Inhibitory Effects of Terminalia catappa on UVB-Induced Photodamage in Fibroblast Cell Line

This study investigated whether Terminalia catappa L. hydrophilic extract (TCLW) prevents photoaging in human dermal fibroblasts after exposure to UVB radiation. TCLW exhibited DPPH free radical scavenging activity and protected erythrocytes against AAPH-induced hemolysis. In the gelatin digestion assay, the rates of collagenase inhibition by TCL methanol extract, TCLW, and its hydrolysates were greater than 100% at the concentration of 1 mg/mL. We found that serial dilutions of TCLW (10–500 μg/mL) inhibited collagenase activity in a dose-dependent manner (82.3% to 101.0%). However, TCLW did not significantly inhibit elastase activity. In addition, TCLW inhibited MMP-1 and MMP-9 protein expression at a concentration of 25 μg/mL and inhibited MMP-3 protein expression at a concentration of 50 μg/mL. TCLW also promoted the protein expression of type I procollagen. We also found that TCLW attenuated the expression of MMP-1, -3, and -9 by inhibiting the phosphorylation of ERK, JNK, and p38. These findings suggest that TCLW increases the production of type I procollagen by inhibiting the activity of MMP-1, -3 and -9, and, therefore, has potential use in anti-aging cosmetics.


Introduction
The leaves of Terminalia catappa L. (Combretaceae) are commonly used as folk medicine in Southeast Asia to treat dermatitis and hepatitis [1]. Extracts of the leaves and bark of the plant have been reported to have chemopreventive [2,3], antioxidant, and superoxide radical scavenger effects [4], anti-HIV reverse transcriptase activity [5], and hepatoprotective [4] and anti-inflammatory effects [6]. Terminalia catappa extract has also been shown to decrease the protein expression of MMP-2 and MMP-9 in lung cancer cells [3]. The phytochemicals in this plant include tannins (punicalagin, punicalin, terflavins A and B, chebulagic acid), flavanoids (isovitexin, vitexin, isoorientin, rutin) and triterpenoids (ursolic acid and asiatic acid) [7][8][9]. The hepatoprotective activity of Terminalia catappa extract has been shown to be due primarily to the superoxide anion and hydroxyl radical scavenging activity of ursolic acid and asiatic acid [10].
Wrinkles, laxity, and hyperpigmentation characterize aging [11]. Skin aging can be divided into two basic processes, intrinsic, and extrinsic aging. Extrinsic aging is generally referred to as photoaging due to chronic exposure to short wavelength UV light (UVB) and is characterized by severe wrinkling and pigmentary changes, such as solar lentigo and mottled pigmentation on exposed areas such as the face, neck, and forearm. The most abundant structural protein in skin connective tissue is type I collagen, which is synthesized primarily by fibroblasts and is responsible for conferring strength and resiliency [12]. Ultraviolet-(UV-) induced skin damage principally manifests as degradation of extracellular matrix (ECM) proteins, including type I collagen, elastin, proteoglycans, and fibronectin [13,14]. It has been shown that UV irradiation leads to the formation of reactive oxygen species (ROS) that activate the mitogenactivated protein (MAP) kinase pathway, which subsequently induces the expression and activation of matrix metalloproteinases (MMPs) in human skin in vivo [15,16]. MMPs are known to be upexpressed in human fibroblasts within hours after exposure to UV irradiation and are, therefore, considered key factors in the photoaging process. Therefore, agents with the ability to elevate ECM protein levels or 2 Evidence-Based Complementary and Alternative Medicine inhibit the major collagen-degrading enzymes like MMPs would prove to be useful in the development of effective antiaging agents.
This study investigated the effects of Terminalia catappa extract and its hydrolysates on protein expression of MMPs, elastase, and type I procollagen in human dermal fibroblasts after exposure to UVB and investigated the mechanism by which the extract protects against photodamage.

Preparation of Terminalia catappa L. Extract and Its
Hydrolysates. The dried leaves were ground and then extracted twice with a 30-fold volume of methanol for 1 h by ultrasonication. The supernatant was filtered and then evaporated to dryness in vaucum to obtain methanol extract of Terminalia catappa L. (TCLM). The hydrophilic extract of Terminalia catappa L. (TCLW) was prepared in the same manner as TCLM except that the solvent was replaced with water. Acid hydrolysis of TCLW was carried out at 80 • C in the presence of 1.2 N HCl (2 mL) for 30 min and 2.4 N HCl (2 mL) for 60 min. After hydrolysis, the solution was partitioned with ethyl acetate (EA). The EA layer was evaporated to dryness in vaucum to obtain TCLW hydrolysate (TCLWH). The abbreviations used for and the hydrolytic conditions of TCLWH are as follows: TCLWH1, 0.6 N HCl for 0.5 h; TCLWH2, 1.2 N HCl for 0.5 h; TCLWH3, 0.6 N HCl for 1 h; TCLWH4, 1.2 N HCl for 1 h. The TCLW extract and TCLW hydrolysates were stored at −20 • C before use.

Total Phenolic Content of TCLW.
Total phenolic content of TCLW was determined by the Folin-Ciocalteu reaction. TCLW was mixed with 2-fold 10% Folin-Ciocalteu phenol reagent. The mixture was allowed to stand at room temperature for 5 min and then sodium carbonate (700 mM) was added to the mixture. The resulting blue complex was then measured at 760 nm. Gallic acid was used as a standard for the calibration curve. The phenolic compound contents were calibrated using the linear equation based on the calibration curve. The contents of phenolic compounds are expressed as μg gallic acid equivalent/mg TCL dry weight.

DPPH Radical Scavenging Activity of TCLW.
Reaction mixtures containing 200 μM DPPH (100 μL) and serial dilutions of sample (concentration of sample ranging from 25 to 1000 μg/mL) were placed in a 96-well microplate at room temperature in the dark for 30 min. After incubation, the absorbance was read at 517 nm by an ELISA reader (Tecan, Grodig, Austria). Ascorbic acid was used as the positive control. Scavenging activity was determined by the following equation: Scavenging effect % capacity to scavenging the DPPH radical (1)

Preparation of Erythrocyte Suspensions and Hemolysis
Assay. Whole blood was obtained from male SD rats via cardiopuncture and collected in an EDTA-containing tube. The erythrocytes were isolated by centrifugation at 3000 × g for 10 min, washed four times with PBS, and then resuspended to the desired hematocrit level using the same buffer. In order to induce free radical chain oxidation in the erythrocytes, aqueous peroxyl radicals were generated by thermal decomposition of AAPH in oxygen. An erythrocyte suspension at 5% hematocrit was incubated with PBS (control) or preincubated with TCLW (10-50 μg/mL) at 37 • C for 30 min, followed by incubation with or without 25 mM AAPH in PBS at pH 7.4. This reaction mixture was shaken gently while being incubated for a fixed interval at 37 • C. A 200-μL aliquot of the reaction mixture was removed and centrifuged at 3000 ×g for 2 min, with absorbance of the supernatant determined at 540 nm. Reference values were determined using the same volume of erythrocytes in a hypotonic buffer (5 mM phosphate buffer at pH 7.4; 100% hemolysis). The hemolysis percentage was calculated using the formula:

Gelatin Digestion Assay.
Agarose solution (1%) was prepared in collagenase buffer (50 mM Tris-HCl, 10 mM CaCl 2 , 0.15 M NaCl, pH 7.8) with 0.15% porcine gelatin (Sigma Aldrich, Cat. G-2500) and allowed to solidify on plates. Various concentrations of TCLW and TCLWH (10 μL) dissolved in 10% DMSO were incubated with 10 μL of bacterial collagenase-1 (0.1 mg/mL) in 80 μL of collagenase buffer for 1 h at room temperature. Doxycycline hyclate was used as positive control. The samples (40 μL) were loaded onto paper disks placed on gelatin-agarose gel and incubated for 18 h at 37 • C. The degree of gelatin digestion in agarose gel was visualized by Coomassie Blue staining after removal of the paper disks. Following destaining, the area of the light translucent zone over blue background was determined by a densitometric program (TINA) to estimate gelatinase activity.

MMP Activity Assays.
Enzyme activity assays were performed in 50 mM tris buffer (pH 7.8), 0.15 M NaCl, and 10 mM CaCl 2 . Various concentrations of TCLW and TCLWH were tested for their ability to digest a synthetic fluorogenic substrate (a general MMP substrate). Each concentration of TCLW and TCLWH was incubated with 1 μM substrate at 37 • C for 20 h. Fluorescence intensity was measured at 320 nm (excitation) and 450 nm (emission) with a fluorescence reader. The rate of collagenase inhibition was calculated by the following equation: where A indicates the absorbance with enzyme but without sample, B indicates the absorbance without enzyme and sample, C indicates the absorbance with enzyme and sample,  and D indicates the absorbance without enzyme but with sample.

Measurement of Elastase Activity.
The elastase inhibition test on TCLW and TCLWH was investigated using elastase derived from porcine pancreas. Elastase (500 U) was dissolved in 5 mL of 10 mM tris buffer solution (pH 6.0) and 5 mg elastase substrate IV was dissolved in 5 mL of 100 mM tris buffer solution (pH 8.0). To measure elastase activity, 100 μL of 100 mM tris buffer solution (pH 8.0), 25 μL of elastase substrate IV solution, 50 μL of sample solution, and 25 μL of elastase solution were dispensed into each well of a 96-well plate and then preincubated for 20 min at room temperature. The elastase activity was quantified by measuring light absorbance at 405 nm using a microplate reader (Tecan, Grodig, Austria). Each assay was carried out in triplicate. The inhibition rate of elastase was calculated by the following equation: where A indicates the absorbance with enzyme but without sample, B indicates the absorbance without enzyme and sample, C indicates the absorbance with enzyme and sample, and D indicates the absorbance without enzyme but with sample.
2.9. Cell Culture. Human foreskin fibroblasts (Hs68) were obtained from neonatal foreskins and cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified  atmosphere of 5% CO 2 at 37 • C. Hs68 cells were plated at 80%-90% confluence in all experiments. was assessed using the MTT assay as previously described [17]. Viability was quantified by measuring the absorbance at 570 nm using a microplate reader (Tecan, Grodig, Austria).
2.11. UV Irradiation. Cells were cultured until 80% confluent, washed twice with phosphate-buffered saline (PBS), and then exposed to UVB irradiation in PBS (302 nm, CL-1000M, UVP, USA). In our previous study, the dose of  80 mJ/cm 2 UVB irradiation was determined to induce MMP without being cytotoxic (data not shown). Subsequently, cells were incubated for 24 h in 37 • C in a humidified atmosphere of 5% CO 2 in serum-free DMEM containing various concentrations of TCLW and TCLWH.

Statistical Analysis.
Each experiment was performed in triplicate and all data are presented as mean ± SD. Significant differences between groups were analyzed by  ANOVA followed by the Scheffe's test. A P value <.05 was considered significant.

Extraction Yield and Total Phenolic Content of TCLW.
The extraction yield of TCLW was 22.5% and that of TCLM was 13.5%. The total phenolic content, expressed as μg gallic acid equivalents (GAE) per mg of dry weight (TCL), was 102.0 ± 0.2 μg GAE/mg. Figure 1 shows the free radical scavenging activity of TCLW (25-1000 μg/mL) and ascorbic acid (25 mg/mL); TCLW exhibited excellent DPPH radical scavenging activity. Our results indicated that DPPH radical scavenging activity of TCLW at 25 μg/mL (95.8 ± 0.3%) was similar to that of ascorbic acid at an equal concentration (96.2 ± 0.2%).

Erythrocyte Hemolysis
Assay. The influence of TCLW (10-500 μg/mL) on in vitro erythrocyte hemolysis was examined by incubating rat erythrocytes in the presence of 25 mM AAPH as an initiator of oxidation. TCLW (50-500 μg/mL) exhibited a strong dose-dependent inhibitory effect against erythrocyte hemolysis (Figure 2).

TCLW and TCLWH Inhibited Bacterial Collagenase-1.
For visual investigation of the inhibitory effect of TCLM, TCLW, and TCLWH on MMPs, an indirect assay was developed using bacterial collagenase-1. Following incubation of bacterial collagenase-1 with various concentrations of TCLW and TCLWH, the inhibition of enzyme activity was compared with enzyme activity of the control. As Figure 3(a) shown, DMSO as the control group reaction with the reagent exhibited the highest gelatinolytic activity in the discrete zone; the doxycycline, a well-known MMP inhibitor, was the positive control resulting clear zone. The collagenase-1 inhibition of TCLW and TCLM was similar to doxycycline. As shown in Figure 3(b), the rates of collagenase-1 inhibition  were 94.3 ± 0.8% for doxycycline (100 μg/mL), 100.7 ± 0.9% for TCLW (1000 μg/mL), 101.4 ± 0.5% for TCLWH1, 102.2 ± 0.4% for TCLWH2, 105.8 ± 3.4% for TCLWH3, and 106.7 ± 3.5% for TCLWH4. The rates for collagenase-1 inhibition for various concentrations (50-1000 μg/mL) of TCLW ranged from 81.9 ± 0.7% to 100.7 ± 0.7%. In addition, TCLW inhibited gelatinolytic activity in a dose-dependent manner (Figure 3(c)).

TCLW Inhibited Bacterial
Collagenase-1. Fluorescenceconjugated gelatin was used to measure the inhibitory effect of TCLW on bacterial collagenase-1 protein expression. Fluorescence-conjugated substrate was incubated with bacterial collagenase-1 for 20 h in the presence of different concentrations of TCLW or doxycycline hyclate (positive control) at 37 • C. TCLW exhibited a significant inhibitory effect on bacterial collagenase-1; the inhibition rate was >95% of the control at concentrations ≥50 μg/mL (Figure 4).

The Effect of TCLW on Elastase
Activity. This assay measures the synthesis and activity of elastase in cells exposed to TCLW. As shown in Figure 5, TCLW (10-500 μg/mL) did not have a significant effect on elastase activity.   of 50 μg/mL and higher significantly inhibited MMP-3 expression ( Figure 6).

Effect of TCLW on MAP Kinase
Expression. UVB irradiation of untreated Hs68 cells resulted in a 1.4-fold increase in p-ERK expression, a 1.2-fold increase in p-JNK expression, and a 1.5-fold increase in phosphorylated p38 relative to control levels. TCLW treatment (5-100 μg/mL), however, significantly inhibited the UVB-induced overexpression of those MAP kinases. P-ERK was suppressed to the basal level at 10 μg/mL TCLW, p-JNK was suppressed to the basal level at 25 μg/mL TCLW, and phosphorylated p38 was suppressed to the basal level at 5 μg/mL TCLW (Figure 7).

Effect of TCLW on Cell Viability.
Hs68 cells were treated with various concentrations of TCLW (5-200 μg/mL) and cell viability was measured. As shown in Figure 8, TCLW did not exhibit cytotoxic effects on the proliferation of cells (cell viability >90% of control). In addition, TCLW promoted cell proliferation in a dose-dependent manner.

Discussion
Aging is a complex phenomenon that is modulated by multiple factors, including genetics, life style, and exposure to sunlight and pollutants. UV radiation produces ROS, which activate MAPkinase pathway to induce the expression and activation of MMPs that degrade extracellular matrix proteins including type I collagen, elastin, and glycosaminoglycans in skin [18,19].
It has been shown that Moringa oleifera extract, which has a total phenolic content of 52.5 μg GAE/mg, has potent DPPH scavenging activity [20]. In addition, Fraxinus chinensis extract scavenges UVA-induced DPPH free radicals and inhibits MMP-1 mRNA and protein expression in human skin fibroblasts [21]. Sorbus commixta Hedl, a compound with a high total phenolic content, exhibits free radical scavenging effect and decreases MMP-1 mRNA expression [22]. Terminalia catappa L. leaves are rich in polyphenol antioxidants. In addition, studies have shown that the antioxidant activities of polyphenols reduce the risk of skin diseases [23]. We speculate that TCLW can protect against oxidative stress-induced photodamage because of its high total phenolic content (102 μg GAE/mg), DPPH free radical scavenging activity, as well as its ability to prevent AAPHinduced hemolysis and activate MMP.
MAP kinases are upstream regulators of MMP activity [34][35][36]. It has been reported that green tea polyphenol, epigallocatechin-3-gallate (EGCG) inhibits the expression of MMPs by attenuating the phosphorylation of ASK-1 and MAPkinase pathway [37]. In addition, treatment of UVirradiated cells with 4-hydroxypanduratin A, isolated from Kaempferia pandurata Roxb, leads to decreased MMP-1 expression by inhibiting c-Fos and c-Jun phosphorylation and MAP kinase pathway activation [38]. In this study, TCLW significantly inhibited UVB-induced p-ERK, p-JNK, and phosphorylated p38 expression at doses of 10, 25, and 5 μg/mL, respectively. It has been shown that UV irradiation induces p38 activation [34]. Furthermore, it has been reported that UVB irradiation induces ERK, JNK, and p38 activation via production of ROS [39,40]. Green tea polyphenol, EGCG, suppresses UV-induced oxygen stress-mediated MAP kinase phosphorylation pathway [41]. The flavonoid, fisetin inhibits UV-induced MAP kinase expression and NF-κB signal transduction [42,43]. In our study, TCLW exhibited potent antioxidant activity, inhibited the expression of MAP kinases and MMPs, and activated type I procollagen. Those findings indicate that TCLW is a potential agent for the treatment or prevention of photodamage. TCLW hampered the activation of MAPKs, and, therefore, may downregulate the down stream proteins c-Fos and c-Jun, as well as the transcription factor AP-1 and protooncogenes involved in photodamage ( Figure 9). The effects of TCLW on transcription of c-Fos, c-Jun, and AP-1 need further study.