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Resveratrol, piperine and apigenin differ in their NADPH-oxidase inhibitory and reactive oxygen species-scavenging properties.

ABSTRACT

Background: Many plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants.

Hypothesis/Purpose: Here we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine.

Study Design: This work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA).

Results: Of the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine.

Conclusion: The results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds.

Keywords:

NADPH oxidase (NOX)

Antioxidant

Reactive oxygen species

Celastrol

Resveratrol

Introduction

Plant extracts have been recognized as having potential health benefits long before the chemical composition or underlying mechanisms were considered. For example, phytochemicals have been used in both traditional and conventional medicine for their antimalarial, anti-inflammatory, anti-infective, anti-tumour, analgesic, neuroprotective and other properties (Abad et al., 2012; Fusco and Giacovazzo, 1997; Negi et al., 2014; Sen and Samanta, 2015; Sun et al., 2008; Wang et al., 2012). The bioactive chemicals found in plant extracts can be classified into 4 main types: aliphatics, alkaloids, phenolics, and terpenoids (Ehrman et al., 2007), each of which can be further subdivided. Multiple mechanisms of action are frequently involved and the complete mechanism of action is often not completely understood (Ehrman et al., 2007). One of the activities commonly exhibited by bioactive compounds, in particular by phenolics, but also by alkaloids and terpenoids is "antioxidant" properties, and occasionally even "prooxidant" qualities (Dai and Mumper, 2010; Matsuura et al., 2014). Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide ([H.sub.2][O.sub.2]). are produced by cells as part of their normal physiological function. ROS generation has an important role in cellular functions, which includes the regulation of certain transcription factors, ion channels, and phosphatases (Sies, 2014; Weidinger and Kozlov, 2015). Cells also contain natural antioxidants and ROS-metabolizing enzymes to control ROS levels and to prevent ROS-induced cellular damage. However, when the amount of ROS generated is increased, or alternatively, when the capacity of the cell to eliminate ROS is decreased, there can be an excess of intracellular ROS leading to a condition referred to as oxidative stress. Excess ROS can lead to inappropriate cell signaling, damage to proteins, lipids and DNA, and ultimately to cell death (Weidinger and Kozlov, 2015). Altering ROS levels may be one of the mechanisms through which plant-derived bioactive compounds produce health benefits.

There are a number of sources of intracellular ROS (Sies, 2014). Members of the NADPH oxidase (NOX) family of enzymes represent a major contribution to ROS production. Other potential sources of ROS include the reaction between oxygen and electrons leaked from the electron transport chain of mitochondria, a by-product of protein folding in the endoplasmic reticulum and metabolic enzymes such as cytochrome P450, or nitric oxide synthetase under some conditions. A central molecule in ROS is the superoxide anion, which is generated when a single electron is transferred to dioxygen. Superoxide does not cross membranes readily; however, it is rapidly converted to [H.sub.2][O.sub.2], either spontaneously or catalyzed by cellular enzymes such as superoxide dismutase. Unlike superoxide, [H.sub.2][O.sub.2] readily crosses membranes, and its range of biological effects are far reaching. Importantly, [H.sub.2][O.sub.2] is able to act as a highly specific signaling molecule by reversibly binding to specific cysteine residues to modify the function of transcriptions factors, ion channels, phosphatases and other cell signaling targets (Sies, 2014). In addition to its role as a signaling molecule, [H.sub.2][O.sub.2] can also be harmful when irreversible oxidation of proteins, DNA and/or lipids occur. Although medicinal plants are often reported to have an "antioxidant" quality, determining whether the effect is obtained by decreasing the generation of ROS by inhibiting the enzymatic production of superoxide, scavenging the initial spatially restricted superoxide molecule, or scavenging the downstream and diffuse [H.sub.2][O.sub.2] molecule, is potentially of great significance.

Anti-cancer activity has been associated with a diverse range of phytochemicals including celastrol, a triterpene compound (Yang et al., 2006); resveratrol, a polyphenol (Jang et al., 1997); apigenin, a flavonoid (Choi et al., 2007); and piperine, an alkaloid (Srinivasan, 2007) (Fig. SI). Celastrol is a component of Tripterygium wilfordii, a plant used in traditional Chinese medicine in the treatment of immunological diseases (Wong et al., 2012). We have previously demonstrated that this compound acts not only as an antioxidant, but also blocks the production of ROS by the NADPH oxidase family of enzymes (Jaquet et al., 2011). Resveratrol is a constituent of red wine, and has been proposed to contribute to health benefits such as cardio-protective effects, anti-diabetic and anti-cancer activities through a range of mechanisms (Baur and Sinclair, 2006; Bhat and Pezzuto, 2002; Szkudelski and Szkudelska, 2011; Wu et al., 2011). There are mixed reports on whether resveratrol acts solely as a ROS scavenger (Derochette et al., 2013) or if it also has an impact on NOX activation (Chen et al., 2013; Gocmen et al., 2013). Apigenin is present in celery and parsley (Manach et al., 2004), and is also abundant in wheatgrass, which has been used in the treatment of cancer, rheumatoid arthritis, and ulcer (Singh et al., 2012). Apigenin has been reported to increase ROS generation (Choi et al., 2007; Harrison et al., 2014) but, conversely, has also been described to have antioxidant properties (Liao et al., 2014) and to increase the expression of antioxidant enzymes within cells (Jung, 2014). Piperine is a constituent of black pepper and has been explored for its potential benefit in a variety of cancers (Samykutty et al., 2013). Piperine has been found to be an antioxidant (Srinivasan, 2007), but part of its biological activity has also been attributed to its ability to promote the production of ROS (Yaffe et al., 2013).

In this study, we used the human lung carcinoma cell line H661 and Epstein-Barr virus (EBV)- transformed B-lymphoblastoid cell line (B-LCL) to investigate the impact of these four bioactive compounds on ROS generation by NOX, and the scavenging of superoxide and/or [H.sub.2][O.sub.2]. Of these, only celastrol inhibited NOX; however, both celastrol and resveratrol scavenged ROS. Apigenin, and piperine had very little impact on ROS.

Materials and methods

Cell lines

The human lung carcinoma cell line H661 (American Type Culture Collection, Manassas, VA) was selected because it contains little or no NADPH oxidase enzymes (von Lohneysen et al., 2008). To generate the S184 cell line functional NOX1 system, H661 cells were transduced using a lentiviral-based gene transfer with vectors encoding the genes for human NOX1 (vector contained green fluorescent protein (GFP) for selection), NOXOl (vector contained the zeocin resistance gene for selection), NOXA1 (the vector contained neomycin resistance gene for selection) and CYBA (the vector contained blasticidin resistance gene for selection). The cytomegalovirus (CMV) promoter was used to drive the expression of the gene of interest in each vector. Cells were selected with the appropriate selection antibiotics, and to isolate NOX1 positive cells, cells were plated at low density in a large plate and GFP positive clones were extracted from the dish.

B-lymphocytes were used as a model for the study of NOX2, as NOX2 has previously been demonstrated to be the main source of ROS in this cell type (Bedard et al., 2009). EBV-transformed Blymphoblastoid cell line (B-LCL) derived from a healthy individual were kindly provided by Dr Marie-Jose Stasia (University Joseph Fourier, Grenoble, France).

Amplex Red (AR) assay

[H.sub.2][O.sub.2] was measured with the Amplex Red[R] fluorescence assay, using the Infinite M200 Pro microplate reader (Tecan Group Ltd., Mannedorf, Switzerland), as previously described (Jaquet et al., 2011). Detached cells were placed in a 96-well plate at a density of 5 x [10.sup.4] in 200 [micro]l of Hank's balanced salt solution (HBSS) containing 25 [micro]M Amplex Red[TM] (Invitrogen Canada Inc., Burlington, ON) and 0.005 U/ml horse radish peroxidase (HRP)(Sigma-Aldrich Canada, Oakvilie, ON). Cells were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Canada), and inhibited with 10 [micro]M diphenyleneiodonium (DPI) (Sigma-Aldrich Canada). The [H.sub.2][O.sub.2] (Fisher Scientific Ltd., Napean, ON) standard curve ranged from 5000 nM to OnM. In order to ensure all superoxide was converted to [H.sub.2][O.sub.2], Cu/Zn SOD from bovine erythrocytes (100 U/well) (Sigma-Aldrich Canada) was added to the appropriate wells. To normalize [H.sub.2][O.sub.2] results to the number of viable cells, calcein (10 [micro]g/ml) (Invitrogen Canada Inc.) was added to cells, and to a serial dilution of control cells with cell densities ranging from 1 x [10.sup.6] to 0 cells per well. For direct scavenging assays, 625 nM of [H.sub.2][O.sub.2] in 200 [micro]l HBSS containing 25 [micro]M Amplex Red[TM] and 0.005 U/ml HRP was combined with various natural compounds at final concentrations ranging from 0.5 [micro]M to 100 [micro]M to determine their antioxidant properties. [H.sub.2][O.sub.2] level was determined by measuring the fluorescence every 2 min for 30 cycles at 37[degrees]C, with excitation and emission wavelengths of 544 nm and 590 nm, respectively. Where inhibition was complete, IC50 values were calculated on normalized data using Prism Graphpad. Curves were fit with the non-linear log(inhibitor) versus normalized response model, which constrains the Hill slope to 1, and the Y values to 100% maximum and zero % minimum. Diphenyleneiodonium (DPI) is a commonly used, nonspecific inhibitor of a wide variety of flavoprotein-dependent electron-transport systems, including NADPH oxidase, xanthine oxidase, nitric oxide synthetase, and those involved in the mitochondrial electron-transport chain (Aldieri et al., 2008; O'Donnell et al., 1993; Riganti et al., 2004). We chose DPI as a positive control due to its strong ability to prevent the formation of superoxide by NADPH oxidases. Trolox, a vitamin E analog, effectively scavenges a wide variety of free radicals via numerous mechanisms, such as hydrogen transfer, radical adduct formation, single electron transfer, and sequential proton loss electron transfer (Alberto et al., 2013). We used trolox as a positive control for scavenging of ROS.

Nitro blue tetrazolium (NBT) assay

Intracellular cellular superoxide generation was measured with the NBT absorbance assay, using the Infinite M200 Pro microplate reader. In a 100 [micro]l volume of HBSS, detached S184 cells or B-LCL were seeded at 4 x [10.sup.4] cells per well in a 96-well plate. Natural compounds, dissolved in dimethyl sulfoxide (DMSO) and diluted in phosphate-buffered saline (PBS), were added to the wells at final concentrations ranging from 0.5 [micro]M to 100 [micro]M. At the final concentrations, DMSO was less than 1% of the total solution volume. NBT (Invitrogen Canada) (0.66 mg/ml) and PMA (100 nM) were added to each well and the plate was placed in a sealed 37[degrees]C incubator for 3 h B-LCLs were pelleted by centrifugation at 2270 x g for 2 min. The well solutions were removed and the cells were washed twice with 100 [micro]l PBS. B-LCLs were centrifuged again for 2 min at 2270 x g between washes. The solution in each well was removed, and the cells were washed with 100 [micro]l methanol, then air dried for 5 min. Next, 112 [micro]l 2 M KOH, 96 [micro]l DMSO (99%) was added to each well, mixed thoroughly by micropipette, and read at 630 nm. Where inhibition was complete, IC50 values were calculated on normalized data using Prism Graphpad. Curves were fit with the non-linear log(inhibitor) versus normalized response model, which constrains the Hill slope to 1, and the Y values to 100% maximum and zero % minimum. For calculation of the amount of superoxide generated by control cells, the monoformazan molar extinction coefficient 15,000 [M.sup.-]1[cm.sup.-1] was used, with a path length of 0.6 cm (Stankovic et al., 2011).

Methyl cypridina luciferin analog (MCLA) assay

Superoxide generation was measured in a cell-free environment with the MCLA (Invitrogen Canada Inc.) chemiluminescence assay using the Infinite M200 Pro microplate reader, as previously described (Jaquet et al., 2011). The generation of superoxide by the xanthine/xanthine oxidase was performed in PBS supplemented with xanthine oxidase (0.1 U/ml). ethylenediaminetetraacetic acid (EDTA) (0.3 mM), MCLA (0.1 mM), xanthine (0.5 mM). Natural compounds at various final concentrations ranging from 0.5 [micro]M to 100 [micro]M were added to the wells. Cu/Zn SOD (100 U/ml) and DPI (10 [micro]M) were added as controls and for verification of xanthine oxidase function and superoxide production. Superoxide generation was determined by measuring the MCLA light emission every 90s for 30 cycles at 37[degrees]C with an integration time of 0.9 s. The antioxidant enzyme SOD was included as a positive control. SOD selectively converts superoxide to [H.sub.2][O.sub.2] (Abreu and Cabelli, 2010).

Results

Effect of plant-derived compounds celastrol, resveratrol, apigenin, and piperine on [H.sub.2][O.sub.2] in NOX1- and N0X2-expressing cells

H661 cells are a human lung cancer cell line selected for this study because they express little to no NADPH oxidase (von Lohneysen et al., 2008). H661 cells were therefore engineered to express NOX1, NOXOl, and NOXA1, as these cells lack endogenous CYBA ([p22.sup.phox]), the assembly of a functional NOX1 complex could only be achieved upon the addition of this subunit. A monoclonal line of H661 cells that expressed all four subunits of the NOX1 NADPH oxidase was selected and referred to as S184 cells. S184 cells produced [H.sub.2][O.sub.2] spontaneously, and production was further increased upon protein kinase C (PKC) activation by the addition of PMA (Fig. S2).

PMA-stimulated activation of NOX1 in S184 led to the generation of [H.sub.2][O.sub.2] at a rate of 35 pmol/h/[10.sup.4] (Fig. 1A). As expected (Jaquet et al., 2011), this increase in NOX1-derived [H.sub.2][O.sub.2] was fully inhibited in a dose-dependent manner by the addition of celastrol (IC50 3.0 [+ or -] 0.1 [micro]M). [H.sub.2][O.sub.2] accumulation was also blocked by resveratrol (IC50 25.7 [+ or -] 4.7 [micro]M) and was partially diminished by piperine. In the case of celastrol, we observed an unexpected large increase in Amplex Red response in the NOX1-expressing S184 cells at higher concentrations of celastrol tested.

PMA-stimulated activation of NOX2 in B-LCLs led to the generation of [H.sub.2][O.sub.2] at a rate of 220 pmol/h/[10.sup.4] cells (Fig. 1A), consistent with the range of ROS expected from B-LCL or neutrophils (Bedard et al., 2009; Mohanty et al., 1997). Again as expected (Jaquet et al., 2011), celastrol fully inhibited the accumulation of [H.sub.2][O.sub.2] generated by NOX2 (IC50 2.8 [+ or -] 0.5 [micro]M). Resveratrol was also able to decrease the accumulation of [H.sub.2][O.sub.2] generated by NOX2 (IC50 24 [+ or -] 18 [micro]M) (Fig. 1B). The unexpected increase in [H.sub.2][O.sub.2] generation by celastrol at high doses that was observed with the NOX1-expressing S184 cells was not observed in B-LCLs. In addition, piperine did not impact the rate of [H.sub.2][O.sub.2] generation in B-LCLs, and apigenin had no effect on PMA-stimulated [H.sub.2][O.sub.2] generation by NOX1 or NOX2. None of the plant derived chemicals displayed auto-fluorescence or interfered with the fluorescence generated by Amplex Red at 100 [micro]M (Fig. S3).

Effect of plant-derived compounds celastrol, resveratrol, apigenin, and piperine on superoxide levels in NOX1- and NOX2-expressing cells

Superoxide was measured by NBT following PMA-stimulated activation of NOX1 (S184 cells) or NOX2 (B-LCL). Out of the four compounds tested, only celastrol showed inhibition of superoxide generation (Fig. 2A and, B), and this was observed for both NOX1 (IC50 15.5 [+ or -] 7.5 [micro]M) and partial inhibition of NOX2. There was no increase in superoxide detected at the higher concentrations of celastrol in either cell line.

[H.sub.2][O.sub.2] scavenging by plant derived compounds celastrol, resveratrol, apigenin, and piperine

Many plant extracts are reported to exhibit antioxidant properties (Dai and Mumper, 2010; Matsuura et al., 2014). Here we set out to evaluate the ability of celastrol, resveratrol, apigenin, and piperine to effectively scavenge [H.sub.2][O.sub.2] and to characterize the concentration--response relationship of this activity. Both celastrol (IC50 16.0 [+ or -] 1.35 [micro]M) and resveratrol (IC50 4.4 [+ or -] 0.2 [micro]M) effectively scavenged [H.sub.2][O.sub.2] in a dose-dependent manner (Fig. 3). Although though there was some scavenging observed with the other compounds, the effect was less pronounced and did not show a strong concentration-response relationship. As a positive control, Trolox (10 pM) was evaluated and effectively scavenged [H.sub.2][O.sub.2]. This was confirmed to not be an effect of merely acting as an inhibitor of HRP (Fig. S4).

Superoxide scavenging by celastrol, resveratrol, apigenin, and pipeline

Both celastrol and resveratrol displayed some scavenging of superoxide at higher concentrations (Fig. 4). Under the same conditions, apigenin and piperine did not act as strong antioxidants against superoxide.

Evaluation of pro-oxidant activity of celastrol, resveratrol, apigenin, and piperine

Piperine has been reported to have pro-oxidant activity at high concentrations (Choi et al., 2007; Harrison et al., 2014; Khajuria et al., 1998; Yaffe et al., 2013). In order to investigate the potential pro-oxidant activity of celastrol, resveratrol, apigenin, and piperine, we exposed H661 cells, S184 (NOX1) cells and B-LCL (NOX2) cells to each of the natural compounds in the absence of PMA stimulation to determine if the compounds affected basal levels of ROS (Fig. 5). S184 cells produce some NOX1-dependent ROS in the absence of stimulation (Fig. 5), which was inhibited by celastrol. In H661 cells that have a very low background level of ROS generation, a small increase in ROS generation was observed with 100 [micro]M apigenin and with 5-100 [micro]M resveratrol but, surprisingly, not with piperine.

Discussion

In this work, we characterized the antioxidant capacity of several medicinal plant extracts. We previously demonstrated that celastro! acts as a direct NOX inhibitor, and possesses some ROS scavenging effects Qaquet et al., 2011). Here we also found that at higher concentrations, treatment with celastrol leads to an apparent elevation in the rate of [H.sub.2][O.sub.2] production. Resveratrol, and to some extent piperine, reduced the rate of [H.sub.2][O.sub.2] generation, but based on the observation that resveratrol did not reduce superoxide levels and showed strong direct antioxidant activity for [H.sub.2][O.sub.2], it is unlikely that resveratrol directly inhibits NOX1 or NOX2 enzyme activity. Apigenin and piperine showed weak antioxidant capacity towards [H.sub.2][O.sub.2] and no significant antioxidant capacity towards superoxide.

Consistent with previous work (Jaquet et al., 2011), celastrol acted as a NOX inhibitor. The IC50 values of ~3 [micro]M reported here for inhibition of [H.sub.2][O.sub.2] generated by NOX1 and NOX2 by celastrol are comparable to IC50 concentrations ranging from 0.3 to 5.4 pM found in other studies (Hirano et al., 2015; Jaquet et al., 2011; Yu et al., 2015). The increased signal in the NOX1-expressing cells observed at the high concentrations of 50 and 100 [micro]M was unexpected and cannot simply be explained by a fluorescence signal from the compound itself as we did not observe an increased fluorescence when the compounds were added to the Amplex Red mixture containing [H.sub.2][O.sub.2] but not cells. Additionally, the Amplex Red assay reports the rate of ROS generation, so the values reflect an increase in fluorescence over time, not simply a high level of fluorescence in the sample. Furthermore, the effect was observed in the S184 cells, but not in the B-LCLs. Together, these argue against a strictly chemical artifact. The S184 cells are derived from H661 cells in which NOX1 has been over-expressed. It is reasonable to expect that these cells may have a greater potential for mitochondrial contribution to ROS generation than B-LCLs in which mitochondrial ROS generation is minimal. However, as there was no corresponding intracellular increase in superoxide at these concentrations detected by NBT, this suggests that neither NOX nor mitochondrial activation were responsible for the observed elevation. One possibility is that high concentrations of celastrol may cause cell death, leading to an increased rate of [H.sub.2][O.sub.2] release by the cells. However, increases in ROS generation in response to celastrol have been reported elsewhere under some conditions (Kim et al., 2013).

There are mixed reports on the ability of resveratrol to act as an antioxidant toward superoxide, with some studies finding inhibition of ROS with IC50 values in the 15-38 [micro]M range (Jang et al., 1999; Wu et al., 2016), comparable to the inhibition of [H.sub.2][O.sub.2] generation by NOX1 and NOX2 found in the present study of 25 [micro]M. Others found inhibition in cell-free systems at higher concentrations of 130 [micro]M (Gulcin, 2010), while others do not report observing this activity (Deby-Dupont et al., 2005; Leiro et al., 2004; Poolman et al., 2005), except possibly at millimolar concentrations (Leonard et al., 2003). We observed a weak antioxidant response with resveratrol towards superoxide in our cell-free system at 100 [micro]M; however, there was no reduction in superoxide at the corresponding concentrations in the cell lines. This might be explained by the restricted ability of resveratrol to enter the cell. While resveratrol can penetrate membranes, it has been reported to become embedded within the lipid bilayer, which could make it less effective as an intracellular antioxidant (Brittes et al., 2010; Lancon et al., 2004). This behavior of resveratrol might explain why we observed some antioxidant effect towards superoxide using the MCLA assay that detected superoxide in the cell-free system, but did not observe a significant effect using the NBT assay that detects intracellular superoxide.

While resveratrol reduces the amount of [H.sub.2][O.sub.2] both in cellular systems and in vitro (Ungvari et al., 2007), there is some debate as to the mechanism of action. In lysates of human umbilical vein endothelial cells, resveratrol prevented NADPH oxidation with an IC50 of 16.0 pM, which was interpreted to reflect an inhibition of a [p47.sup.phox]-dependent NADPH oxidase (Steffen et al., 2008). However, in the present study, we observed no effect at that resveratrol concentration on superoxide generation by NOX1- or NOX2-expressing cells. Thus NOX1 and NOX2 enzymes do not appear to be direct molecular targets of resveratrol, although our study did not address the possibility that resveratrol may have indirect effects on NOX expression or activity or the expression of cellular antioxidants. Resveratrol inhibits LPS-induced NOX1 expression, and inhibits tumour necrosis factor-or-induced NOX activation (Park et al., 2009). Resveratrol can decrease expression of NOX1, NOX2, and NOX4, in addition to increasing SOD1 and glutathione peroxidase 1 expression (Spanier et al., 2009). PKC can activate NOX1 and NOX2, and resveratrol is known to directly inhibit PKC (Poolman et al., 2005). In the H661 cells, which were selected because they have little NOX expression (von Lohneysen et al., 2008), we observed that the [H.sub.2][O.sub.2] level increased when cells were exposed to concentrations of 5-50 pM resveratrol. This was not seen in the other unstimulated cells, although the unstimulated S184 cells displayed some level of constitutive NOX1 activation.

Piperine has been shown in other studies to reduce levels of oxidative stress in cells (Ma et al., 2014) and animal models (Arcaro et al., 2014; Umar et al., 2013). Although piperine is sometimes considered to be an antioxidant, the present study indicates that it does not act to directly scavenge either [H.sub.2][O.sub.2] or superoxide. This finding is consistent with an earlier report (Krishnakantha and Lokesh, 1993). The effect of piperine on reducing oxidative stress is more likely to be an indirect mechanism since the current study suggests that piperine does not directly inhibit either NOX1 or NOX2 enzymes.

No significant impact of apigenin was observed either in cells expressing NOX1 or NOX2, or in scavenging assays for [H.sub.2][O.sub.2] or superoxide. Although apigenin is a member of the flavone subclass of flavonoids which can often function as antioxidants (Bubols et al., 2013), apigenin did not appear to display any antioxidant capacity for [H.sub.2][O.sub.2] or superoxide. A comparative analysis of the structures of various flavonoids predicted that apigenin might fail to display good antioxidant qualities due to the lack of a H-donating catechol group that characterizes other effective flavonoid antioxidants (Leopoldini et al., 2004). Indeed, apigenin is reported to act rather as a pro-oxidant (Harrison et al., 2014; Skerget et al., 2005; Zhang et al., 2015). As expected, apigenin had little or no effect on ROS scavenging in either the [H.sub.2][O.sub.2] or the superoxide generating systems. No further increase in ROS generation was observed in the presence of apigenin in either the NOX1 or NOX2 expressing cells when NOX was already activated; however, a slight increase was detected at 100 [micro]M apigenin in unstimulated H661 cells. The intention of antioxidant therapeutics is to reduce the damaging oxidizing or unwanted signaling effects elicited by ROS. This outcome could be achieved by disabling the mechanisms that create ROS, or by neutralizing ROS after their formation.

While theoretically very promising, in practice, results from antioxidant therapy are mixed at best, and have often been disappointing (Margaritelis, 2016; Tinkel et al., 2012; Wu et al., 2014). One of the difficulties of applying antioxidant therapeutics is that while the accumulation of ROS is determined by the balance between the rate of production versus the rate of elimination, the action of antioxidants is determined largely by how they get distributed in the body and what compartments they are able to access (Margaritelis, 2016). An advantage of inhibiting ROS-forming or ROS-toxifying enzymes through the use of pharmacological agents is the potential to quickly, specifically, and reversibly affect the levels of various ROS molecules. Another advantage is the potential to target particular sources of ROS production by selected ROS-producing enzymes, such as NOX enzymes (Bedard and Krause, 2007). While completely inhibiting NOX2 over a prolonged period may have the side effect of suppressing the immune system, potentially resulting in symptoms of chronic granulomatous disease (Casas et al., 2015), a targeted reduction in unwanted excess ROS during specific pathologic conditions remains an important potential therapeutic strategy.

In summary, the results of this study add to our knowledge of the pro-oxidant, antioxidant, and ROS-production inhibiting activities of several plant-derived compounds and draws distinctions between cell-free and cellular effects.

ARTICLE INFO

Article history:

Received 6 October 2015

Revised 25 August 2016

Accepted 29 August 2016

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

Funding for this study was provided by the Nova Scotia Health Research Foundation. The authors would like to thank Dr. Marie-Jose Stasia for kindly providing the control B-lymphocyte cell line used in this study, and Dr. Emilie Lefort and Dr. Jonathan Blay for helpful discussions and providing the apigenin.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.08.011.

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Chemical compounds

Celastrol ([greater than or equal to] 98%. Sigma Aldrich Canada): (2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11 -oxo-1,3,4,5,6,13,14,14b-octahydropicene-2-carboxylic acid

Resveratrol ([greater than or equal to] 99%. Sigma Aldrich Canada): 5-[(E)-2- (4-hydroxyphenyl)ethenyl]benzene-1,3-diol

Apigenin ([greater than or equal to] 97%, Sigma Aldrich Canada): 5,7-dihydroxy-2- (4-hydroxyphenyl)chromen-4-one

Piperine ([greater than or equal to] 97%, Sigma Aldrich Canada): (2E, 4E)-5-(1,3-benzodioxol-5-yl)-1 -piperidin-1-ylpenta-2,4-dien-1-one

Abbreviations: B-LCL, B-lymphoblastoid cell line; CMV. cytomegalovirus; DMSO. dimethyl sulfoxide: DPI. diphenyleneiodonium; EBV, Epstein-Barr virus; EDTA. Ethylenediaminetetraacetic acid; GFP, green fluorescent protein; [H.sub.2][O.sub.2]. hydrogen peroxide; HBSS. Hank's balanced salt solution; HRP, horse radish peroxidase; IC50. Inhibitory concentration for 50*; LPS. Lipopolysaccharide; MCLA. methyl cypridina luciferin analog: NADPH. nicotinamide adenine dinudeotide phosphate; NBT. nitroblue tetrazolium; NOX. NADPH oxidase; PKC, protein kinase C; PMA. phorbol 12-myristate 13-acetate; ROS. reactive oxygen species; S184. H661 cell line modified to express NOX1. NOXOI. NOXA1 and CYBA; SOD, superoxide dismutase.

Scott Whitehouse (a), Pei-Lin Chen (a), Anna L. Greenshields (a), Mat Nightingale (a), David W. Hoskin (a,b), Karen Bedard (a), *

(a) Department of Pathology, Dalhousie University, Halifax, Nova Scotia Canada, B3H 4R2

(b) Department of Microbiology and Immunology. Dalhousie University. Halifax, Nova Scotia, Canada. B3H 4R2

* Corresponding author: Department of Pathology, Dalhousie University. 5850 College St., Sir Charles Tupper Medical Building. Room 11-F, Halifax, Nova Scotia, Canada. Fax: +01 902 494 2519.

E-mail addresses: kbedard@dal.ca. kbedard@gmail.com (K. Bedard).

http://dx.doi.org/10.1016/j.phymed.2016.08.011

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Title Annotation:nicotinamide adenine dinucleotide phosphate
Author:Whitehouse, Scott; Chen, Pei-Lin; Greenshields, Anna L.; Nightingale, Mat; Hoskin, David W.; Bedard,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Nov 15, 2016
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