Antioxidant Effects of the Ethanol Extract from Flower of Camellia japonica via Scavenging of Reactive Oxygen Species and Induction of Antioxidant Enzymes

The aim of this study was to investigate the antioxidant properties of the ethanol extract of the flower of Camellia japonica (Camellia extract). Camellia extract exhibited 1,1-diphenyl-2-picrylhydrazyl radical and intracellular reactive oxygen species (ROS) scavenging activity in human HaCaT keratinocytes. In addition, Camellia extract scavenged superoxide anion generated by xanthine/xanthine oxidase and hydroxyl radical generated by the Fenton reaction (FeSO4 + H2O2) in a cell-free system, which was detected by electron spin resonance spectrometry. Furthermore, Camellia extract increased the protein expressions and activity of cellular antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. These results suggest that Camellia extract exhibits antioxidant properties by scavenging ROS and enhancing antioxidant enzymes. Camellia extract contained quercetin, quercetin-3-O-glucoside, quercitrin and kaempferol, which are antioxidant compounds.


Introduction
Oxidative damage initiated by reactive oxygen species (ROS) is a major contributor to the aging process [1]. Skin is a major candidate and target of oxidative stress, and during the skin aging process, ROS levels rise and antioxidant defenses decline [2]. Due to the high prevalence of potential biological targets for oxidative damage, skin is very susceptible to such reactions. For example, skin is rich in lipids, proteins, and DNA, all of which are extremely sensitive to the oxidation process [3]. The antioxidant system is an important defense mechanism against oxidative cell damage [4]. The antioxidant system is consisted of intrinsic enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase, and extrinsic antioxidant nutrients, which serve to reduce free radicals to less toxic states [5]. Some studies suggest that age-related decrease in antioxidant enzyme activity is consistent with increased free radical damage that contributes to aging [6].
Camellia japonica is a broad-leaved evergreen woody species, widely distributed in Japan and Korea. The flowers and flower buds of Camellia japonica have been used for the treatment of bleeding and as an anti-inflammation in oriental traditional medicine [7]. Studies have been conducted on the constituents of Camellia japonica, including saponins in fruits [8] and seeds [9], flavonol glycosides in leaves [10], and triterpenes, flavonols and phenolic compounds in flowers [11,12]. However, the biological effects of C. japonica have been less frequently studied. In the present study, we focused on investigating the in vitro antioxidant effect of the ethanol extract from flower of Camellia extract in human keratinocytes.
We chose 50 µg/mL as the optimal dose of Camellia extract for further investigations. Control or Camellia extract at 50 µg/mL showed no specific signal of superoxide anion, while in the xanthine/xanthine oxidase system, superoxide anion signal increased to 3239. Camellia extract treatment decreased superoxide anion signal in the xanthine/xanthine oxidase system to 1875 (Figure 2A    Similarly, Camellia extract decreased generation of hydroxyl radical in the FeSO 4 + H 2 O 2 system from 3921 to 1533 ( Figures 3A and B). Taken together, these results suggest that Camellia extract can directly scavenge ROS.
In order to investigate whether the radical scavenging activity of Camellia extract was mediated by antioxidant enzyme activities, the protein expressions and activities of SOD, CAT and GPx in Camellia extract-treated cells were measured. As shown in Figure 4A

Discussion
The antioxidant properties of Camellia extract were evaluated in two categories: direct action on superoxide and hydroxyl radical scavenging in a cell-free system, and indirect action through induction of antioxidant enzymes. Camellia extract exerted direct scavenging activity on superoxide and hydroxyl radical as shown by ESR spectrometry. It was reported that the flower of C. japonica contained flavonols, such as quercetin, kaempferol, and sexangularetin, and phenolic compounds, such as p-hydroxybenzoic acid, protocatechuic acid and gallic acid [12]. The antioxidant compounds of Camellia extract in our study contained quercetin, quercetin-3-O-glucoside, quercitrin (a glycoside rhamnose of quercetin) and kaempferol. Quercetin exerts its antioxidant activity through scavenging ROS and preventing ROS formation with chelating transition metal ions, such as iron and copper [13,14]. Quercitrin has a free radical scavenging activity in a model of auto-oxidation of rat cerebral membranes [15]. Kaempferol has shown strong inhibitory/scavenging activity on ROS generation with numerous hydroxyl groups on their structures [16]. Moreover, it has been found to be a particularly potent blocker of extracellular ROS production, and to inhibit the ascorbate-dependent NADH oxidase and superoxide anion production activities of the neuronal plasma membrane redox chain [17]. Therefore, the ROS scavenging effect of Camellia extract demonstrated in this study might have been associated with flavonol or phenolic compounds.
Antioxidant enzymes, such as SOD, CAT, and GPx are the main antioxidant defense system. SOD plays an important role in scavenging superoxide anion which are formed during the early stages of oxidative stress, and in preventing aging [18]. SOD catalyzes the conversion of superoxide to hydrogen peroxide plus dioxygen. SOD can be classified into three groups, Cu/Zn SOD, Mn SOD, and Fe SOD, by the metals that they contain at their active sites. Cu/Zn SOD is usually found in the cytoplasm of eukaryotic cells and Mn SOD in mitochondria, whereas prokaryotic cells contain Fe SOD and Mn SOD [19]. Hydrogen peroxide is a harmful by-product of many normal metabolic processes. However, CAT and GPx are frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide [20]. CAT catalyzes the formation of water and oxygen from hydrogen peroxide and prevents oxidative damage [21]. GPx is a well-known selenoenzyme that functions as an antioxidant, and catalyzes the reduction of hydroperoxides, including hydrogen peroxides, by reduced glutathione and protect cells from oxidative damage [22].
Quercetin has been reported to reduce or prevent oxidative stresses induced by ultraviolet A through activation of antioxidant enzymes and induction of their expressions [23]. Also, quercetin has antioxidant and cytoprotective properties against renal ischemia-reperfusion injury by inducing SOD, CAT, GPx expression and activating their activities [24]. Kaempferol enhances the antioxidant properties by raised antioxidant proteins expression, such as metallothionein, CAT, and SOD [25]. Moreover, kaempferol inhibits H 2 O 2 -induced lipid peroxidation and enhances the activity of SOD and CAT [26,27]. Quercitrin enhances intracellular antioxidant defense against free radicals by increasing production of antioxidant enzymes [28]. Camellia extract increased the activity and protein levels of these enzymes, which may mediate its inhibition of ROS production. Thus, Camellia extract not only directly quenches ROS, but also indirectly induces antioxidant enzymes. In conclusion, Camellia extract exerts in vitro its antioxidant properties by scavenging ROS and enhancing antioxidant enzymes activities ( Figure 6).

Cell Viability Assay
Cells were seeded in a 96 well plate at a concentration of 1  10 5 cells/mL, and after plating for 16 h, Camellia extract at 6.25, 12.5, 25, and 50 μg/mL was added to the plate. After incubating for 24 h at 37 C, fifty microliter of MTT stock solution (2 mg/mL) was added to each well of a total reaction volume of 200 L. After incubating for 4 h, the plate was centrifuged at 800  g for 5 min and the supernatants aspirated. The formazan crystals in each well were dissolved in 150 L dimethylsulfoxide and the absorbance at 540 nm read on a scanning multi-well spectrophotometer.

Intracellular ROS Scavenging Activity
The DCF-DA method was used to detect the intracellular ROS levels [29]. Cells were seeded 1.5 × 10 5 cells/well and 16 h after plating, cells were treated with Camellia extract at 6.25, 12.5, 25, and 50 μg/mL (10 μL). After 30 min, 1 mM of H 2 O 2 (10 μL) was added to the plate. Cells were incubated for an additional 30 min at 37 °C . After the addition of 25 µM of DCF-DA solution for 10 min, fluorescence of 2',7'-dichlorofluorescein was detected using a PerkinElmer LS-5B spectrofluorometer. Image analysis for the generation of intracellular ROS was achieved by seeding cells on a cover-slip loaded six well plate at 2  10 5 cells/well. Sixteen hours after plating, cells were treated with Camellia extract. One hour later, 1 mM of H 2 O 2 was added to the plate. Twenty four hours later, 100 M DCF-DA was added to each well and the cells were incubated for an additional 30 min at 37 C. After washing with phosphate buffered saline (PBS), the stained cells were mounted onto a microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Images were collected using the laser scanning microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) on a confocal microscope.

Detection of Superoxide Anion
Superoxide anion was generated by xanthine and xanthine oxidase, which were then quickly reacted with a nitrone spin trap, DMPO. The resultant DMPO/· OOH adducts were detected using an electron spin resonance (ESR) spectrometer. The ESR spectrum was recorded using JES-FA ESR spectrometer (JEOL, Tokyo, Japan), at 2.

Detection of Hydroxyl Radical
Hydroxyl radicals were generated by the Fenton reaction (H 2 O 2 +FeSO 4 ), which were then quickly reacted with DMPO. The resultant DMPO/ . OH adducts were detected using an ESR spectrometer. The ESR spectrum was recorded using JES-FA ESR spectrometer at 2.

Western Blot Analysis
Cells were lysed in 100 L of a lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40]. Aliquots of the lysates (40 g of protein) were boiled for 5 min and electrophoresed in 10% SDS-polyacrylamide gel. The blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and subsequently incubated with anti-primary antibodies. The membranes were further incubated with secondary anti-immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA), followed by exposure to X-ray film. The protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK).

Measurement of SOD Activity
Cells were seeded in a culture dish at 1 × 10 5 cells/mL, and were treated with Camellia extract at 50 µg/mL. After 1 h, 150 µM of H 2 O 2 was added to the plate, which was incubated for a further 24 h. The harvested cells were suspended in 10 mM phosphate buffer (pH 7.5) and then lysed on ice by sonicating twice for 15 sec. Triton X-100 (1%) was then added to the lysates and was incubated for 10 min on ice. The lysates were centrifuged at 5,000 × g for 10 min at 4 °C to remove the cellular debris. Fifty micrograms of protein was added to 500 mM phosphate buffer (pH 10.2) and 1 mM epinephrine. Epinephrine rapidly undergoes auto-oxidation at pH 10 to produce adrenochrome, a pink-colored product, which was assayed at 480 nm using an ultraviolet/visible spectrophotometer in kinetic mode. SOD inhibits auto-oxidation of epinephrine. The rate of inhibition was monitored at 480 nm and the amount of enzyme required to produce 50% inhibition was expressed as units/mg protein.

Measurement of CAT Activity
Fifty milligrams of protein was added to 50 mM phosphate buffer (pH 7) containing 100 mM H 2 O 2 . The reaction mixture was incubated for 2 min at 37 °C and the absorbance was monitored at 240 nm for 5 min. The change in absorbance with time was proportional to the breakdown of H 2 O 2 . The amount of enzyme required to breakdown 1 mM of H 2 O 2 was expressed as units/mg protein.

Measurement of GPx Activity
Fifty milligrams of protein was added to 25 mM of phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM NaN 3 , 1 mM glutathione, 0.25 units of glutathione reductase and 0.1 mM NADPH. After incubation for 10 min at 37 °C, H 2 O 2 was added to the reaction mixture at a final concentration of 1 mM. Absorbance was monitored at 340 nm for 5 min. GPx activity was measured as the rate of NADPH oxidation by change in absorbance at 340 nm. The amount of enzyme required to oxidize 1 mM NADPH was expressed as units/mg protein.

Statistical Analysis
All the measurements were made in triplicate and all values are represented as means ± standard error (SE). The results were subjected to an analysis of the variance (ANOVA) using the Tukey test to analyze the difference. The p < 0.05 was considered significant.