Protective Effect of Mangifera indica Linn., Cocos nucifera Linn., and Averrhoa carambola Linn. Extracts against Ultraviolet B-Induced Damage in Human Keratinocytes

This study was aimed at investigating the antioxidant activity of Mangifera indica Linn., Cocos nucifera Linn., and Averrhoa carambola Linn. and their biological effect on human keratinocytes affected by the ultraviolet B (UVB), a major cause of cell damage and skin cancer through induction of DNA damage, production of reactive oxygen species (ROS), and apoptosis. The richest antioxidant activity was found in ethanol fraction of M. indica (21.32 ± 0.66 mg QE/g dry weight), while the lowest one was found in aqueous fractions of M. indica and C. nucifera (1.76 ± 2.10 and 1.65 ± 0.38 mg QE/g dry weight, respectively). Ethanol and aqueous fractions of A. carambola (250 µg/mL) significantly reduced the number of apoptotic cells. The expression of cleaved caspase 3 in UVB-treated group was significantly greater than that in untreated group. Both fractions of A. carambola (50, 100, and 250 µg/mL) significantly decreased the expression of cleaved caspase 3. Regarding the induction of DNA repair, ethanol (100 and 250 µg/mL) and aqueous (50, 100 and 250 µg/mL) fractions of A. carambola significantly decreased the percentage of cyclobutane pyrimidine dimers (CPD). Taken together, our results suggest that both fractions of A. carambola may be potentially developed for dermal applications.


Introduction
Ultraviolet B (UVB) is a well-known risk factor playing a role in photoaging and skin cancer in epidermis through triggering DNA damage or generating reactive oxygen species (ROS). ROS are chemically reactive molecules containing oxygen and play the important roles in cell signaling and homeostasis. In environmental stress such as UV or heat exposure, ROS levels can dramatically increase causing cell structures and DNA damage and apoptosis [1,2]. Prevention of UVB-induced damage in skin by lowering ROS production is an evidence-based strategy against photoaging and skin cancer.
Thailand is rich in fruits that are not only diversified but also inexpensive and delicious. Unfortunately, there have been a few researches with evidence-based findings that demonstrate the health benefits of these fruits. For example, resveratrol mostly found in grapes and red wine could exert photoprotective properties on UVB-irradiated cells. To reduce cell death in UVB-damaged skin, resveratrol reduced the production of ROS and attenuated the activation of caspase 3 and caspase 8 that play a major role in apoptosis [3]. Moreover, the extracts of Elaeocarpus hygrophilus (makoknum) and Phyllanthus emblica (makampom) had high antimicrobial and strong antioxidant activities [4].
Caspase 3 is an effector caspase protein frequently activated in mammalian cell apoptosis [5][6][7]. It is associated with the initiation of the death cascade. Pathways to caspase 3 activation are either extrinsic or intrinsic apoptotic pathways by interacting with caspase 8 and caspase 9, respectively. Besides apoptotic pathway, caspase 3 is essential for cell survival that converges on many events such as cell shrinkage, blebbing, chromatin condensation, and DNA fragmentation [7][8][9][10]. The other pathway through which UVB damages cells is DNA damage. UV induction of DNA damage is a factor that influences the normal life process of all organisms. Minor DNA damage is to allow effective repair, while more severe damage can induce apoptosis and cell cycle arrest. There are two types of UVB-induced DNA damage such as cyclobutane pyrimidine dimers (CPD) and pyrimidine  pyrimidone photoproducts . Two results (CPDs and 6-4PPs) are the transition of C to T and CC to TT. CPD contains a fourmembered ring which is from the coupling of the double bonds (C=C) of pyrimidines. CPD is the major source of UVinduced mutations because these dimers interfere with base pairing during DNA replication. CPD is usually at a 5 to 10 folding higher frequency than 6-4PPs. In minor DNA damage, CPD is repaired by exogenous CPD photolyase [1,2,11].
Previous study provided the evidence to support the protective effect of Thailand native herb extracts on UVBinduced toxicity in human keratinocyte. It found that the extracts of turmeric and ginger could protect human keratinocyte from UVB-induced DNA damage and apoptosis through the attenuation of caspase 3 activity and CPD formation [12].
This objective of this study was to evaluate the protective effect of three Thai fruit species, Mangifera indica Linn., Cocos nucifera Linn., and Averrhoa carambola Linn., on UVBinduced damage in human keratinocytes. All fruits selected in this investigation were evaluated for their antioxidant activities as potential mechanisms for antiapoptotic activity and induction of DNA repair in human keratinocyte cell line (HaCaT). Development of natural products for dermal applications is our future goal based on the findings of this work.
In traditional medicine, M. indica was used to clear digestion and acidity. It is antidiuretic, antidiarrheal, antiemetic, and cardiac herb. Its fruits are known as a potential source of natural antioxidants containing phenolic compounds, ascorbate, and -carotene [13].
The aqueous extract of C. nucifera was found to contain a free amino acid, L-arginine, which reduced the free radical generation. Moreover, vitamin C significantly reduced lipid peroxidation and increased antioxidant enzymes. C. nucifera could reduce lipid peroxidation content due to the high content of L-arginine. Besides, the high content of polyphenol could maintain the normal levels of lipid in tissue and serum. The aqueous extract of C. nucifera may be a new source of antineoplastic and antimultidrug resistance activities [14].
A. carambola or star fruits contain high polyphenol contents which were contributed significantly in ferric reducing capacity and radical scavenging capacity. Their antioxidant capacities were significantly increased with ripening and associated with flavonol, flavones, and hydrolysable tannins [15].

Chemicals and Reagents.
All reagents used in this study were of analytical grade. Dimethyl sulfoxide (DMSO) and ethanol were purchased from Merck (Darmstadt, Germany). 1,4-Dithiothreitol (DTT) was purchased from Bio Basic Inc. (Ontario, Canada). Phenylmethyl sulphonyl fluoride (PMSF) was purchased from United States Biochemicals (Cleveland, OH, USA). Kodak processing chemicals for autoradiography films, Amersham ECL Select Western blotting detection reagent, and Hyperfilm ECL were purchased from GE Healthcare (Piscataway, NJ, USA). Dulbecco's modified Eagle medium (DMEM)/high glucose were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and penicillin-streptomycin solution (10,000 units/mL of penicillin and 10,000 g/mL of streptomycin) were purchased from HyClone (Logan, UT, USA).

Cell Line.
HaCaT cells, an immortalized human epidermal keratinocyte cell line, were purchased from cell line service (Heidelberg, Germany). They were cultured in DMEM/high glucose containing 10% FBS and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at 37 ∘ C in a humidified atmosphere at 5% CO 2 .

Plant Materials.
Thai fruits were collected from Pathumthani and Nakornpathom provinces. They were authenticated based on their characteristics by Professor Dr. Thaweesakdi Boonkerd (Department of Botany, Faculty of Science, Chulalongkorn University). The voucher specimens deposited at Professor Kasin Suvatabhandhu Herbarium (Department of Botany, Faculty of Science, Chulalongkorn University) were A015246 (BCU), A015247 (BCU), and A015251 (BCU) for A. carambola, M. indica, and C. nucifera, respectively.

Thai Fruit Extraction.
The dried fruits were extracted by maceration method using absolute ethanol (ratio 1 : 2) at 4 ∘ C for 48 h and filtered. For extraction using water, the mixture (ratio 1 : 2) was incubated at 100 ∘ C for 30 min and filtered. The residues were extracted twice. The two filtrates were combined and concentrated by evaporation at 45 ∘ C. The crude extracts were dissolved in DMSO or kept at −80 ∘ C until further investigation.

Antioxidant Determination by Folin Ciocalteu Phenol
Assay and Total Flavonoid of Determination

Fixation and Denaturation.
Media were removed and 100 L of 75% methanol/25% acetic acid was added. Cells were incubated at room temperature for 30 min. Wells were aspirated and 100 L of 70% ethanol was added and then they were incubated at room temperature for 30 min. Wells were aspirated and 100 L of Denaturation Solution A was added and then they were incubated at room temperature for 5 min. Cells were gently washed with 200 L of Dulbecco's Phosphate-Buffered Saline (DPBS) containing magnesium and calcium. After aspirating wells, 100 L of Denaturation Solution B was added and then they were incubated at room temperature for 10 min. Wells were aspirated and 200 L of Assay Diluent was added and they were incubated at room temperature for 30 min.

CPD
Detection. 100 L of diluted anti-CPD antibody was added to wells and they were incubated at room temperature for 1 hour on the orbital shaker. Wells were washed with 1x wash buffer.

Statistical
Analysis. Data were presented as the mean ± standard error (SD). Means were from three or more independent experiments. Data were analyzed by one-way analysis of variance (one-way ANOVA), followed by post hoc Dunnett's test ( value < 0.05).

Total Phenol and Flavonoid Contents of M. indica, C. nucifera, and A. carambola Extracts.
Results of phenol and flavonoid of M. indica, C. nucifera, and A. carambola extracts were shown in Table 1. In all assays, the richest antioxidant activity was found in ethanol fraction of M. indica (21.32 ± 0.66 mg QE/g dry weight by total flavonoid determination). The lowest antioxidant activities were found in aqueous fractions of both M. indica and C. nucifera (1.76 ± 2.10 and 1.65 ± 0.38 mg QE/g dry weight, resp.).

The Effect of of M. indica, C. nucifera, and A. carambola
Extracts on Cell Viability. To evaluate the effect of Thai fruit extracts on HaCaT cell viability, MTT assay was employed. Cell was treated with the different concentrations of extracts (0-500 g/mL). Results of cytotoxicity of all extracts were shown in Figure 1. The aqueous extract of A. carambola at the concentration of 500 g/mL could significantly decrease cell viability (73.42% ± 3.66, < 0.05). Therefore, three concentrations of all extracts used in this study were 50, 100, and 250 g/mL.

The Effect of UVB Intensity on Cell Viability.
Evaluating the effect of UVB intensity on cell viability was employed by Trypan blue assay. Cells were treated with different intensities of UVB (0-1,600 mJ/cm 2 ). The lowest intensity that could significantly decrease cell viability was 200 mJ/cm 2 . Results of the effect of all intensity of UVB on cell viability were shown in Figure 2.

The Protective Effect of All Fractions of M. indica, C. nucifera, and A. carambola Extracts on UVB-Induced
Apoptosis by Flow Cytometry. Results of the protective effect of all fractions on UVB-induced apoptosis were shown in Figures 3(a) and 3(b). Ethanol and aqueous fractions of A. carambola (250 g/mL) could significantly decrease the

The Effect of M. indica, C. nucifera, and A. carambola Extracts on Caspase 3 Expression by Western Blot.
Since caspase 3 plays a major role in caspase-dependent apoptosis, the effect of Thai fruit extracts on the reduction of cleaved caspase 3 expression was investigated in this investigation. Using Western blot analysis, the aqueous extract (Figure 4(a)) Evidence-Based Complementary and Alternative Medicine

Discussion
UVB is a major cause of cell damage and skin cancer through inducing DNA damage and apoptosis. There are two pathways that decrease UVB-induced cell damage such as antiapoptosis and DNA damage repair. The effect of these extracts on apoptosis was detected by flow cytometry and Western blot analysis.
According to the flow cytometric data, the percentage of apoptotic cells in the untreated group was significantly different from that in the UVB-treated group, suggesting that UVB at 200 mJ/cm 2 could lead to the increase in apoptotic cells. In addition, both ethanol and aqueous extracts of A. carambola at the concentration of 250 g/mL could significantly decrease the percentage of the number of apoptotic cells ( < 0.05).
To confirm the effect of both extracts on protecting UVinduced apoptosis, the expression of caspase 3 was detected by Western blot. Many studies indicated that caspase 3 (35 kDa) in UVB-treated cells was cleaved. Cleaved caspase 3 (17 and 19 kDa) is an important factor which plays a role in the induction of cell apoptosis through apoptotic pathway [7,[16][17][18][19][20][21].
It was recently reported that vitamin C exerted antiapoptotic activity by attenuating caspase 3 expression [22,23]. Therefore, vitamin C was used as a control in this study. Results of the expression of cleaved caspase 3 decreasing in both ethanol and aqueous fractions of A. carambola-treated and vitamin C-treated cells implied that the attenuation of cleaved caspase 3 was involved in cell survival after UVB irradiation [20,24,25]. The results showed that the level of CPD expression was increased when treated with UVB. After UVB radiation, CPD level in A. carambola-treated group was significantly decreased ( < 0.05). The extract of A. carambola has been used in the traditional medicine for treating many diseases such as diabetes and diabetic nephropathy. Many studies indicated that it could inhibit apoptotic pathway by attenuating the activation of caspase 3, caspase 8, and caspase 9 [26,27]. Level of active caspase 3 can affect the formation of DNA fragmentation, since caspase 3 is a primary activator which induces the cleavage of DNA fragmentation factor (DFF) complex. Cleaved DFF causes DNA damage and cell death [9,28]. To date, there are not many studies to investigate the effect of A. carambola extract on DNA damage and cytotoxicity.
Collectively, our results showed that both ethanol and aqueous fractions of A. carambola could attenuate UVBinduced damage in human keratinocytes by inhibiting the cleavage of caspase 3 and CPD formation in the HaCaT keratinocyte cell line. The present study is the first to provide the evidence of potent protective effect of A. carambola extract against ultraviolet B-induced damage in human keratinocytes. The extracts of A. carambola may be developed as the agent for the protection of UVB-induced damage in skin.