Background
Reactive oxygen species (ROS) are produced in oxidation processes essential to most living organisms and are essential to produce the energy required to fuel other biological processes. However, excessive production of ROS is damaging to cells because ROS destroy molecules such as DNA and proteins. Thus, ROS play an important role in the pathogenesis of various serious diseases, such as neurodegenerative disorders, cancer, cardiovascular diseases, atherosclerosis, cataracts, and inflammation [
1‐
5]. The mechanism of inflammation injury partially involves the release of ROS from activated neutrophils and macrophages. ROS propagate inflammation by stimulating the release of cytokines such as interleukin-1, tumor necrosis factor-α, and interferon-α, which stimulate recruitment of additional neutrophils and macrophages. Free radicals are important mediators that provoke or sustain inflammatory processes, and consequently, their neutralization by antioxidants and radical scavengers can attenuate inflammation [
6,
7]. Therefore, compounds that have scavenging activities toward these radicals could have therapeutic potential.
There are two methods of suppressing ROS. First, natural defense mechanisms present in the human/mammalian system counteract the potential deleterious effects of ROS. Normally, cells have antioxidant systems that protect against the harmful effects of ROS, including superoxide dismutase (SOD), which converts superoxide anions to hydrogen peroxide (H
2O
2) for rapid removal by detoxifying enzymes such as glutathione peroxidase (GPx). Similarly, glutathione (GSH) can reduce ROS for GPx-catalyzed H
2O
2 reductions [
8,
9]. Second, functional components from the external environment such as flavonoids, L-ascorbic acid (Vit C), and α-tocopherol (Vit E), act as antioxidants [
10,
11]. Therefore, a diet rich in antioxidants could help the body defend itself against the molecular effects of free radicals and ROS and hinder the development of many chronic diseases. Numerous studies have shown that natural dietary compounds can potently modulate oxidative stress. Plant phenolics, flavonoids, tannins, and anthocyanins have useful properties such as antioxidant, immune, and anticancer activities. The presence of these antioxidants increases the efficacy of the protection system against ROS [
12‐
14].
Cornus officinalis (Cornaceae) is a deciduous tree native to eastern Asia (e.g., Korea, China, and Japan). The fruit of
C. officinalis, known as “Sansuyu” in Korean, is mainly harvested in the central and southern regions of Korea [
15].
C. officinalis is often included in traditional treatments for conditions such as backache, polyuria, hypertension, and nervous breakdown [
16]. Pharmacological studies have demonstrated that
C. officinalis possesses antioxidant [
17], antihyperglycemic [
18], immune regulatory [
19] and anti-inflammatory effects [
20].
Furthermore, many functional compounds such as ursolic acid, tartaric acid, malic acid, glucosides, and fatty acids are present in the fruit. Several studies have also reported that these compounds have antioxidant and anti-inflammatory effects [
21‐
24].
Reports on the antioxidant activity of C. officinalis have been restricted to in vitro radical scavenging studies. Its mechanism of action within the cell at the genetic level has not yet been clearly defined. Therefore, the aims of this study were to identify the effect of C. officinalis on antioxidant activity, inhibition of ROS production, and antioxidant-related gene expression in RAW 264.7 cells (murine macrophage cell line). This study suggests that the ethanol extract of C. officinalis could be used as a natural source of antioxidants in the food and pharmaceutical industries.
Methods
Reagents
Folin-Denis reagent, sodium carbonate, aluminum chloride, potassium acetate, potassium persulfate, 1,1-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinibis 3-ethyl benzothiazoline-6-sulfonic acid (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate, gallic acid, acetic acid, lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ascorbic acid (Vit C), and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iron (II) sulfate heptahydrate (FeSO4) was purchased from Junsei (Tokyo, Japan). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin (P/S), and trypsin-EDTA were obtained from Gibco (Waltham, MA, USA). The xanthine oxidase (XO) assay kit was purchased from Abcam (Cambridge, MA, USA). The other reagents used were of analytical grade.
Sample preparation and extraction
C. officianalis was purchased from Korea Medicine Herbal Association, which is under the jurisdiction of the Ministry of Agriculture, Food and Rural Affairs (Seoul, Korea). The plant was identified and authenticated by the Korea Medicine Herbal Association. Voucher specimens (NAAS-15-03) were deposited at the Department of Agrofood Resources Herbarium, National Academy of Agricultural Science, Korea. C. officianalis (20 g) was extracted twice with 70 % ethanol at 70 °C for 6 h. The 70 % ethanol extract was filtered using filter paper (Advantec, Tokyo, Japan). Subsequently, the filtrates were combined and evaporated under vacuum (EYELA CCA-1110, Tokyo Rikakikai Co., Tokyo, Japan) and then lyophilized with a freeze dryer (Ilshine Lab, Suwon, Korea) at −70 °C under reduced pressure (<20 Pa). The dry residue was stored at −70 °C. For further analysis, the dry extract was reconstituted with dimethyl sulfoxide (DMSO).
Total phenolic content
The total phenol content of
C. officianalis extract was determined by the Folin-Ciocalteau method [
25]. The extract was oxidized with Folin-Ciocalteau reagents, and then the reaction was neutralized with saturated sodium carbonate. After incubation at room temperature for 1 h, the absorbance of the reaction mixture was measured at 725 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The total phenolic content is expressed as gallic acid equivalents in milligrams per gram (mg GAE/g) of dry extract.
Total flavonoid content
A sample solution was mixed with 100 % ethanol, 10 % aluminum chloride, 1 M potassium acetate, and distilled water. The reagents were thoroughly mixed and allowed to stand for 40 min at room temperature, and the absorbance of the supernatant was measured at 415 nm [
26]. Quercetin was used to plot a standard calibration curve, and the results are expressed as quercetin equivalents in milligram per gram (mg QE/g) of dried extract.
DPPH radical-scavenging activity
The DPPH radical-scavenging activity was carried out according to the Blois method [
27]. DPPH (0.3 mM) was added to each sample. After incubation for 30 min in the dark at room temperature, the absorbance was measured at 518 nm using a microplate reader. Vit C was used as a positive control. The free radical-scavenging capacity was expressed by IC
50.
ABTS radical cation-scavenging activity
The ABTS assay was performed to evaluate the ability of the
C. officianalis extract to scavenge the ABTS radical cation in comparison to that of a standard (Vit C) [
28]. The radical cation was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate (1:1 v/v) and leaving the mixture for 24 h until the reaction was completed and the absorbance was stable. The ABTS radical solution was diluted with PBS to an absorbance of 0.7 (±0.02) at 732 nm. The assay was conducted with diluted ABTS radical solution mixed with samples, and the measurements were taken at 734 nm after 30 min. The antioxidative activity of the samples was calculated by determining the decrease in absorbance. The free radical-scavenging capacity was expressed by IC
50.
Ferric-reducing antioxidant power (FRAP) activity
FRAP activity was determined using manual assay methods [
29]. The working fluid was freshly prepared by mixing acetate buffer (300 mM, pH 3.6) with 10 mM TPTZ in HCl and 20 mM iron (III) chloride hexahydrate. The sample solution or Vit C was added to working fluid, and the mixture was left for 4 min at room temperature. The absorbance was measured at 593 nm. The results are expressed as FeSO
4 equivalents.
Cells and culture
RAW 264.7 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). The cell lines were grown in DMEM with 10 % FBS and 1 % P/S, and incubated at 37 °C in 5 % CO2.
Cell cytotoxicity assay
RAW 264.7 cells were plated at 1 × 104 cells/well. The C. officianalis ethanol extract in DMSO was diluted in PBS to obtain final concentrations of 10, 50, and 100 μg/mL. Cells were treated with samples for 24 h and MTT solution was added. After 4 h, the media were removed and DMSO was added to each well. The resulting absorbance was measured at 540 nm.
Xanthine oxidase inhibitory activity assay
XO inhibitory activity was assayed using a commercial xanthine oxidase assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Briefly, RAW 264.7 cells were plated at 5 × 105 cells/well. After 4 h, the cells were treated with 10, 50, and 100 μg/mL of C. officianalis for 24 h. The treated cell pellets were mixed with assay buffer and the supernatants were isolated. The working solutions were added to the supernatants and incubated at 37 °C. After 1 h, the absorbance was measured at 570 nm.
Intracellular reactive oxygen species scavenging activity
RAW 264.7 cells were plated at 1 × 106 cells/well. After 4 h, the cells were treated with 10, 50, and 100 μg/mL C. officianalis and LPS for 24 h. After incubation, the cells were washed with PBS and harvested. The cells were then incubated with dichlorofluorescein diacetate (DCF-DA) (25 μM) for 30 min at 37 °C in the dark. After several washings with PBS, the fluorescence was captured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). DCF fluorescence was measured at an excitation wavelength of 488 nm and emission wavelength of 515–540 nm.
Real-time reverse transcription polymerase chain analysis
To determine the expression levels of Cu/Zn SOD, Mn SOD, catalase, and GPx, real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using a real-time thermal cycler Qiagen Rotorgene Q (Valencia, CA, USA), in accordance with the manufacturer’s instructions. The cells were treated with C. officianalis extracts and cultured for 24 h. Thereafter, cDNA was synthesized from the total RNA isolated from cells. The PCR reaction was performed using the 2X SYBR Green mix (Qiagen, Valencia, CA, USA). All results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The following primer sequences were used for real-time RT-PCR: GAPDH, 5′-GAG CCA AAA GGG TCA TCA TC-3′ (forward), 5′-TAA GCA GTT GGT GGT GCA GG-3′ (reverse); Cu/Zn SOD, 5′-CAG CAT GGG TTC CAC GTC CA-3′ (forward), 5′-CAC ATT GGC CAC ACC GTC CT-3′ (reverse); Mn SOD, 5′-GGG TTG GCT TGG TTT CAA TAA GGA A-3′ (forward), 5′-AGG TAG TAA GCG TGC TCC CAC ACA T-3′ (reverse); catalase, 5′-AAG ACA ATG TCA CTC AGG TGC GGA-3′ (forward), 5′-GGC AAT GTT CTC ACA CAG GCG TTT-3′ (reverse); and GPx, 5′-CTC GGT TTC CCG TGC AAT CAG-3′ (forward), 5′-GTG CAG CCA GTA ATC ACC AAG-3′ (reverse).
Statistical analysis
Statistical analysis was performed using SPSS (version 17.0; SPSS Inc., Chicago, IL, USA). Descriptive statistics were used to calculate the mean and standard error of the mean (SEM). One-way analysis of variance was performed, and when significance (p < 0.05) was found, the differences of mean values were identified with Duncan’s multiple range tests.
Abbreviations
ABTS, 2,2’-azinibis 3-ethyl benzothiazoline-6-sulfonic acid; DCFH-DA, dichlorofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DPPH, 1,1-diphenyl-1-picrylhydrazyl; FBS, fetal bovine serum; FeSO4, sulfate heptahydrate; GAE, gallic acid equivalent; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPx, glutathione peroxidase; LPS, lipopolysaccharides; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; P/S, penicillin-streptomycin; QE, quercetin equivalent; ROS, reactive oxygen species; RT-PCR, real-time reverse transcription polymerase chain reaction; SEM, standard error of the mean; SOD, superoxide dismutase; TPTZ, 2,4,6-tris(2-pyridyl)-s-triazine; XO, xanthine oxidase.
Acknowledgements
This study was supported by the “Research Program for Agricultural Science & Technology Development (Project No. PJ010946),” National Academy of Agricultural Science, Rural Development Administration, Korea.