature. After washing the membranes with TBST three times for 10 min each time, we detected their chemiluminescence using ECL reagent [66]. Relative band intensities were measured using ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).

Statistical Analyses
All data are presented as the mean ± standard deviation of at least three independent experiments. A Mann-Whitney test was used to compare statistical differences between experimental and control groups. p-values < 0.05 were considered statistically significant. All statistical analyses were conducted using SPSS (SPSS, Chicago, IL, USA).

Conclusions
Herein, we demonstrated the anti-aging and antioxidant capacity of Bv-EE by evaluating its inhibitory effect on ROS in human keratinocytes and dermal fibroblasts exposed to UVB irradiation and H2O2. Bv-EE was able to downregulate aging factors (COX-2 and MMPs). Correspondingly, the AP-1 (c-Jun/c-Fos) signaling activity was reduced upon Bv-EE treatment, as summarized in Figure 7. On the other hand, Bv-EE upregulated Col1A1 expression in human dermal fibroblasts.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.