http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–7 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.931362
RESEARCH ARTICLE
Flavonols and flavan-3-ols as modulators of xanthine oxidase and manganese superoxide dismutase activity
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Danila Di Majo1, Maurizio La Guardia2, Gaetano Leto1, Marilena Crescimanno1, Carla Flandina1, and Marco Giammanco1 1
Unit of Physiology and Pharmacology, Department DIGISPO, University of Palermo, Italy and 2Division of Cell Biology, Department STEBICEF, University of Palermo, Italy Abstract
Keywords
Experiments were performed to assess the dose-dependent effects of quercetin, kaempferol, (+) catechin, and () epicatechin on superoxide radical production through the modulation of manganese superoxide dismutase and xanthine oxidase activities. The experiments were carried out at flavanoid concentrations ranging from 1 mM to 100 mM. This investigation highlighted that flavonols induced opposite effects on superoxide radical production at different doses, i.e. pro-oxidant at the highest concentration (100 mM) and anti-oxidant at the lowest concentration (1 mM). Similar behaviors were observed for xanthine oxidase with flavan3ols. The diastereoisomer (the catechin) acted as a stronger radical scavenger than the epicatechin. However, flavan-3ols were less pro-oxidant than flavonols: in fact, the addition of the superoxide dismutase enzyme was able to cancel the flavan-3ols’ pro-oxidant effect. This study also shows that the absence of the 4-carbonyl group conjugated with the 2–3 double bonds in the heterocyclic ring cancelled the pro-oxidant effect of flavan-3ols. The opposite dose-dependent effects of flavonols suggest that they may be used as either a pro-oxidant or antioxidant.
Antioxidant, dose response, functional food, inhibitor, pro-oxidant, structure-activity relationship
Introduction Flavonoids are a subclass of polyphenols that are devoid of calories. However, growing evidence indicates that the introduction of these substances into the human diet may have physiological benefits. Therefore, according to the definition of ‘‘nutraceutical’’ (Dillard & German, 2000), the use of polyphenols as ‘‘nutraceuticals’’ in food preparations (Sajilata et al., 2008) or as dietary-ingested compounds has been suggested. In particular, clinical observations have reported the therapeutic benefits of these molecules in some pathological conditions such as myocardial ischemia, liver disease, and atherosclerosis (Siasos et al., 2013; Williamson & Manach, 2005). Furthermore, experimental findings suggest that these molecules are endowed with chemopreventive and antitumor properties (Del Rio et al., 2013; Gonza`lez-Vallinas et al., 2013; Huang et al., 2010). The therapeutic effects of flavonoids may be explained in part by their antioxidant activity due to their ability to (i) inhibit specific enzymes involved in reactive oxygen and nitrogen species (ROS/RNS) formation, (ii) act as scavenger of ROS/RNS, (iii) chelate trace elements, and/or (iv) up-regulate antioxidant genes (Gong & Chen, 2003; Joven et al., 2014).
Correspondence: Danila Di Majo, Unit of Physiology and Pharmacology, Department DIGISPO, University of Palermo, Italy. E-mail: danila. [email protected]
History Received 15 May 2014 Accepted 22 May 2014 Published online 27 June 2014
The antioxidant properties of food are differently influenced by the presence of a specific polyphenolic molecule (Di Majo et al., 2005, 2008). Among the enzymes involved in ROS formation and modulation, manganese superoxide dismutase (Mn-SOD) is of pivotal importance in an oxygen-rich atmosphere, as demonstrated by several in vivo studies in various models (Duttaroy et al., 2003; Wicks et al., 2009). Altered Mn-SOD expression or activity has been observed in neurodegenerative diseases, such as Alzheimer’s disease (Esposito et al., 2006) and cancer (Dhar & St Clair, 2012; Oberley, 2005). Many other studies report that flavonoids appear to contribute to the regulation of the expression and/or activity of enzymes such as cyclooxygenase, lipoxygenase, and nitric oxide synthase (Santangelo et al., 2007). However, studies regarding the possible dose-dependent effect of these molecules on the activity of these antioxidant enzymes are currently lacking, even though clinical evidence suggests that chemoprevention via phenolic phytochemicals may be an inexpensive, readily applicable, acceptable, and effective approach to the clinical management of cancer progression (Luk et al., 2007). Although the consumption of flavonoid/ phenolics is considered safe, their use for therapeutic or chemopreventive purposes has been reported to occasionally induce side effects such as liver failure, dermatitis, anemia, male reproductive disorders, and, in cases of an estrogen-like chemical structure of the compound, breast cancer (Galati & O’Brien, 2004). As these effects were found to be dose-dependent, this phenomenon must be taken into consideration when considering any long-term preventive or therapeutic use of these molecules
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(Fresco et al., 2006). Therefore, more information about the health benefits and the possible risks of dietary supplements or herbal medicines are needed to better define their pharmacological profile and their clinical usefulness. On the basis of these findings, we have undertaken some investigations to evaluate the effects of two flavonols (kaempferol and quercetin) and two flavan-3ols (catechin and epicatechin) on superoxide radical production through the concentration-dependent modulation of Mn-SOD and XO to better define the potential nutraceutical interest of these compounds. Furthermore, some studies have shown that the chemical structure of flavonoid compounds may affect the antioxidant activity of food and that there is a clear relationship between a planar flavonoid structure and antioxidant activity (Di Majo et al., 2005). In particular, these studies highlight that the total number of hydroxyl and methoxyl groups and their position on the ring may influence the extent and the mechanism of antioxidant and pro-oxidant activities (Finotti & Di Majo, 2003). We have therefore undertaken experiments with molecules of two classes of flavonoids, i.e. flavonols and flavan-3ols, to specifically clarify the influence of the carboxyl group at the C4 position of the heterocyclic ring conjugated with the C2-C3 double bond on enzymatic activities (Figure 1). As data for the influence of the stereochemical configuration of flavanols on their biological activities are scant, parallel studies were performed to assess whether both flavan-3-ol diastereoisomers have the same effect on the XO and Mn-SOD enzymes. These studies were performed using the aglycone forms but not on the methylated, glucurono-, and sulfo-conjugated metabolites. According to some authors, the studies on quercetin aglycone and other flavonoids are questionable because only metabolites were determined in the plasma after the oral
administration of these molecules (Kroon et al., 2004, Manach et al., 2004). The current available evidence indicates that although quercetin is not found in the plasma after oral administration, it exerts biologically demonstrable systemic effects, whereas its metabolites show weak activity in vitro and often appear to be completely inactive (Perez-Vizcaino et al., 2012). According to Perez-Vizcaino, glucuronidated derivatives transport quercetin and deliver it to tissues as the free aglycone, which is the final effector. A similar phenomenon may be observed with kaempferol (O’Leary et al., 2001) and flavanols (Serra et al., 2011; Ottaviani et al., 2011), suggesting that the effects described above may also be extended to other flavonoids.
Materials and methods Sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), ethylene diaminetetraacetic acid (EDTA), XTT (2,3-bis[2-methoxy-4-nitro-sulfo-phenyl]-2H-tetrazolium-5-carboxanilide), xanthine (X), xanthine oxidase (EC 1.1.3.22) from buttermilk (0.6 units/mg protein), Mn-SOD from bovine erythrocytes (EC 1.15.1.1), quercetin, kaempferol, and (+) catechin and () epicatechin were purchased from Sigma-Aldrich (St Louis, MO). All the stock solutions were prepared with water purified using a Milli-Q system (Millipore, Billerica, MA). XTT and X were dissolved in a 50 mM sodium carbonate buffer (pH 9.8) at room temperature and at 45 C, respectively. To allow complete superoxide radical consumption, Mn-SOD was used at a concentration of 0.3 mM. Stock solutions of flavonoids were prepared in ethanol at 1 mM concentration and then diluted just before the assay in a range of concentrations between 1 mM and 100 mM. Flavonols
Figure 1. Flavonoid backbone and chemical structure of flavonols and flavan-3ol sub-groups.
OH OH
OH HO
HO
O
O
O
OH
OH
O
Kaempferol
Quercetin 3' 4'
2' 8 7 6
1 9 O
A 5
2
B
1'
5' 6'
C 10
4
3
Flavan-3ols OH
OH
OH
OH HO
O
R
HO
O R
R OH
S OH OH
OH
(+) catechin
(−) epicatechin
DOI: 10.3109/09637486.2014.931362
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SOD activity assay The effects of different compounds on XO and Mn-SOD activities were determined by a spectrophotometric assay using the X/XO/ XTT system, as previously reported (Farines et al., 2004). Briefly, at the same time, three quartz cuvettes (a, b, and c) were prepared. In the first cuvette (a), each tested compound and XO without SOD were added to the reagents to evaluate the capacity of the compounds to scavenge superoxide radicals directly or to inhibit xanthine oxidase. In the second (b), XO and SOD without the sample (flavonoids) were added to the mixture of reagents to analyze the capacity of SOD to abolish the radical production of the X/XO-XTT system. In the last cuvette (c), SOD was added to the reagents of the first cuvette to evaluate the combined action of the compounds on XO and SOD. The reagents were distributed to all three quartz cuvettes (final volume 3 mL) by adding 50 mM sodium carbonate buffer (pH 9.8), 67 mL of NaEDTA (3 mM), 67 mL of XTT (0.8 mM), 67 mL of xanthine (3 mM), and 67 mL of xanthine oxidase, with 400 mL of SOD only added to the c and b cuvettes. Ethanol was added to the SOD-free (control) cuvette. The reaction was initiated by the addition of XO solution at a concentration of 60 mUmL1. The change in absorbance at 470 nm was monitored for 30 minutes with a spectrophotometer Beckman 640 set at 25 C. The modulation of relative SOD activity (RA) due to the tested compounds was expressed as: ðc bÞ 100 RA ¼ 1 ða bÞ where a is the absorbance without SOD in the presence of the compound tested, b is the absorbance of SOD in the system (X/ XO-XTT) w/o the tested compound, c is the absorbance of SOD on the system (X/XO-XTT) in presence of the tested compound. If RA 0, the absorbance of the solution containing SOD in the presence of the compound (c) is lower than that determined in the solution without SOD and in the presence of tested compound (a). This means that the compound has no effect on SOD activity. In fact, the enzyme is completely or partially able to neutralise the superoxide radicals produced by the X/XO/compound system. Conversely, RA values 0 indicates that they act as potent prooxidants or as inhibitors of the SOD enzyme.
Flavonols and flavan-3-ols as modulators of XO and MnSOD
3
samples. The statistical analysis was performed using the XLSTAT 2012 statistics package for Excel. p Values 0.05 were considered statistically significant.
Results Effect of flavonols on XO and Mn-SOD Table 1 summarizes the dose-dependent pro-oxidant or antioxidant effects of kaempferol and quercetin on the XO enzyme. These compounds at concentrations between 25 and 100 mM resulted in a significant increase in superoxide anion levels when compared with the controls. Consequently, they revealed a prooxidant action. This phenomenon is most likely the consequence of an intrinsic pro-oxidant activity of flavonols. These results are in good agreement with the data reported by Lo`pez-Lo`pez et al. (2004). In fact, according to these authors, quercetin undergoes auto-oxidation and generates superoxide radicals in a dosedependent manner. A marked production of radicals was also observed at concentrations of quercetin higher than 25 mM. The maximal production of superoxide radicals occurred at 100 mM (Table 1), and kaempferol induced effects similar as quercetin. However, kaempferol at concentrations between 25 mM and 75 mM induced a more consistent production of radicals compared to quercetin. Table 1 shows that kaempferol produced the same amount of superoxide radicals (79%) at concentrations of 50 and 100 mM. When Mn-SOD was added at the same concentration of 0.3 mM, the amount of superoxide radicals was reduced to 20% (Table 2) with 50 mM kaempferol. However, when kaempferol was used at the concentration of 100 mM, the addition of Mn-SOD was not able to cancel the production of radicals (Table 2), and the system (X/XO/XTT/ SOD) with the compound showed a higher production of Table 1. Dose-dependent effects of quercetin, kaempferol, (+) catechin, and () epicatechin on xanthine oxidase. The values are expressed as the mean ± standard deviation of least three independent determinations. RA is the relative activity of compounds on the enzyme. When RA 40, the compound tested increased superoxide radical production, in contrast to RA 50. Activity on the XO (% RA)
Xanthine oxidase activity assay
Molecules
Concentrations (mM)
RA 0 " [O 2 ]
The assay uses the X/XO/XTT system but without SOD. The reagents and their concentrations were the same as used in the superoxide dismutase assay. The relative activity of the compounds, expressed in percentage, was calculated with the following equation: ðb aÞ 100 RA ¼ a
Kaempferol
100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1
79.46 ± 1.56 69.56 ± 0.95 76.90 ± 3.17 43.03 ± 4.20
where (a) is the absorbance of the X/XO-XTT system without the compounds (control) and (b) is the absorbance of the solution containing flavonoids. When RA 40, the absorbance of the solution containing the compounds is higher than the control. When RA 50, the absorbance of formazan is lower in the presence of the compound than in the control.
Quercetin
Catechin
Epicatechin
Statistical analysis Data are reported as the mean value ± standard deviation (SD). The significance of differences between the parameters considered in this study was assessed by Student’s t-test for unpaired
RA 0 # [O 2 ]
8.47 ± 0.02 18.75 ± 4.44 74.00 ± 0.46 61.76 ± 2.63 25.85 ± 6.05 22.61 ± 3.38 9.06 ± 2.35 37.08 ± 3.17 69.32 ± 3.67 22.01 ± 2.36 26.04 ± 1.99 81.67 ± 6.37 108.65 ± 6.74 36.67 ± 5.72 75.67 ± 3.07 65.29 ± 1.31 44.77 ± 1.29 55.19 ± 3.20 40.52 ± 1.60 55.07 ± 3.39
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Table 2. Dose-dependent effects of quercetin, kaempferol, (+) catechin, and () epicatechin on both enzymes. The values are expressed as the mean ± standard deviation of least three independent determinations. RA is the relative activity of compounds on the enzyme. When RA 40, the modulating action of the compound tested on both enzymes reduced superoxide radical production, in contrast to RA 50. Relative activity on both the enzymes (% RA) Molecules
Concentrations (mM)
Kaempferol
100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1
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Quercetin
Catechin
Epicatechin
RA 0 # [O 2 ]
RA 0 " [O 2 ] 32.41 ± 3.46 2.18 ± 0.15
20.26 ± 8.27 36.06 ± 3.04 46.29 ± 8.63 97.99 ± 3.30 9.97 ± 3.11 8.51 ± 3.17 26.90 ± 10.87 44.92 ± 3.06 53.31 ± 0.12 95.10 ± 3.55 37.85 ± 3.96 28.61 ± 4.79 51.98 ± 7.66 31.26 ± 5.73 32.18 ± 3.44 93.52 ± 7.7 25.16 ± 6.63 22.69 ± 1.37 57.80 ± 3.07 39.11 ± 3.19 52.14 ± 6.47 105.54 ± 5.91
Figure 3. Dose-dependent effects of flavonols (+) catechin ( ) and () epicatechin ( ) on superoxide anion production, as evaluated by the SOD assay. Data are expressed as the mean ± standard deviation of last three independent determinations.
superoxide radicals. According to Pauff & Hille, (2009), the antioxidant effects may be due either to the direct reaction with superoxide radicals (radical scavenger effect) or to the inhibition of XO enzyme activity. Our results demonstrate that quercetin had a greater inhibitory effect on XO than kaempferol (Table 1). These findings are consistent with those of Cos et al. (1998), which suggests that quercetin might be classified into category C (xanthine oxidase inhibitors with an additional superoxidescavenging activity), whereas kaempferol falls into category B, namely, xanthine oxidase inhibitors without any additional superoxide-scavenging activity. Consistent with the results of Lin et al. (2002), the inhibiting effect of flavonols can be in part explained by the high capacity of these molecules to interact with the molybdopterin moiety present in the active site of the XO enzyme. In addition, this study showed that when SOD was added to the system (Table 2), no significant difference between the two molecules was observed at the lowest concentrations (p40.05). Modulator effects of flavan-3ols on XO and Mn-SOD
Figure 2. Dose-dependent effects of flavonols quercetin ( ) and kaempferol ( ) on superoxide anion production, as evaluated by the SOD assay. Data are expressed as the mean ± standard deviation of last three independent determinations.
superoxide radicals than the control (system without the compound). This effect may be explained by an inhibition of SOD activity that is not the consequence of a saturation effect of the enzyme. The dose-dependent effects of flavonols on superoxide radical production via the modulation of XO and Mn-SOD are shown in Figure 2. The differences observed between the two compounds further confirm the findings by Cao et al. (1997), indicating that the number of OH groups in the aromatic ring and their positions are of pivotal importance in determining the anti/pro-oxidant action of polyphenols. Quercetin, which carries two electrondonating groups on the B-ring, showed a pro-oxidant activity lower than that of kaempferol, which has only one OH group. At concentrations lower than 25 mM, quercetin and kaempferol acted as antioxidants. In fact, these molecules were able to reduce
Regarding flavan-3ols, it is important to note that catechin and epicatechin are stereoisomers, each of which exists in two enantiomeric forms. The four distinct stereochemical configurations of these flavanols are () epicatechin, (+) epicatechin, () catechin, and (+) catechin. It has been reported that the stereochemical configuration of flavan-3ols deeply influences their uptake and metabolism in humans (Ottaviani et al., 2011) and that the plasma levels of () epicatechin and (+) catechin are higher than those of the other two stereoisomers. Therefore, we focused our attention on the () epicatechin and (+) catechin diastereoisomers. Figure 3 shows the dose-dependent effects of (+) catechin and () epicatechin on superoxide anion production, as evaluated by the SOD assay. The results in Table 1 show the effects of flavan-3-ols on XO activity. Similarly to flavonols, these compounds increased the production of superoxide radicals at the highest concentrations (50 mM), an effect that is likely due to an intrinsic pro-oxidant activity. Catechin was found to be less pro-oxidant than kaempferol. In fact, the latter molecule increased radical production at 25 mM. Conversely, at the same concentration, catechin reduced the radical production by the system (Table 1). These results are in agreement with those of Marozien_e (2012), reporting that flavonoids exhibit prooxidant cytotoxicity in mammalian cells due to the formation of free radicals and that this effect increases as the redox potential of the phenoxyl radical/phenol couple decreases. These data indicate that flavonols induce a stronger cytotoxic effect on mammalian cells than flavan-3ols. These results may be in part explained as follows: due to auto-oxidation, flavonols may produce a more
Flavonols and flavan-3-ols as modulators of XO and MnSOD
DOI: 10.3109/09637486.2014.931362
Kaempferol
O-semiquinone radical intermediate (K.−) OH
O−
.
HO
O
HO
+ O2
O
O2.− + 2H+ +
OH OH
5
O
O
O
OH
O HO
O
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C OH
O−
O O
O−
O
C
OH
OH
O2 −e− O
O
O
O2.− + O OH
OH
O-Quinone Figure 4. Auto-oxidation of kaempferol and quinone/quinomethide tautomerisation of kaempferol oxidate.
stable radical that can be delocalized on the C and A rings (Figure 4); instead, the semiquinone radical of catechins remains localized on the B ring. Hence, the semiquinone radical of flavan3-ols is less stabilized by resonance, and, as a consequence, the auto-oxidation reaction is less promoted (Figure 5a). According to Mochizuki et al. (2002), catechins undergo multi-step autooxidation at concentrations higher than 40 mM (Figure 5a), with the formation of a semiquinone radical on the B ring, as confirmed by ESR analysis. This radical may continue to react until it transforms into a quinone. The superoxide radical generated by the first reaction (Figure 5a) can react with other molecules of catechin (Figure 5b). This reaction is fostered by the strong oxidant activity of the superoxide radical (Ered ¼ 0.89 V) versus O2 (Ered ¼ 0.16 V). For this reason, O 2 itself participates into the auto-oxidation of catechins. At low concentrations (525 mM), catechin behaved as a radical scavenger of superoxide anion. At these concentrations, it is possible that the auto-oxidation of catechin (Figure 5a) was not complete but may be stopped at the level of semiquinone radical formation because the generated superoxide radical reacted with more molecules of catechin (Figure 5b), thus producing hydrogen peroxide and other semiquinone radicals (faster reaction). Among flavan-3-ols, catechins showed a radical scavenger activity that was stronger than epicatechins (Table 1). In fact, this effect was observed in the entire range of 25 mM to 1 mM.
Furthermore, epicatechin behaved as a radical scavenger only at a lower concentration (1 mM). The addition of SOD was able to inhibit the pro-oxidant activity of flavan-3-ols in a dosedependent manner (Table 2). At low concentrations, the effect of SOD was stronger because the radical-scavenging reaction (Figure 5b) might be accelerated by the disproportion of superoxide radical (Figure 5c).
Conclusions The results presented here show that marked differences can be observed between flavonoids with different structural characteristics with regard to their ability to inhibit or stimulate superoxide radical production through a modulation of XO and Mn-SOD. The study of the structure-activity relationship on these compounds showed that flavonols (quercetin and kaempferol) differ from flavan-3ols (catechin and epicatechin) in their pro-oxidant effect. The absence of the carbonyl group at C-4 and of the double bond between C-2 and C-3 of the heterocyclic ring resulted in a dose-dependent loss of the pro-oxidant effect. This may be due to the saturation of the double bond, which destroys the conjugation and coplanarity of the flavan-3ols structure. These data further demonstrate the opposite dose-dependent effects of flavonols on XO and Mn-SOD. At concentrations 75 mM, these molecules showed a high pro-oxidant activity, and the addition of Mn-SOD
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Figure 5. (a) Auto-oxidation reaction of catechin at higher concentrations; (b) free radical-scavenging behavior of catechin at low concentrations; (c) the dismutation of superoxide free radical by the enzyme superoxide dismutase.
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(a)
OH
O.
OH
HO
HO
O
O OH
O−
+ 2O2
O OH
OH
OH
Semiquinone form
+ 2O2.− + 2H+
Quinone form
.
OH
O
OH
HO
O
OH
OH
(b)
O
HO
HO
O
O
O−
.−
+ 2O2
H2O2
OH
OH OH
OH
Semiquinone form
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(c)
2O2.− + 2H+
SOD
was not able to abolish this effect. However, they acted as antioxidants at concentrations lower than 25 mM. These opposite dose-dependent effects we observed suggest exercising caution in the use of flavonols for clinical purposes. Unlike flavonols, flavan-3-ols showed antioxidant activity at all the tested concentrations. Our study demonstrates that two diastereoisomers exhibit different properties on XO but that no difference was observed following the addition of Mn-SOD. This study also shows that the a-hydroxyl on the C-3 of heterocyclic ring (catechin) is important for the radical-scavenging activity of flavan-3ols. Therefore, our results suggest that flavan-3ols, at low concentrations, may act as radical scavengers and therefore show strong antioxidant effects, whereas they may undergo auto-oxidation at higher concentrations, showing a weaker antioxidant potential.
Declaration of interest The authors declare that they have no conflicts of interest.
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Serra A, Macia` A, Romero MP, Angle`s N, Morello` JR, Motilva MJ. 2011. Distribution of procyanidins and their metabolites in rat plasma and tissue after an acute intake of hazelnut extract. Food Funct 2:562–568. Siasos G, Oikonomou E, Chrysohoou C, Tousoulis D, Panagiotakos D, Zaromitidou M, Zisimos K, et al. 2013. Consumption of a boiled Greek type of coffee is associated with improved endothelial function: The Ikaria Study. Vasc Med 18:55–62. Santangelo C, Varı` R, Scazzocchio B, Di Benedetto R, Filesi C, Masella R. 2007. Polyphenols, intracellular signalling and inflammation. Ann Ist Sup Sanita` 43:394–405. Wicks P, Abrahams S, Papps B, Al-Chalabi A, Shaw CE, Leigh PN, Goldstein LH. 2009. SOD1 and cognitive dysfunction in familial amyotrophic lateral sclerosis. J Neurol 256:234–241. Williamson G, Manach G. 2005. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am J Clin Nutr 81:243S–255S.
RESEARCH ARTICLE
Flavonols and flavan-3-ols as modulators of xanthine oxidase and manganese superoxide dismutase activity
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Danila Di Majo1, Maurizio La Guardia2, Gaetano Leto1, Marilena Crescimanno1, Carla Flandina1, and Marco Giammanco1 1
Unit of Physiology and Pharmacology, Department DIGISPO, University of Palermo, Italy and 2Division of Cell Biology, Department STEBICEF, University of Palermo, Italy Abstract
Keywords
Experiments were performed to assess the dose-dependent effects of quercetin, kaempferol, (+) catechin, and () epicatechin on superoxide radical production through the modulation of manganese superoxide dismutase and xanthine oxidase activities. The experiments were carried out at flavanoid concentrations ranging from 1 mM to 100 mM. This investigation highlighted that flavonols induced opposite effects on superoxide radical production at different doses, i.e. pro-oxidant at the highest concentration (100 mM) and anti-oxidant at the lowest concentration (1 mM). Similar behaviors were observed for xanthine oxidase with flavan3ols. The diastereoisomer (the catechin) acted as a stronger radical scavenger than the epicatechin. However, flavan-3ols were less pro-oxidant than flavonols: in fact, the addition of the superoxide dismutase enzyme was able to cancel the flavan-3ols’ pro-oxidant effect. This study also shows that the absence of the 4-carbonyl group conjugated with the 2–3 double bonds in the heterocyclic ring cancelled the pro-oxidant effect of flavan-3ols. The opposite dose-dependent effects of flavonols suggest that they may be used as either a pro-oxidant or antioxidant.
Antioxidant, dose response, functional food, inhibitor, pro-oxidant, structure-activity relationship
Introduction Flavonoids are a subclass of polyphenols that are devoid of calories. However, growing evidence indicates that the introduction of these substances into the human diet may have physiological benefits. Therefore, according to the definition of ‘‘nutraceutical’’ (Dillard & German, 2000), the use of polyphenols as ‘‘nutraceuticals’’ in food preparations (Sajilata et al., 2008) or as dietary-ingested compounds has been suggested. In particular, clinical observations have reported the therapeutic benefits of these molecules in some pathological conditions such as myocardial ischemia, liver disease, and atherosclerosis (Siasos et al., 2013; Williamson & Manach, 2005). Furthermore, experimental findings suggest that these molecules are endowed with chemopreventive and antitumor properties (Del Rio et al., 2013; Gonza`lez-Vallinas et al., 2013; Huang et al., 2010). The therapeutic effects of flavonoids may be explained in part by their antioxidant activity due to their ability to (i) inhibit specific enzymes involved in reactive oxygen and nitrogen species (ROS/RNS) formation, (ii) act as scavenger of ROS/RNS, (iii) chelate trace elements, and/or (iv) up-regulate antioxidant genes (Gong & Chen, 2003; Joven et al., 2014).
Correspondence: Danila Di Majo, Unit of Physiology and Pharmacology, Department DIGISPO, University of Palermo, Italy. E-mail: danila. [email protected]
History Received 15 May 2014 Accepted 22 May 2014 Published online 27 June 2014
The antioxidant properties of food are differently influenced by the presence of a specific polyphenolic molecule (Di Majo et al., 2005, 2008). Among the enzymes involved in ROS formation and modulation, manganese superoxide dismutase (Mn-SOD) is of pivotal importance in an oxygen-rich atmosphere, as demonstrated by several in vivo studies in various models (Duttaroy et al., 2003; Wicks et al., 2009). Altered Mn-SOD expression or activity has been observed in neurodegenerative diseases, such as Alzheimer’s disease (Esposito et al., 2006) and cancer (Dhar & St Clair, 2012; Oberley, 2005). Many other studies report that flavonoids appear to contribute to the regulation of the expression and/or activity of enzymes such as cyclooxygenase, lipoxygenase, and nitric oxide synthase (Santangelo et al., 2007). However, studies regarding the possible dose-dependent effect of these molecules on the activity of these antioxidant enzymes are currently lacking, even though clinical evidence suggests that chemoprevention via phenolic phytochemicals may be an inexpensive, readily applicable, acceptable, and effective approach to the clinical management of cancer progression (Luk et al., 2007). Although the consumption of flavonoid/ phenolics is considered safe, their use for therapeutic or chemopreventive purposes has been reported to occasionally induce side effects such as liver failure, dermatitis, anemia, male reproductive disorders, and, in cases of an estrogen-like chemical structure of the compound, breast cancer (Galati & O’Brien, 2004). As these effects were found to be dose-dependent, this phenomenon must be taken into consideration when considering any long-term preventive or therapeutic use of these molecules
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(Fresco et al., 2006). Therefore, more information about the health benefits and the possible risks of dietary supplements or herbal medicines are needed to better define their pharmacological profile and their clinical usefulness. On the basis of these findings, we have undertaken some investigations to evaluate the effects of two flavonols (kaempferol and quercetin) and two flavan-3ols (catechin and epicatechin) on superoxide radical production through the concentration-dependent modulation of Mn-SOD and XO to better define the potential nutraceutical interest of these compounds. Furthermore, some studies have shown that the chemical structure of flavonoid compounds may affect the antioxidant activity of food and that there is a clear relationship between a planar flavonoid structure and antioxidant activity (Di Majo et al., 2005). In particular, these studies highlight that the total number of hydroxyl and methoxyl groups and their position on the ring may influence the extent and the mechanism of antioxidant and pro-oxidant activities (Finotti & Di Majo, 2003). We have therefore undertaken experiments with molecules of two classes of flavonoids, i.e. flavonols and flavan-3ols, to specifically clarify the influence of the carboxyl group at the C4 position of the heterocyclic ring conjugated with the C2-C3 double bond on enzymatic activities (Figure 1). As data for the influence of the stereochemical configuration of flavanols on their biological activities are scant, parallel studies were performed to assess whether both flavan-3-ol diastereoisomers have the same effect on the XO and Mn-SOD enzymes. These studies were performed using the aglycone forms but not on the methylated, glucurono-, and sulfo-conjugated metabolites. According to some authors, the studies on quercetin aglycone and other flavonoids are questionable because only metabolites were determined in the plasma after the oral
administration of these molecules (Kroon et al., 2004, Manach et al., 2004). The current available evidence indicates that although quercetin is not found in the plasma after oral administration, it exerts biologically demonstrable systemic effects, whereas its metabolites show weak activity in vitro and often appear to be completely inactive (Perez-Vizcaino et al., 2012). According to Perez-Vizcaino, glucuronidated derivatives transport quercetin and deliver it to tissues as the free aglycone, which is the final effector. A similar phenomenon may be observed with kaempferol (O’Leary et al., 2001) and flavanols (Serra et al., 2011; Ottaviani et al., 2011), suggesting that the effects described above may also be extended to other flavonoids.
Materials and methods Sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), ethylene diaminetetraacetic acid (EDTA), XTT (2,3-bis[2-methoxy-4-nitro-sulfo-phenyl]-2H-tetrazolium-5-carboxanilide), xanthine (X), xanthine oxidase (EC 1.1.3.22) from buttermilk (0.6 units/mg protein), Mn-SOD from bovine erythrocytes (EC 1.15.1.1), quercetin, kaempferol, and (+) catechin and () epicatechin were purchased from Sigma-Aldrich (St Louis, MO). All the stock solutions were prepared with water purified using a Milli-Q system (Millipore, Billerica, MA). XTT and X were dissolved in a 50 mM sodium carbonate buffer (pH 9.8) at room temperature and at 45 C, respectively. To allow complete superoxide radical consumption, Mn-SOD was used at a concentration of 0.3 mM. Stock solutions of flavonoids were prepared in ethanol at 1 mM concentration and then diluted just before the assay in a range of concentrations between 1 mM and 100 mM. Flavonols
Figure 1. Flavonoid backbone and chemical structure of flavonols and flavan-3ol sub-groups.
OH OH
OH HO
HO
O
O
O
OH
OH
O
Kaempferol
Quercetin 3' 4'
2' 8 7 6
1 9 O
A 5
2
B
1'
5' 6'
C 10
4
3
Flavan-3ols OH
OH
OH
OH HO
O
R
HO
O R
R OH
S OH OH
OH
(+) catechin
(−) epicatechin
DOI: 10.3109/09637486.2014.931362
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SOD activity assay The effects of different compounds on XO and Mn-SOD activities were determined by a spectrophotometric assay using the X/XO/ XTT system, as previously reported (Farines et al., 2004). Briefly, at the same time, three quartz cuvettes (a, b, and c) were prepared. In the first cuvette (a), each tested compound and XO without SOD were added to the reagents to evaluate the capacity of the compounds to scavenge superoxide radicals directly or to inhibit xanthine oxidase. In the second (b), XO and SOD without the sample (flavonoids) were added to the mixture of reagents to analyze the capacity of SOD to abolish the radical production of the X/XO-XTT system. In the last cuvette (c), SOD was added to the reagents of the first cuvette to evaluate the combined action of the compounds on XO and SOD. The reagents were distributed to all three quartz cuvettes (final volume 3 mL) by adding 50 mM sodium carbonate buffer (pH 9.8), 67 mL of NaEDTA (3 mM), 67 mL of XTT (0.8 mM), 67 mL of xanthine (3 mM), and 67 mL of xanthine oxidase, with 400 mL of SOD only added to the c and b cuvettes. Ethanol was added to the SOD-free (control) cuvette. The reaction was initiated by the addition of XO solution at a concentration of 60 mUmL1. The change in absorbance at 470 nm was monitored for 30 minutes with a spectrophotometer Beckman 640 set at 25 C. The modulation of relative SOD activity (RA) due to the tested compounds was expressed as: ðc bÞ 100 RA ¼ 1 ða bÞ where a is the absorbance without SOD in the presence of the compound tested, b is the absorbance of SOD in the system (X/ XO-XTT) w/o the tested compound, c is the absorbance of SOD on the system (X/XO-XTT) in presence of the tested compound. If RA 0, the absorbance of the solution containing SOD in the presence of the compound (c) is lower than that determined in the solution without SOD and in the presence of tested compound (a). This means that the compound has no effect on SOD activity. In fact, the enzyme is completely or partially able to neutralise the superoxide radicals produced by the X/XO/compound system. Conversely, RA values 0 indicates that they act as potent prooxidants or as inhibitors of the SOD enzyme.
Flavonols and flavan-3-ols as modulators of XO and MnSOD
3
samples. The statistical analysis was performed using the XLSTAT 2012 statistics package for Excel. p Values 0.05 were considered statistically significant.
Results Effect of flavonols on XO and Mn-SOD Table 1 summarizes the dose-dependent pro-oxidant or antioxidant effects of kaempferol and quercetin on the XO enzyme. These compounds at concentrations between 25 and 100 mM resulted in a significant increase in superoxide anion levels when compared with the controls. Consequently, they revealed a prooxidant action. This phenomenon is most likely the consequence of an intrinsic pro-oxidant activity of flavonols. These results are in good agreement with the data reported by Lo`pez-Lo`pez et al. (2004). In fact, according to these authors, quercetin undergoes auto-oxidation and generates superoxide radicals in a dosedependent manner. A marked production of radicals was also observed at concentrations of quercetin higher than 25 mM. The maximal production of superoxide radicals occurred at 100 mM (Table 1), and kaempferol induced effects similar as quercetin. However, kaempferol at concentrations between 25 mM and 75 mM induced a more consistent production of radicals compared to quercetin. Table 1 shows that kaempferol produced the same amount of superoxide radicals (79%) at concentrations of 50 and 100 mM. When Mn-SOD was added at the same concentration of 0.3 mM, the amount of superoxide radicals was reduced to 20% (Table 2) with 50 mM kaempferol. However, when kaempferol was used at the concentration of 100 mM, the addition of Mn-SOD was not able to cancel the production of radicals (Table 2), and the system (X/XO/XTT/ SOD) with the compound showed a higher production of Table 1. Dose-dependent effects of quercetin, kaempferol, (+) catechin, and () epicatechin on xanthine oxidase. The values are expressed as the mean ± standard deviation of least three independent determinations. RA is the relative activity of compounds on the enzyme. When RA 40, the compound tested increased superoxide radical production, in contrast to RA 50. Activity on the XO (% RA)
Xanthine oxidase activity assay
Molecules
Concentrations (mM)
RA 0 " [O 2 ]
The assay uses the X/XO/XTT system but without SOD. The reagents and their concentrations were the same as used in the superoxide dismutase assay. The relative activity of the compounds, expressed in percentage, was calculated with the following equation: ðb aÞ 100 RA ¼ a
Kaempferol
100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1
79.46 ± 1.56 69.56 ± 0.95 76.90 ± 3.17 43.03 ± 4.20
where (a) is the absorbance of the X/XO-XTT system without the compounds (control) and (b) is the absorbance of the solution containing flavonoids. When RA 40, the absorbance of the solution containing the compounds is higher than the control. When RA 50, the absorbance of formazan is lower in the presence of the compound than in the control.
Quercetin
Catechin
Epicatechin
Statistical analysis Data are reported as the mean value ± standard deviation (SD). The significance of differences between the parameters considered in this study was assessed by Student’s t-test for unpaired
RA 0 # [O 2 ]
8.47 ± 0.02 18.75 ± 4.44 74.00 ± 0.46 61.76 ± 2.63 25.85 ± 6.05 22.61 ± 3.38 9.06 ± 2.35 37.08 ± 3.17 69.32 ± 3.67 22.01 ± 2.36 26.04 ± 1.99 81.67 ± 6.37 108.65 ± 6.74 36.67 ± 5.72 75.67 ± 3.07 65.29 ± 1.31 44.77 ± 1.29 55.19 ± 3.20 40.52 ± 1.60 55.07 ± 3.39
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Table 2. Dose-dependent effects of quercetin, kaempferol, (+) catechin, and () epicatechin on both enzymes. The values are expressed as the mean ± standard deviation of least three independent determinations. RA is the relative activity of compounds on the enzyme. When RA 40, the modulating action of the compound tested on both enzymes reduced superoxide radical production, in contrast to RA 50. Relative activity on both the enzymes (% RA) Molecules
Concentrations (mM)
Kaempferol
100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1 100 75 50 25 10 1
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Quercetin
Catechin
Epicatechin
RA 0 # [O 2 ]
RA 0 " [O 2 ] 32.41 ± 3.46 2.18 ± 0.15
20.26 ± 8.27 36.06 ± 3.04 46.29 ± 8.63 97.99 ± 3.30 9.97 ± 3.11 8.51 ± 3.17 26.90 ± 10.87 44.92 ± 3.06 53.31 ± 0.12 95.10 ± 3.55 37.85 ± 3.96 28.61 ± 4.79 51.98 ± 7.66 31.26 ± 5.73 32.18 ± 3.44 93.52 ± 7.7 25.16 ± 6.63 22.69 ± 1.37 57.80 ± 3.07 39.11 ± 3.19 52.14 ± 6.47 105.54 ± 5.91
Figure 3. Dose-dependent effects of flavonols (+) catechin ( ) and () epicatechin ( ) on superoxide anion production, as evaluated by the SOD assay. Data are expressed as the mean ± standard deviation of last three independent determinations.
superoxide radicals. According to Pauff & Hille, (2009), the antioxidant effects may be due either to the direct reaction with superoxide radicals (radical scavenger effect) or to the inhibition of XO enzyme activity. Our results demonstrate that quercetin had a greater inhibitory effect on XO than kaempferol (Table 1). These findings are consistent with those of Cos et al. (1998), which suggests that quercetin might be classified into category C (xanthine oxidase inhibitors with an additional superoxidescavenging activity), whereas kaempferol falls into category B, namely, xanthine oxidase inhibitors without any additional superoxide-scavenging activity. Consistent with the results of Lin et al. (2002), the inhibiting effect of flavonols can be in part explained by the high capacity of these molecules to interact with the molybdopterin moiety present in the active site of the XO enzyme. In addition, this study showed that when SOD was added to the system (Table 2), no significant difference between the two molecules was observed at the lowest concentrations (p40.05). Modulator effects of flavan-3ols on XO and Mn-SOD
Figure 2. Dose-dependent effects of flavonols quercetin ( ) and kaempferol ( ) on superoxide anion production, as evaluated by the SOD assay. Data are expressed as the mean ± standard deviation of last three independent determinations.
superoxide radicals than the control (system without the compound). This effect may be explained by an inhibition of SOD activity that is not the consequence of a saturation effect of the enzyme. The dose-dependent effects of flavonols on superoxide radical production via the modulation of XO and Mn-SOD are shown in Figure 2. The differences observed between the two compounds further confirm the findings by Cao et al. (1997), indicating that the number of OH groups in the aromatic ring and their positions are of pivotal importance in determining the anti/pro-oxidant action of polyphenols. Quercetin, which carries two electrondonating groups on the B-ring, showed a pro-oxidant activity lower than that of kaempferol, which has only one OH group. At concentrations lower than 25 mM, quercetin and kaempferol acted as antioxidants. In fact, these molecules were able to reduce
Regarding flavan-3ols, it is important to note that catechin and epicatechin are stereoisomers, each of which exists in two enantiomeric forms. The four distinct stereochemical configurations of these flavanols are () epicatechin, (+) epicatechin, () catechin, and (+) catechin. It has been reported that the stereochemical configuration of flavan-3ols deeply influences their uptake and metabolism in humans (Ottaviani et al., 2011) and that the plasma levels of () epicatechin and (+) catechin are higher than those of the other two stereoisomers. Therefore, we focused our attention on the () epicatechin and (+) catechin diastereoisomers. Figure 3 shows the dose-dependent effects of (+) catechin and () epicatechin on superoxide anion production, as evaluated by the SOD assay. The results in Table 1 show the effects of flavan-3-ols on XO activity. Similarly to flavonols, these compounds increased the production of superoxide radicals at the highest concentrations (50 mM), an effect that is likely due to an intrinsic pro-oxidant activity. Catechin was found to be less pro-oxidant than kaempferol. In fact, the latter molecule increased radical production at 25 mM. Conversely, at the same concentration, catechin reduced the radical production by the system (Table 1). These results are in agreement with those of Marozien_e (2012), reporting that flavonoids exhibit prooxidant cytotoxicity in mammalian cells due to the formation of free radicals and that this effect increases as the redox potential of the phenoxyl radical/phenol couple decreases. These data indicate that flavonols induce a stronger cytotoxic effect on mammalian cells than flavan-3ols. These results may be in part explained as follows: due to auto-oxidation, flavonols may produce a more
Flavonols and flavan-3-ols as modulators of XO and MnSOD
DOI: 10.3109/09637486.2014.931362
Kaempferol
O-semiquinone radical intermediate (K.−) OH
O−
.
HO
O
HO
+ O2
O
O2.− + 2H+ +
OH OH
5
O
O
O
OH
O HO
O
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C OH
O−
O O
O−
O
C
OH
OH
O2 −e− O
O
O
O2.− + O OH
OH
O-Quinone Figure 4. Auto-oxidation of kaempferol and quinone/quinomethide tautomerisation of kaempferol oxidate.
stable radical that can be delocalized on the C and A rings (Figure 4); instead, the semiquinone radical of catechins remains localized on the B ring. Hence, the semiquinone radical of flavan3-ols is less stabilized by resonance, and, as a consequence, the auto-oxidation reaction is less promoted (Figure 5a). According to Mochizuki et al. (2002), catechins undergo multi-step autooxidation at concentrations higher than 40 mM (Figure 5a), with the formation of a semiquinone radical on the B ring, as confirmed by ESR analysis. This radical may continue to react until it transforms into a quinone. The superoxide radical generated by the first reaction (Figure 5a) can react with other molecules of catechin (Figure 5b). This reaction is fostered by the strong oxidant activity of the superoxide radical (Ered ¼ 0.89 V) versus O2 (Ered ¼ 0.16 V). For this reason, O 2 itself participates into the auto-oxidation of catechins. At low concentrations (525 mM), catechin behaved as a radical scavenger of superoxide anion. At these concentrations, it is possible that the auto-oxidation of catechin (Figure 5a) was not complete but may be stopped at the level of semiquinone radical formation because the generated superoxide radical reacted with more molecules of catechin (Figure 5b), thus producing hydrogen peroxide and other semiquinone radicals (faster reaction). Among flavan-3-ols, catechins showed a radical scavenger activity that was stronger than epicatechins (Table 1). In fact, this effect was observed in the entire range of 25 mM to 1 mM.
Furthermore, epicatechin behaved as a radical scavenger only at a lower concentration (1 mM). The addition of SOD was able to inhibit the pro-oxidant activity of flavan-3-ols in a dosedependent manner (Table 2). At low concentrations, the effect of SOD was stronger because the radical-scavenging reaction (Figure 5b) might be accelerated by the disproportion of superoxide radical (Figure 5c).
Conclusions The results presented here show that marked differences can be observed between flavonoids with different structural characteristics with regard to their ability to inhibit or stimulate superoxide radical production through a modulation of XO and Mn-SOD. The study of the structure-activity relationship on these compounds showed that flavonols (quercetin and kaempferol) differ from flavan-3ols (catechin and epicatechin) in their pro-oxidant effect. The absence of the carbonyl group at C-4 and of the double bond between C-2 and C-3 of the heterocyclic ring resulted in a dose-dependent loss of the pro-oxidant effect. This may be due to the saturation of the double bond, which destroys the conjugation and coplanarity of the flavan-3ols structure. These data further demonstrate the opposite dose-dependent effects of flavonols on XO and Mn-SOD. At concentrations 75 mM, these molecules showed a high pro-oxidant activity, and the addition of Mn-SOD
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Figure 5. (a) Auto-oxidation reaction of catechin at higher concentrations; (b) free radical-scavenging behavior of catechin at low concentrations; (c) the dismutation of superoxide free radical by the enzyme superoxide dismutase.
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(a)
OH
O.
OH
HO
HO
O
O OH
O−
+ 2O2
O OH
OH
OH
Semiquinone form
+ 2O2.− + 2H+
Quinone form
.
OH
O
OH
HO
O
OH
OH
(b)
O
HO
HO
O
O
O−
.−
+ 2O2
H2O2
OH
OH OH
OH
Semiquinone form
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(c)
2O2.− + 2H+
SOD
was not able to abolish this effect. However, they acted as antioxidants at concentrations lower than 25 mM. These opposite dose-dependent effects we observed suggest exercising caution in the use of flavonols for clinical purposes. Unlike flavonols, flavan-3-ols showed antioxidant activity at all the tested concentrations. Our study demonstrates that two diastereoisomers exhibit different properties on XO but that no difference was observed following the addition of Mn-SOD. This study also shows that the a-hydroxyl on the C-3 of heterocyclic ring (catechin) is important for the radical-scavenging activity of flavan-3ols. Therefore, our results suggest that flavan-3ols, at low concentrations, may act as radical scavengers and therefore show strong antioxidant effects, whereas they may undergo auto-oxidation at higher concentrations, showing a weaker antioxidant potential.
Declaration of interest The authors declare that they have no conflicts of interest.
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