Taxifolin Potently Diminishes Levels of Reactive Oxygen Species in Living Cells Possibly by Scavenging Peroxyl Radicals

Corresponding Author: Yohko Fujimoto Department of Physiological Chemistry, Osaka University of Pharmaceutical Sciences, 4-201 Nasahara, Takatsuki, Osaka 569-1094, Japan Tell: +81 726 9


Introduction
Excessive amounts of Reactive Oxygen Species (ROS) contribute to the aging process and the development of various diseases such as atherosclerosis, diabetes, cancer, neurodegenerative diseases and liver cirrhosis (Basaga, 1990). To prevent the damages caused by ROS, organisms have developed an antioxidant defense system that includes non-enzymatic antioxidants and enzymes such as superoxide dismutase, catalase and glutathione peroxidase (Sorg, 2004). A second level of prevention against ROS-induced damage is constituted by other scavenging compounds present in the diet, especially from medicinal plants (Valko et al., 2007;Xia et al., 2010;Jorge et al., 2016).
From the perspective of free radical biology, plants encounter serious oxidative stress from strong UV-Vis light, atmospheric ROS, temperature changes and the processes of oxygen consumption for photosynthesis. Flavonoids are a main class of phenolic compounds and secondary plant metabolites generally located in leaves as water soluble glycosides in the vacuoles of plant cells (Bimpilas et al., 2015). Flavonoids are not only present in plants as constitutive agents but they also accumulate in plant tissues in response to microbial attack (Harborne and Williams, 2000).
Taxifolin is a dihydroflavonol that is abundant in citrus fruits and onions. Taxifolin is arguably the most interesting and promising component of dietary supplements or antioxidant-rich functional foods in the last two decades (Rice Evans et al., 1996;Topal et al., 2016;Li et al., 2017). Importantly, taxifolin exerts significant antioxidant effects that are critical in preventing the onset of apoptosis (Vladimirov et al., 2009). Moreover, taxifolin has been found to inhibit oxidative enzymes and the overproduction of ROS, thus ameliorating cerebral ischemia-reperfusion injury (Voulgari et al., 2010).
Until now, there has been little information concerning the antioxidant potency of taxifolin as a flavonoid. Therefore, in the present study, the efficacy of taxifolin to reduce ROS levels in living cells was compared with that of catechin, which is a well-known, potent radical scavenger (Harborne and Williams, 2000; 2 Bimpilas et al., 2015), by using flow cytometry with the oxidation-sensitive fluorescent dye 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA). The ability of taxifolin to scavenge peroxyl radicals (ROO·) by use of an Electron Spin Resonance (ESR) spin-trapping technique were also compared with that of catechin.

Measurement of ROS in Caco-2 Cells
Measurements of ROS in Caco-2 cells were performed by the method previously reported (Kohda et al., 2016). Caco-2 cells were purchased from the European Collection of Cell Cultures (Salisbury, Wilts, UK) and cultured in Minimum Essential Medium (Life Technologies Corporation) supplemented with 10% fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan) and 1% non-essential amino acids (Life Technologies Corporation). The cells were maintained in a humidified atmosphere of 5% carbon dioxide at 37°C.
The measurement of ROS was performed by flow cytometry with DCFH-DA. The cells (1.0×10 6 cells/28 cm 2 dish) were incubated with the test reagents for 6 h and then DCFH-DA was added at a final concentration of 10 µM. After incubating for 30 min, the cells were collected by centrifugation (4°C and 200 × g for 5 min) and washed twice. The samples were filtered through a nylon mesh (37 µm) and subjected to flow cytometry (FACSAria™ III flow cytometer, Becton Dickinson, Basel, Switzerland).

ESR Measurement
An ESR spectrometer, TE-2100 (JEOL, Tokyo, Japan) and a JEOL flat quartz cell were used. The conditions were: field, 336±5 mT; power, 4 mW; field modulation, 0.200 mT; time constant, 0.1; and amplitude, 300. A manganese signal was used for the external standard.

Observation of POBN-Signal Adducts Reflecting t-BOO·
The Ce 4+ /t-BOOH reaction was started by adding t-BOOH (final concentration, 0.4 M) to a mixture of POBN (final concentration, 10 mM) and Ce(SO 4 ) 2 ·4H 2 O (final concentration, 0.2 mM) in 0.1 M sodium phosphate buffer (pH 7.4) in a total volume of 0.5 mL. The POBN-signal adducts reflecting t-BOO· were measured 1 min after the addition of t-BOOH.

Statistical Analysis
The results are expressed as means ± standard errors of the mean. Significant differences between two groups were assessed using t-tests, whereas differences between multiple groups were assessed by one-way analysis of variance, followed by Scheffé's multiple comparison tests. P-values less than 0.05 were considered statistically significant. Figure 1A shows the effects of t-BOOH with or without taxifolin on the intracellular ROS generation of Caco-2 cells, measured by flow cytometry with the redox-sensitive fluorescent dye, DCFH-DA. The addition of t-BOOH (50 µM) to the Caco-2 cells shifted the Mean Fluorescence Intensity (MFI, dashed line) to the right, which indicates an increase in ROS levels measured by the DCF fluorescence. The increment in MFI induced by t-BOOH was reduced by the addition of 50 µM taxifolin. Figure 2B summarizes the MFI data measured by the method in Fig. 1A. The addition of NAC, which is often used as an antioxidant in cell experiments (Lasram et al., 2015), diminished the t-BOOH-induced increase in MFI (2 mM, 50% inhibition). Fifty micromolar of both taxifolin (61% inhibition) and catechin (39% inhibition), but not chrysin, reduced the t-BOOH-induced increase in MFI. The taxifolin effect was stronger than that of catechin and their difference was statistically significant. This means that taxifolin reduces the t-BOOH-induced increase in ROS levels in living cells stronger than catechin does.

Taxifolin Quenches t-BOO·
A direct method for measuring free radicals in aqueous conditions is detection by ESR spectroscopy (Venkataraman et al., 2004). Figure 2A shows the ESR spectra of the spin signal adduct from t-BOO· [a(N) = 1.51 mT, a(H) = 0.23 mT] by the reaction of the Ce 4+ /t-BOOH system with POBN. The hyperfine fit parameters are identical to those previously reported (Panasenko et al., 2002;). An obvious quenching by 0.5 mM Trolox, which is known as a potent peroxyl radical scavenger 3 (Barclay et al., 1995;Stefek et al., 2005), supported the identity of the product. A positive correlation between the disappearance of the signal intensity of POBN signal adducts of t-BOO· and trolox concentrations was observed ( Supplementary Fig. 1). Figure 2A and Supplementary Fig. 1 collectively demonstrate that this experimental condition using the ESR apparatus can assess the diminishing efficacy of specific substances against t-BOO·. Figure 2B shows the effects of taxifolin, catechin and chrysin on the spin signal adduct of t-BOO· detected by the methods used in Fig. 2A. Both Taxifolin and catechin, but not chrysin, from 5 to 200 µM concentration-dependently diminished the signal adduct of t-BOO· [taxifolin, 34-90% inhibition; catechin, 3-82% inhibition). The scavenging effect of 200 µM taxifolin was stronger than that of 200 µM catechin and the difference was statistically significant. The results of Fig. 2 show that taxifolin diminishes t-BOO· and the t-BOO· scavenging efficacy seems to be somewhat greater than that of catechin.

Discussion
ROS contributes to the development of various diseases such as atherosclerosis, diabetes, cancer, neurodegenerative diseases and liver cirrhosis. It also contributes to the aging process (Basaga, 1990). The use of antioxidant compounds, such as radical scavengers, might prevent the development and progression of these diseases to maintain health.
As shown in Fig. 1, taxifolin suppressed t-BOOHinduced increases in ROS levels in Caco-2 cells and the effect of 200 µM taxifolin was significantly stronger than that of catechin. Based on an ESR technique, Fig. 2 showed that taxifolin could diminish ROO· and 200 µM taxifolin's effect was significantly stronger than that of catechin. Chrysin was without effects on both ROS levels in Caco-2 cells and ESR radical intensity of the spin signal adduct of t-BOO·. Both taxifolin and catechin have a catechol group in ring B and taxifolin possesses an additional 4-oxo group in ring C ( Supplementary Fig. 2). Thus, it is possible that the catechol group in ring B plays an important role for the ROO· scavenging activity and the 4-oxo group in ring C determines the strength of the ROO· scavenging capacity of taxifolin. The belief that the catechol group in ring B of flavonoids is the major structural feature imparting antioxidant activity has been supported by the work of Jovanovic et al. (1996). Topal et al. (2016) and Li et al. (2017) have reported that taxifolin is an effective antioxidant and antiradical by using indirect in vitro bioanalytical methods, including 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging, Fe 3+reducing and Cu 2+ -reducing assays. However, to our knowledge, there is no information concerning the types of ROS scavenged by taxifolin by using an ESR method. In the present study, we showed that taxifolin scavenged ROO·. Li et al. (2017) has reported that taxifolin is an effective scavenger of hydroxyl radical (·OH). The present study together with the report by Li et al. (2017) indicates that taxifolin can be a scavenger of both ROO· and ·OH.
Determining the significance of the present finding that taxifolin quenches t-BOO· will require further study. Additional studies are also needed to clarify the mechanism by which taxifolin scavenges ROO·. However, to the best of our knowledge, the results of this study show for the first time that taxifolin diminishes ROS levels in living cells possibly by scavenging ROO·. This provides new mechanistic insight into the preventive effects of taxifolin in various disorders.

Conclusion
Taxifolin significantly reduced t-BOOH-induced increases in ROS levels in Caco-2 cells and this effect was stronger than that of another flavonoid, catechin. Taxifolin also scavenged ROO· by using an ESR method. We believe that our study makes a significant contribution to the literature because, to the best of our knowledge, this is the first time that taxifolin has the potential to diminish ROS levels in living cells possibly by scavenging ROO·.

Authors Contributions
Satoru Sakuma: Participated in all experiments, coordinated the data-analysis and contributed to the writing of the manuscript.

Ethics
This article is original and contains unpublished material. The corresponding author confirms that all of the other authors have read and approved the manuscript and there are no ethical issues involved.
Supplementary Fig. 1: Effects of trolox on the amounts of spin signal adduct of t-BOO• generated by the chemical reaction system The radical intensity was defined as the ratio of the peak height of signal [indicated as an arrow in Fig. 2(A)] to that of Manganese (Mn). The data are presented as means ± standard errors of the mean (n = 3). α p < 0.05, β p < 0.01 vs. Control. Electron-spin resonance (ESR) coupled with spin-trapping technique used in this study is shown in Material and Methods and Results and Discussion sections in the manuscript.