Protective effects and mechanisms of Terminalia catappa L. methenolic extract on hydrogen-peroxide-induced oxidative stress in human skin fibroblasts

Background Oxidative stress plays a crucial role in aging-related phenomenon, including skin aging and photoaging. This study investigated the protective role and possible mechanism of Terminalia catappa L. methanolic extract (TCE) in human fibroblasts (Hs68) against hydrogen peroxide (H2O2)-induced oxidative damage. Methods Various in vitro antioxidant assays were performed in this study. The effect and mechanisms of TCE on oxidative stress-induced oxidative damage were studied by using western blotting. Results The IC50 of TCE was 8.2 μg/mL for 1,1-diphenyl-2-picrylhydrazyl radical scavenging, 20.7 μg/mL for superoxide anion radical scavenging, 173.0 μg/mL for H2O2 scavenging, 44.8 μg/mL for hydroxyl radical scavenging, and 427.6 μg/mL for ferrous chelation activities. Moreover, TCE inhibited the H2O2-induced mitogen-activated protein kinase signaling pathway, resulting in the inhibition of c-Jun, c-Fos, matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, and cyclooxygenase-2 expression. TCE also increased hemeoxygenase-1 expression inhibited by H2O2. Finally, TCE was demonstrated reverse type I procollagen expression in fibroblasts after H2O2 treatment. Conclusions According to our findings, TCE is a potent antioxidant and protective agent that can be used in antioxidative stress-induced skin aging. Electronic supplementary material The online version of this article (10.1186/s12906-018-2308-4) contains supplementary material, which is available to authorized users.


Background
Aging can be divided into two basic processes: intrinsic aging, which is related to age, and extrinsic aging, which is generally due to long-term exposure to environmental factors, including ultraviolet (UV) light and pollutants. Oxidative stress plays a crucial role in aging-related disorders, including atherosclerosis, cardiovascular diseases and skin aging [1]. High levels of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide anion, and singlet oxygen, can cause oxidative damage to cellular DNA, protein, and lipids, resulting in the initiation or development of various disorders and diseases such as cardiovascular diseases, type 2 diabetes mellitus, and cancer [2]. In addition, free transition metal ions combine with H 2 O 2 and can cause extensive oxidative damage to biomolecules such as lipids, proteins, and nucleic acids, leading to age-related disorders [3]. Skin aging is characterized by a sagging appearance, wrinkles, and pigmentary changes, and principally manifests as the degradation of extracellular matrix (ECM) proteins, including type I collagen, elastin, proteoglycans, and fibronectin [4,5]. Type I collagen is the most abundant structural protein in skin connective tissue and is primarily synthesized by fibroblasts, whereas collagen in the dermis is responsible for skin strength and resiliency [6,7]. Oxidative stress or inflammation can cause collagen degradation resulting in wrinkle formation and sagging skin [8]. In addition, ROS activate the mitogen-activated protein kinase (MAPK) pathway, which subsequently induces the expression and activation of matrix metalloproteinases (MMPs) in human skin [9]. The activation of MAPK and MMPs may cause damage and aging of the skin [10,11]. Agents that can elevate ECM protein levels or downregulate collagen-degrading enzymes, such as MMPs, may prove useful in the development of effective antiaging agents [12,13].
Terminalia catappa L. belongs to the family Combretaceae, and in Southeast Asia, it is commonly used as a folk medicine for treating hepatoma and hepatitis [14,15]. The leaf and bark extracts of T. catappa have been reported to exhibit chemopreventive, antioxidant, hepatoprotective, and anti-inflammatory activities [16,17]. T. catappa includes the phytochemicals of flavanoids (rutin, isoorientin, vitexin, and isovitexin), tannins (chebulagic acid, punicalagin, punicalin, and terflavins A and B), and triterpenoids (asiatic acid and ursolic acid) [14,18]. In addition, the T. catappa extract exhibits antifungal and antidepressant activities [19,20]. Topical application of ointment containing T. catappa was shown to promote wound healing in rats [21], and our previous study demonstrated that the T. catappa L. hydrophilic extract exerts protective effects on UVB-induced photoaging by inhibiting MMPs expression and upregulating type I procollagen expression [22]. However, the activity and related mechanisms of T. catappa against oxidative stress-induced skin damaging are unclear. Therefore, this study investigated the effects of T. catappa methanolic extract (TCE) on H 2 O 2 -induced skin damage and on the protein expression of MAPKs, which activate protein-1 (AP-1), MMPs, and type I procollagen in human skin fibroblasts (Hs68).

Preparation and quantitation of TCE
T. catappa leaves were collected in Wufeng, Taichung City, Taiwan, as previously described [22]. The leaves were identified by Professor KC Wen, a professor in Department of Cosmeceutics, China Medical University and a voucher specimen of this material (FCRDSAL-Plants-0003) has been deposited in Functional Cosmeceutics Research & Development and Safety Assessment Laboratory, China Medical University, Taiwan. The dried leaves (150 g) were ground and then extracted twice with 2 L of methanol for 1 h by using ultrasonication. The extraction liquid was filtrated, and the filtrate was evaporated to dryness in a vacuum to obtain TCE.
The total phenolic content of TCE was measured using the Folin-Ciocalteu reaction, as previously described [23]. Briefly, TCE was mixed with the Folin-Ciocalteu phenol reagent and sodium carbonate, and absorbance was measured at 760 nm. The phenolic content is expressed as microgram GAE/microgram T. catappa leaf dry weight herein.
The total flavonoid content of TCE was determined using the aluminum chloride colorimetric assay, as described elsewhere [23]. Briefly, TCE was mixed with aluminum chloride hexahydrate, potassium acetate, and deionized water, and the absorbance of the mixture was measured at 405 nm on an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Grödig, Austria). The flavonoid content is expressed as microgram QE/microgram T. catappa leaf dry weight herein.
DPPH radical scavenging activity assay DPPH was mixed with various concentrations of TCE. The mixture was added to a 96-well microplate and incubated at room temperature for 30 min in the dark. Subsequently, absorbance was measured at 492 nm on the ELISA reader. Ascorbic acid was used as a positive control [24,25].

Superoxide anion radical scavenging activity assay
Dihydronicotinamide-adenine dinucleotide, phenazinemethosulfate, and nitroblue tetrazolium were prepared in 0.1 M phosphate buffered saline (PBS), after which TCE was added. Absorbance was measured at 560 nm on the ELISA reader.

Determination of peroxide scavenging activity
The peroxide scavenging activity of TCE was spectrophotometrically detected using a previously described method [23,26]. H 2 O 2 was prepared in PBS and mixed with various concentrations of TCE. Then, after incubation, absorption was measured at 230 nm on the ELISA reader.

Hydroxyl radical scavenging activity assay
The hydroxyl radical scavenging activity assay was performed by mixing TCE, ascorbic acid, deoxyribose, iron (III) chloride, EDTA, H 2 O 2 , a monopotassium phosphate-potassium hydroxide buffer, and distilled water; the mixture was then incubated at 100°C for 15 min and centrifuged. The absorbance of the supernatant was subsequently measured at 532 nm on a microplate reader (BioTek, Winooski, VT, USA). Mannitol was used as a positive control, and the hydroxyl radical scavenging activity of TCE was obtained as the percentage inhibition of deoxyribose degradation [3,27].

Ferrous ion chelating activity assay
Various concentrations of TCE were mixed with an iron (II) chloride solution. The reaction was initiated after ferrozine was added. Absorbance was then spectrophotometrically measured at 562 nm on the microplate reader. The results are expressed as the percentage inhibition of the generation of the ferrozine-ferrous complex herein [24].

Measurement of reducing power
The reducing power of TCE was determined using a previously described method [24,28]. Various concentrations of TCE were mixed with ferrocyanate and trichloroacetic acid. After centrifugation, the supernatant was mixed with ferric chloride and absorbance was measured at 700 nm. Ascorbic acid and distilled water were used as the positive and negative controls, respectively.

Cell cultures
Hs68, HaCaT cells, and B16F0 cells were purchased from the Bioresource Collection and Research Center in Hsinchu, Taiwan. These cells were maintained in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in an incubator set at 37°C.

Cell viability assay for three skin cell lines
To understand the cytotoxicity of TCE on the skin, Hs68, HaCaT cells, and B16F0 cells were applied to study the cell viability. The cells were seeded in the plate, allowed to attach overnight, and were treated with 1 mL of various concentrations of TCE dissolved in DMEM for 24 h. The cytotoxicity of TCE was then evaluated using the MTT assay, as described elsewhere [22].

Fluorescence assay for IntracellularROS generation in fibroblasts
Intracellular ROS generation was measured using a previously detailed method [22]. In brief, fibroblasts were added to a 24-well plate and then incubated with various concentrations of TCE for 24 h. The cells were washed with PBS and incubated with 150 μM H 2 O 2 for 1 h. Subsequently, the cells were incubated with 10 μM DCFDA in DMEM for 30 min, after which they were examined under a fluorescence microscope (Leica DMIL, Wetzlar, Germany). Fluorescence (emission wavelength: 520 nm; excitation wavelength: 488 nm) was measured on a microplate reader (Thermo Electron Corporation, Vantaa, Finland).

Western blotting
The cells were incubated with TCE (5-50 μg/mL) for 4 h, followed by incubation with 150 μM H 2 O 2 for 1 h. The cells were collected and lysed with protein extraction buffer, as previously described [22]. An equal amount of protein was loaded, separated on 10% sodium dodecyl sulfate polyacrylamide gels, and then electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was incubated with specific antibodies against MMP-1, − 3, and − 9; type I procollagen; HO-1; MAPKs; c-Jun; c-Fos; and COX-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The blots were then incubated with anti-immunoglobulin G-horseradish peroxidase and chemiluminescent detection reagent (Amersham Biosciences, Buckinghamshire, United Kingdom). Finally, immunoreactive bands were detected using a chemiluminescent detection system (LAS-4000, Fujifilm, Tokyo, Japan), and the density of the bands was determined using a densitometric program (Multi Gauge V2.2, Fujifilm, Tokyo, Japan).

Statistical analyses
Values are presented as the mean ± standard deviation of at least three independent experiments. The results were analyzed using one-way analysis of variance, followed by Scheffe's test. Statistical significance was set at p < 0.05.

Extraction yield and quantitation of TCE
The extraction yield of TCE from leaves was 11.5%. The total phenolic content of the extract was determined using the Folin-Ciocalteu method, and the regression coefficient of the calibration curve was 0.9995. Specifically, the total phenolic content of TCE was 220.2 ± 0.2 μg/mg gallic acid equivalent (GAE). Additionally, the total flavonoid content of TCE was determined using the aluminum chloride colorimetric method, and the regression coefficient of the calibration curve was 0.9991. The total flavonoid content was 109.0 ± 0.8 μg/mg quercetin equivalent (QE). The content of gallic acid was 74.62 μg/mL by HPLC/UV analysis (Additional file 1: S1).
The 2′,7′-dichlorofluorescin diacetate (DCFDA) fluorescence assay was used to qualitatively characterize intracellular ROS generation. As shown in Fig. 2c, ROS levels were markedly higher in H 2 O 2 -exposed fibroblasts than in control cells. Moreover, this increase in ROS generation was attenuated in H 2 O 2 -exposed fibroblasts pretreated with various concentrations of TCE (5-50 μg/mL). ROS generation in H 2 O 2 -exposed fibroblasts increased to 1.7-fold compared with control cells, and significantly decreased to 1.3-fold compared with control cells. TCE at 50 μg/mL decreased H 2 O 2 -induced intracellular ROS generation by 23.1%. Thus, TCE protects the skin from ROS damage.

Inhibition of MAPK phosphorylation through TCE
As shown in Fig. 3a, H 2 O 2 induced the phosphorylation of p38, extracellular signal-regulated kinase (ERK), and Fig. 2 The cytotoxicity and effect on intracellular oxidative stress in Hs68 of TCE. a Cell viability (%) of human fibroblasts (Hs68), human keratinocytes, and mouse melanoma cells treated with TCE; b Cell viability of Hs68 after treatment with TCE with or without 150 μM hydrogen peroxide (H 2 O 2 ) exposure. c Repressive effect of TCE on H 2 O 2 -induced intracellular oxidative stress in Hs68. Significant difference versus control: *p < 0.05; **p < 0.01; ***p < 0.001. Significant inhibition versus H 2 O 2 -exposed group: # p < 0.05; ## p < 0.01 c-Jun N-terminal kinase (JNK). TCE (5-50 μg/mL) dose-dependently inhibited the phosphorylation of ERK, and the effect was significant in the cells treated with > 20 μg/mL TCE. Similar to the effect on ERK, TCE inhibited JNK and p38 activation, which were significantly suppressed when TCE concentration was 20 μg/mL.

TCE inhibited phosphorylation of AP-1 in Hs68
As shown in Fig. 3b, H 2 O 2 increased c-Jun and p-c-Jun expression to 1.5-and 2.9-fold that of the control, respectively, whereas > 5 μg/mL TCE significantly reduced the effect. In addition, H 2 O 2 induced c-Fos expression, but TCE reduced the effect. These results further indicated that TCE protects the skin from oxidative stress-induced damage.

Effect of TCE on H 2 O 2 -induced Hemeoxygenase-1 expression
The hemeoxygenase (HO)-1 gene and protein play a pivotal role in the modulation of antioxidant, anti-inflammatory, and antiapoptotic activities. This study revealed that H 2 O 2 significantly reduces HO-1 protein expression in Hs68, whereas TCE treatment dose-dependently increases HO-1 expression (Fig. 3d).

Effect of TCE on H 2 O 2 -induced Cyclooxygenase-2 expression in Hs68
Cyclooxygenase (COX)-2 levels were 1.3-fold higher in fibroblasts exposed to 150 μM H 2 O 2 than in control cells (Fig. 3e). In addition, various concentrations of TCE (5-50 μg/mL) reduced COX-2 expression; the effect was significant in the cells treated with > 5 μg/mL TCE. These results further confirmed that TCE protects the skin from damage by inhibiting inflammation.

Reversal of H 2 O 2 -induced upregulation of type I procollagen expression in Hs68 through TCE
After treatment with 150 μM H 2 O 2 , the expression of type I procollagen increased to 1.3-fold compared with that in control cells, whereas TCE inhibited this effect (Fig. 3f ). Notably, treatment of the cells with 50 μg/mL TCE decreased type I procollagen expression to a level similarly expressed in the control cells.

Discussion
Polyphenols are the second most abundant metabolic products in plants. Notably, plants with high polyphenolic content exhibit potent antioxidant activity [29]. Free radical scavenging activity is related to the polyphenic and flavonoid content of plants. In a previous study, the total phenol content of Rosa hemisphaerica was 138.3 μg/mg GAE [30]. In the present study, the total phenolic content was 220.2 μg/mg GAE dry leaves, the total flavonoid content was 109.0 μg/mg QE dry leaves, and the IC 50 of TCE for DPPH radical scavenging was 5.6 μg/mL. In addition, TCE exhibited strong scavenging activity for ROS including superoxide, peroxide, and hydroxyl radicals. Peroxide is the primary product of initial oxidation, and it can react with ferrous ions, producing more toxic hydroxyl radicals. Iron also has high reactivity and is the pivotal factor in lipid peroxidation catalyzed by transition metals [3]. Furthermore, TCE exhibits potent metal chelating activity and reducing power attenuating features; in the present study, TCE attenuated H 2 O 2 -induced metal chelation, reducing power, ROS generation, and free radical scavenging. Our results suggest that the high polyphenic and flavonoid content of TCE contribute to it potent antioxidant activity.
Molecules such as glutathione, catalase, and HO-1 provide cells, and the body overall, with defense systems against intrinsic and extrinsic oxidative stress. Nuclear factor E2-related factor 2 (Nrf2) and Keap1 are redox-sensitive transcription factors and key intracellular modulators of antioxidant defense against environmental stresses. For example, Nrf2 has been reported to protect skin cells from UV-and pollutant-induced oxidative damage and cellular dysfunction [31]. On exposure to oxidative stress, Nrf2 is translocated to the cell nucleus and binds to antioxidant elements, activating phase II detoxification enzymes such as HO-1 and glutathione [32]. In the present study, H 2 O 2 was found to reduce HO-1 expression; however, TCE treatment increased HO-1 expression, alleviating H 2 O 2 -induced oxidative stress in the skin cells. In other words, TCE may repair or protect skin from the damage caused by superoxide peroxide and hydroxyl radicals.
Exposure of the skin to UV induces ROS generation and regulates the expression of genes and proteins, resulting in photodamage and photocarcinogenesis [7]. In addition, H 2 O 2 has been reported to cause skin aging by inducing oxidative stress and MMP expression [33], while UVB-induced ROS generation triggers ERK, JNK, and p38 phosphorylation, AP-1 activation, and MMP expression, leading to collagen degradation [7]. In addition, H 2 O 2 disrupts transforming growth factor beta transduction and subsequently inhibits collagen biosynthesis, inducing skin aging [8]. In the present study, H 2 O 2 was determined to upregulate the phosphorylation of MAPKs, c-Jun, c-Fos, and MMP-1, − 3, and − 9 proteins, whereas TCE inhibited these effects. This finding suggests that TCE activity is dependent on this signaling transduction. MMPs mediate degradation of ECM and play an important role in tissue homeostasis and remodeling including angiogenesis and tissue repair. Over suppression of MMPs may cause abnormal accumulation of ECM.
The results are consistent with those of our previous study, in which the T. catappa water extract protected skin from photodamage by inhibiting the MAPK/AP-1/MMP pathway [22]. UVB rays inhibited collagen synthesis and induced collagen degradation, whereas T. catappa water extract elevated the collagen content in Hs68 [22]. Similarly, in the present study, H 2 O 2 was found to increase the collagen content in Hs68, whereas TCE reversed the effect. One previous study showed that H 2 O 2 also reduces mRNA expression of type I collagen (COL1A1) in fibroblasts [34], although these results are inconsistent with those reported elsewhere. For example, researchers demonstrated that H 2 O 2 induces oxidative stress damage to cells and the body, which can trigger the repair process of the skin, thereby increasing the collagen content. However, excessive collagen synthesis may cause collagen fibrosis and scleroderma [35]. Overall, our results here indicate that TCE can regulate the collagen content within a normal range.

Conclusion
The present study indicated that TCE with high polyphenic and flavonoid content exhibits potent free radical scavenging and antioxidant activity. Specifically, we determined that TCE protects against H 2 O 2 -induced skin damage by inhibiting the protein expression of MMP, AP-1, MAPKs, and COX-2 (Fig. 4). The antioxidant and antiaging activities of TCE make it suitable for application in skin care products.

Additional file
Additional file 1: S1. The active components in Terminalia catappa L.