Antioxidant Activity and Effect of Tradescantia palida Methanol Extract on Viability of UVA-irradiated Human Dermal Fibroblasts

Abstract

This study aimed to investigate the antioxidant activity of Tradescantia palida methanol extract (TPME) and its effect on viability of UVA-irradiated human dermal fibroblasts (HDFs). The total polyphenol and flavonoid contents of TPME were 418.4 tannic acid equivalent (TAE) mg/g, and 247.1 rutin equivalent (RE) mg/g, respectively. The electron-donating abilities of TPME and ascorbic acid (AA) at 1,000 ㎍/mL were 66.8% and 97.5%, respectively. The 50% scavenging concentration of free radicals (SC₅₀) from TPME and AA were 660.8 ㎍/mL and <125 ㎍/mL, respectively. The maximum permissible concentration of TPME in treating HDFs was 50 ㎍/mL. Treatment of 10, 15, and 20 mJ/cm² UVA-irradiated HDFs with 50 ㎍/mL TPME increased cellular viability by 5.40% (p<0.01), 15.0% (p<0.001), and 20.8% (p<0.001), respectively. Treatment with 50 ㎍/mL AA increased cellular viability by 11.5% (p<0.001), 20.6% (p<0.001), and 15.4% (p<0.001) in 10, 15, and 20 mJ/cm² UVA-irradiated HDFs, respectively. These results indicate that TPME contains abundant antioxidants, which confer TPME with a high degree of antioxidant activity. Thus, TPME is a natural antioxidant that can protect HDFs against UVA-irradiation.

1. Introduction

The perception of age and beauty largely depends on the appearance of the exposed skin [1], and its condition is partly controlled by environmental effects, especially Ultraviolet(UV) light [2]. Both epidermal keratinocytes and dermal fibroblasts are targets of solar irradiation, and the expression pattern and activity of many proteins is altered in response to exposure on UVA and UVB light [3]. Alterations in collagen, the major structural component of skin, have been suggested as a cause of the clinical changes observed in photoaged and naturally aged skin [4].

UV radiation is one of the major physical stress factors that affect human skin [5]. UVA light, which accounts for 95% of solar UV light, is primarily responsible for photoaging, which is characterized by wrinkles and loss of skin elasticity [6]. Photoaging is caused by an imbalance between the accumulation and degradation of extracellular matrix (ECM) components that provide structural and functional support to skin tissue [7].

Plants contain a wide array of antioxidant compounds including polyphenols, flavonoids, tocopherols, phenolic acids and tannins [8]. Crude plant extracts that are rich in phenolic content are of interest to the cosmetic industry because of their anti-oxidation and anti-photoaging activities [9]. Tradescantia pallida is a species of spider-wort (a genus of New World plants) native to the gulf coast region of eastern Mexico [10]. These plants are often grown for ornamental purposes due to their bluish or purplish leaves and/or flowers. They are also used ethnobotanically to treat many diseases, including mucosal infections [11], wounds, and gastrointestinal disorders, which may be linked to their antibacterial and antioxidant properties [12]. Although there one recent study demonstrated the antioxidant and antimicrobial activity of T. pallida [13], little is known about the antioxidant activity and cytotoxicity of T. pallida methanol extract (TPME) on UVA-irradiated human dermal fibroblasts(HDFs).

2. Materials and Methods

2.1 Reagents and apparatus

The following reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA): dimethyl sulfoxide (DMSO), 1,1-diphenyl-2-picrylhydrazyl (DPPH), tannic acid, ascorbic acid, Folin-Ciocalteu’s phenol reagent, and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). The following reagents were obtained from the Lonza Company (Cascade, CA, USA): Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin mixture (P/S). UV irradiation was performed with a UVA sunlamp (Sankyo Denki, Kanagawa, Japan). An inverted microscope (CKX41; Olympus Corporation, Tokyo, Japan) was used to observe cell growth, and a CO₂ incubator (MCO-17AC; Sanyo Electric, Osaka, Japan) was used for cell culture.

2.2 Plant materials

T. pallida extract was collected from the greenhouse at the College of Applied Life Sciences, Jeju National University in December, 2018, and the above-ground portion of plant was dried in a dryer. Dried T. pallida was crushed with a blender, filtered through a 500 μm sieve, extracted with 100 mL of methanol per 10 g of plant for 24 h twice, and then concentrated under a reduced pressure concentrator. Concentrates were dried with a freeze dryer and used as TPME samples. Each sample was dissolved in DMSO, diluted with distilled water to an appropriate concentration, and used in an experiment.

2.3 Antioxidant activity

2.3.1 Total polyphenol content

The total polyphenol content of TPME was measured using the Folin and Denis assay [14]. One milliliter of TPME dissolved in distilled water at concentrations of 25, 50, 100, 200, and 400 μg/mL was placed in a test tube. Folin reagent (1 mL) was added, and the tubes were allowed to stand for 3 min. One mL of 10% Na₂CO₃ was added and the mixture was shaken vigorously and allowed to stand for 1 h. Absorbance was measured at 725 nm and polyphenol content was quantified using a standard curve of tannic acid.

2.3.2 Total flavonoid content

The total flavonoid content of TPME was measured using a method modified from Davies et al. [15]. One hundred microliters of TPME dissolved in DMSO at concentrations of 25, 50, 100, 200, and 400 μg/mL was placed in a test tube. One milliliter of di-ethylene glycol reagent and 100 μL of 1 N NaOH were added to the test tubes. The mixture was shaken vigorously and reacted at 37℃ for 1 h before absorbance at 420 nm was measured. The standard curve was prepared using rutin.

2.3.3 Electron-donating ability

Electron-donating ability was evaluated as previously described [16]. One hundred microliters of TPME dissolved in methanol at concentrations of 125, 250, 500, or 1,000 μg/mL was placed in a test tube, to which 50 μL of 0.2 mM DPPH was added. The mixture was shaken vigorously and allowed to stand for 30 min in the dark before absorbance was measured at 517 nm. Ascorbic acid (AA) was used as the positive control. The DPPH radical scavenging activity of each solution was calculated as percent inhibition with the following equation:

% donating ability=[1-(A_sample/A_control)]×100

where A_sample and A_control represent the absorbance of the sample and the control, respectively. The SC₅₀ (50% free radical scavenging concentration) was calculated using GraphPrism software Ver. 7 (Graphpad Software, Inc, La Jolla, CA, USA).

2.4 Cell experiments

2.4.1 Cell culture

Human dermal fibroblasts (HDFs) [Korean Cell Line Bank (KCLB), Seoul, Korea] were maintained at 37℃ in a humidifier with 95% air and 5% CO₂; the cells were kept in DMEM supplemented with 10% heat-inactivated FBS and 1% P/S.

2.4.2 Cell viability assay

HDFs were grown in 96-well plates(1×10⁴ cells/well) overnight. The cells were then treated without or with TPME or AA at the indicated concentrations in the absence or presence of UVA. At each time point, cells were incubated with MTT (20 μL/well) for 4 h at 37℃. Subsequent measurements were made using a flow cytometer(Beckman Coulter, Miami, FL, USA).

2.4.3 Morphology of HDFs

HDFs were treated with TPME or AA at concentrations of 12.5, 25, and 50 μg/mL, and the cells were incubated as described above for 48 h. After the medium was changed, the cells were observed with a microscope and photographed.

2.5 Statistical analysis

Significance of data was analyzed by Student’s t-test (SPSS 21.0 for Windows, IBM, Armonk, NY, USA). Differences between experimental groups were considered to be significant when p<0.05.

3. Results and Discussion

3.1 Antioxidant activity of TPME

Fruit and plants contain polyphenol compounds, including gallic acid and hydrolyzable tannin, which are known to be metal chelators and, thus, may bind to a Zn ion active site and prevent the substrate from enzyme digestion [17]. Flavonoids derived from plants have the potential to bind metalloenzymes, thus inhibiting metabolic pathways by forming complexes with metal ions [18]. The antioxidant capacities of our test extracts were determined by DPPH-based methods, which are widely used in plant and food research to screen for antioxidant activity [19].

In this study, the total polyphenol and flavonoid contents of the extracts were 418.4 TAE mg/g and 247.1 RE mg/g, respectively (Fig. 1). The electron-donating abilities of TPME and AA at 1,000 μg/mL were 66.8% and 97.5%, respectively (Fig. 2). TPME showed electron-donating ability in a concentration-dependent manner and the SC₅₀ of free radicals for TPME was 660.8 μg/mL. The SC₅₀ for AA was lower than 125 μg/mL. These results indicate that TPME contains abundant antioxidants and exerts a high degree of antioxidant activity.

Fig. 1. Total polyphenol and flavonoid contents of TPME. Total polyphenol and flavonoid contents were quantified by using standard curves of tannic acid and rutin, respectively. Values represent the means±SD of three independent measurements. TPME: T. pallida methanol extract.

Fig. 2. Electron-donating ability of TPME relative to the positive control, ascorbic acid (AA). Each substance was evaluated on its ability to provide electrons to the free radical DPPH. Values represent the means±SD of three independent measurements. TPME: T. pallida methanol extract. ***p<0.001 compared to AA by Student’s t-test.

3.2 Effects of TPME treatment on HDFs

Oxidative stress plays a major role in the induction of photoaging [20]. The antioxidants protect against the potentially damaging oxidative stress, which is a result of an imbalance between the formation of reactive oxygen species (ROS) and the body antioxidant defense [21]. UVA rays are absorbed by skin leading to the generation of ROS in dermal fibroblasts [22]. Fibroblasts play an important role in production of elastin, collagen, and ground substances such as glycosaminoglycans and proteoglycans[23]. The reduction of collagen is due to a reduced synthetic activity of dermal fibroblasts or an increased rate of degradation by collagenase [24].

Compared with normal control cells, the viability of HDFs treated with 50 μg/mL TPME was 80.2% (Fig. 3). The viability of 10, 15, and 20 mJ/cm² UVA-irradiated HDFs decreased by 18.3% (p<0.001), 32.1% (p<0.001), and 42.9% (p<0.001), respectively. Treatment with 50 μg/mL TPME increased cellular viability by 5.4% (p<0.01), 15.0% (p<0.001), and 20.8% (p<0.001) in 10, 15, and 20 mJ/cm² UVA-irradiated HDFs, respectively (Fig. 4). Treatment with 50 μg/mL AA increased cellular viability by 11.5% (p<0.001), 20.6% (p<0.001), and 15.4% (p<0.001) in 10, 15, and 20 mJ/cm² UVA-irradiated HDFs, respectively. As shown in Fig. 5, treatment with TPME at concentrations of 12.5, 25 and 50 μg/mL did not affect the morphology of HDFs as compared with the normal control. However, treatment with TPME at concentrations of 100 and 200 μg/mL reduced cell density compared with the normal control. These results indicate that TPME seems to protect HDFs against UVA-irradiation, suggesting that TPME could be an effective natural antioxidant to prevent and/or alleviate a skin photo-aging.

Fig. 3. Effects of TPME or AA treatment on the viability of HDFs. Values represent the means±SD of three independent measurements. AA: ascorbic acid, TPME: T. pallida methanol extract. N: non-treated.

Fig. 4. Effects of TPME or AA treatment on the viability of UVA-irradiated HDFs. Values represent the means±SD of three independent measurements. AA: ascorbic acid, TPME: T. pallida methanol extract. (A) 10 mJ/cm² UVA-irradiated HDFs, (B) 15 mJ/cm² UVA-irradiated HDFs, (C) 20 mJ/cm² UVA-irradiated HDFs. *p<0.05, **p<0.01, ***p<0.001, statistically significant differences versus control (UVA irradiation only) by ANOVA and Duncan’s multiple range test.

Fig. 5. Morphological observation of HDFs treated with TPME at the indicated concentrations for 48 h ( × 200). AA: ascorbic acid, TPME: T. pallida methanol extract. (A) Non-treated, (B) TPME 12.5 μg/mL, (C) TPME 25 μg/mL, (D) TPME 50 μg/mL, (E) TPME 100 μg/mL, (F) TPME 200 μg/mL.