interest in the redox properties of natural products has stimulated many studies focusing on natural products that prevent the destructive effects caused by reactive oxygen species ( ros ) on many biomolecules ( 1 ) .
ros are also involved in controlling the pathogenesis of many degenerative diseases ( 2,3 ) and may activate redox sensitive transcription factors ( 4 ) . as a result , the maintenance of redox homeostasis is a crucial asset for survival of stress scenarios , in the tissues of both plants ( 5 ) and animals ( 2).the maintenance of physiological redox homeostasis is as essential for a cell as that of osmosis or ph . during evolution
, cells have developed complex interacting regulatory mechanisms that include enzymes and reducing metabolites that help to maintain the redox homeostasis ( 6 ) .
consequently , natural products have been and are screened for their antioxidant activity and , in this context , especially for their ros - scavenging properties .
this broad interest has led to the development of various assays to determine antioxidant activity ( 710 ) .
the hydroxyl radical ( oh ) occupies an exceptional position among ros because of its extreme reactivity and oxidative potential ; it attacks even inert compound such as alkanes ( 11 ) which are normally considered to be stable under physiological conditions .
one route to hydroxyl radicals is outlined by the fenton reaction ( reaction 1 ) which is catalyzed by transition metals ( e.g. , fe , cr , cu , or mn ) .
gutteridge and co - workers introduced an assay that allowed the detection of interactions of the test compounds with the formation of hydroxyl radicals by the fenton reaction ( 1214 ) .
this assay uses 2-deoxy - d - ribose as detection molecule , and it is utilized in phytochemistry and food chemistry to assess antioxidant properties of various compounds or extracts ( 10,1517 ) .
2-deoxy - d - ribose is degraded by hydroxyl radicals that are generated in the reaction mixture by the fenton reaction .
the amount of the degradation product , malonyldialdehyde ( mda ) , can be determined photometrically after a reaction with 2-thiobarbituric acid yielding a pink pigment .
benzoic or formic acids , rhodamine b and other substances were used as substrates to detect hydroxyl radical attack ( 18,19 ) ; they show , however , no special advantage compared to 2-deoxy - d - ribose .
the originally described reaction mixture contains hydrogen peroxide , iron(iii ) ions , ascorbic acid and 2-deoxy - d - ribose .
iron is added in two forms into the reaction mixture , either as fecl3 that can be chelated by tested substances or in the form of a fe(iii)edta complex .
the complex of iron with edta avoids complex formation with the tested substances , 2-deoxy - d - ribose ( 13 ) or ascorbic acid ( 20 ) , but does not prevent the participation of the iron in the fenton reaction . according to gutteridge ( 12,13 ) , redox active scavengers inhibit efficiently 2-deoxy - d - ribose degradation by hydroxyl radicals that were formed in the solution : the iron ions were complexed by edta . in the absence of edta , a portion of the iron ions is complexed by 2-deoxy - d - ribose .
accordingly , compounds with ligand properties compete for iron ions with the 2-deoxy - d - ribose molecules and thus decrease 2-deoxy - d - ribose degradation that is caused by iron - catalyzed hydroxyl radical attack .
the reactants can be dissolved only in water or inorganic buffers , because the hydroxyl radical reacts with most of the organic solvents and substances employed for buffer preparations ( 21 ) .
the kinetics of this iron(iii ) reduction may be the decisive factor ( 22,23 ) . thus ,
if ascorbic acid is omitted in the reaction mixture , the capability of the test compound itself to reduce iron(iii ) and start the fenton reaction can be also assessed ( 10 ) .
the aim of this paper is to introduce additional modifications of the 2-deoxy - d - ribose degradation assay that facilitate even more detailed insights into the possible reactions of the test compound than those that are already described in the literature .
the main innovations that these modifications offer are that ( i ) the omission of hydrogen peroxide allows determination of interactions with the autoxidation of ascorbic acid , and ( ii ) if ascorbic acid is also left out , the possible autoxidation of the tested substance itself may become evident .
the quality of the information that may be obtained by the newly introduced modifications of the 2-deoxy - d - ribose degradation assay are exemplified by juglone and quercetin ( figure 1 ) . for comparative reasons ,
the chosen ph is exactly the same as of the cytoplasm ( ph = 7.4 ) .
juglone ( 5-hydoxy-1,4-naphthoquinone ) is known as a phytotoxin and redox cycler ( 17,24 ) , and quercetin is a flavonoid aglycon widespread within the plant kingdom and well - known for its antioxidative and iron chelating properties ( 25,26 ) .
reputedly , quinones are good acceptors of electrons ( 27 ) whereas phenols are donors of electrons ( 28 ) .
the o - hydroxyl groups of ring b are usually the initial target of oxidants .
structural features that are required for complex stability with transition metals include the 3-hydroxy-4-keto grouping in ring c , the 5-hydroxy-4-keto arrangement of rings a and c , and the o - hydroxyl groups of ring b ( figure 2 ) ( 26,29 ) .
consequently , the antioxidant activities of flavonoids depend on their redox properties and chelation of transition metals
juglone , hydrogen peroxide , and 2-deoxy - d - ribose ( deoxyribose ) were obtained from fluka ( buchs , switzerland ) .
all other chemicals and organic solvents used were of analytical grade and purchased from sigma - aldrich inc .
juglone or quercetin was dissolved in an aqueous kh2po4/koh buffer solution ( 50 mm , ph 7.4 ) to yield final concentrations from 2 to 500 m ; to 125 l of this solution , 25 l of a 10.4 mm 2-deoxy - d - ribose solution in the same buffer system and 50 l of an aqueous solution of fecl3 ( 50 m ) were added . in one series ,
those 50 l contained 52 m edta dissolved in buffer , which was premixed with the aqueous fecl3 solution ( 1:1 v / v ) . in the other series , the edta solution was replaced by the same volume of the buffer . in the first series , edta complexed the iron ions , preventing them from being chelated by the test compound ; in the second series , the iron ions were complexed by the test compound . to start the fenton reaction ,
various reactants dissolved in the above - mentioned buffer systems or in water were added : 25 l of 10.0 mm aqueous solution of h2o2 and 25 l of 1.0 mm ascorbic acid in buffer .
standard 1.5 ml sample vials ( la - pha - pack , werner reifferscheidt gmbh , langerwehe , germany ) were used as reaction vials .
thereafter , 10 l of 2.5% ethanolic butylated hydroxytoluene solution ( w / v ) followed by 250 l of 1.0% 2-thiobarbituric acid dissolved in 3% trichloroacetic acid ( w / v ) was added to each vial to detect malonyldialdehyde , the decomposition product of 2-deoxy - d - ribose caused by the attack of hydroxyl radicals .
the vials were vortexed and heated in a water bath at 85 c for 30 min .
the reaction was stopped by transferring the vials into an ice water bath for 3 min . to extract the reaction product of mda and thiobarbituric acid ,
600 l of n - butanol was added , and the mixture was rigorously vortexed .
the butanol layers of the vials , each 350 l , were pipetted into flat bottomed 96 well plates ( greiner , kremsmnster , austria ) and the absorbance was determined with a microplate reader ( tecan infinite m200 , mnnedorf , switzerland ) at 532 nm .
the blank contained the full reaction mixture except 2-deoxy - d - ribose ( negative control ) .
this modification was carried out without the addition of ascorbic acid , which was replaced by the same volume of the buffer .
scoring was performed after 1 h. the blank contained the full reaction mixture except 2-deoxy - d - ribose ( negative control ) .
the positive control was the h2o2/fe / ascorbic acid system mixture lacking the test compound ( 100% mda ) .
consequently , scoring was performed only after 16 h. the blank ( negative control ) contained the full reaction mixture without 2-deoxy - d - ribose ( 0% mda ) .
the positive control was the h2o2/fe / ascorbic acid system mixture lacking the test compound ( 100% mda ) .
this modification of the deoxyribose assay was carried out without the addition of h2o2 and ascorbic acid , which were replaced by the same volume of the buffer or water .
the blank contained the full reaction mixture except 2-deoxy - d - ribose ( negative control ) .
the positive control was the h2o2/fe / ascorbic acid system mixture lacking the test compound ( 100% mda ) .
for all systems , possible interferences of tested compounds with the mda detection procedure in the assay systems were checked before the experiments ( 8) .
rockville , md ) was used to perform analyses of variance ( anova ) with duncan s multiple range tests at a confidence level of 95% .
all reactions that are presented in the ongoing text are hypotheses that aim to explain the results which were obtained from the deoxyribose degradation assay . these reactions
the fenton reaction generates hydroxyl radicals after reduction of iron(iii ) by ascorbic acid ( 21 ) .
juglone and quercetin proved to be strong inhibitors of 2-deoxy - d - ribose degradation in both variants of this experiment , with and without addition of edta ( figure 3a , b ) .
additionally , juglone showed a very weak pro - oxidative effect in the concentration 500 m in the variant without edta ( figure 3a ) .
several interactions are possible : ( i ) the hydroxyl group can reduce hydroxyl radical to water by an one electron transfer ( figure 4 ) ; ( ii ) the quinone part can oxidize hydrogen peroxide to molecular oxygen and/or ascorbic acid to dehydroascorbic acid by two electron transfers ( figure 4 ) ; ( iii ) juglone may be reduced to trihydroxynaphthalene by two electron transfers , a further reducing agent that scavenges any ros by either one or two electron transfers ( figures 4 and 5 ) .
the absence of hydrogen peroxide and ascorbic acid prevented the initialization of fenton reaction in both variants ( with and without edta ) when the scoring was performed within one hour .
juglone showed pronounced antioxidative effects by decreasing the deoxyribose degradation in both systems , either with or without addition of edta . in the latter system , however , in 500 m , juglone again increased the concentration of mda compared to the concentration of 250 m .
the following chemistry is possible : in the concentrations 2250 m juglone removes the iron ions from the competive ligand 2-d - deoxyribose and concomitantly scavenges arising ros .
juglone is a 5-hydroxy-1,4-naphthoquinone , and the 5-hydroxy-4-carbonyl moiety of the juglone molecule complexes iron ions ( figure 2 ) ( 29 ) .
most flavonoids show the same combination of functional groups that are well - known for chelation of transition metals ( 30 ) .
activities of juglone and quercetin in various systems of the deoxyribose degradation assay , quantified in % malonyldialdehyde , an oxidative decomposition product of 2-deoxy - d - ribose ; bars are means ; error bars , standard deviation ; n = 3 , letters indicate different levels of significance ( 95% probability , duncan s multiple range test ) .
selected possible one electron and two electron redox reactions of phenolic and quinone groups of juglone .
aa , ascorbic acid ; daa , dehydroascorbic acid . selected possible one electron redox reactions of quinone and semiquinone groups of juglone .
the weak pro - oxidative effect observed ( figure 3a ) in the concentration 500 m in the variant without addition of edta most probably is caused by the different redox properties of ironjuglone complex .
the reduction of iron(iii ) in the ironjuglone complex might proceed more easily compared to the complex of fe(iii)edta if trihydroxynaphthalene or higher concentrations of juglone are present ( figure 6 ) .
this interpretation offers a possible explanation for the different behavior of juglone in the two variants of the assay at the highest concentration tested , 500 m .
low solubility of higher concentrations than that precluded the testing of higher dosages of juglone .
possible one electron and two electrons reactions of juglone are shown in figures 4 and 5 . reduction of the iron(iii)juglone complex by trihydroxynaphthalene or juglone .
quercetin is well - known as an efficient reducing agent and chelator of transition metals ( 31 ) .
both characteristics are visible in the results of the assay : the decrease of the relative mda concentrations depends on the quercetin concentrations in both variants of the assay ( figure 3b ) ( 12,13 ) .
quercetin can reduce not only the highly reactive hydroxyl radical but also the more stable hydrogen peroxide to water ( figures 7 and 8) .
scavenging of free radicals and chelation of iron both contribute to the antioxidant activity of flavonoids .
this assay suggests that the redox properties of quercetin are more responsible for the observed antioxidative effect than the chelation of iron .
the protection of deoxyribose against attack by hydroxyl radicals was more efficient in the variant with edta added ( figure 3b ) .
oxidation products of quercetin , such as phenolic acids , their esters and other derivatives ( 32 ) with similar antioxidant and/or chelation properties , however , could also participate in the previously outlined reactions .
this assay system explores if the tested compound can substitute for the function of ascorbic acid and start the fenton reaction by reduction of iron(iii ) ( 10 ) .
hydrogen peroxide may also reduce iron(iii ) ions ( reaction 3 ) and initiate the fenton reaction
superoxide anion radicals ( o2 ) undergo spontaneous dismutation to hydrogen peroxide ( reaction 4 )
. the speed of this reaction may be increased by phenolic complexes of the metals that function as catalysts ( 33 ) .
juglone demonstrated negligible antioxidant activity in the variant of the assay without addition of edta , especially in the concentrations 500 and 250 m ( figure 3c ) .
in the variant with addition of edta , the concentration of 250 m indicates the emergence of a weak pro - oxidative effect .
quercetin definitely had no effects in either variant of this assay ( figure 3d ) . in summary ,
the obtained results suggest that neither juglone nor quercetin can promote the 2-deoxy - d - ribose degradation in the presence of hydrogen peroxide . here , ascorbic acid undergoes autoxidation in the presence of transition metals and , as a result , produces ros ( reactions 7 and 8) ( 20,34 ) , but compared to the previously described systems the speed of the whole process is slower because it depends on the diffusion rate of atmospheric oxygen into the reaction liquid .
the time point for scoring thus was extended to 16 h. if the iron ions form a complex with ascorbic acid  this is the case if no edta is added 
the reduction of iron ions and molecular oxygen increases ( 0 concentrations in figure 3e , f ) , and , thus , more ros are formed that may enter the haberweiss and fenton reactions . by contrast , buettner reported the opposite phenomenon ( 20 ) : the iron(iii)edta complex increased the speed of the ascorbic acid autoxidation reaction .
it is important to pay attention to the fact that buettner studied the decrease of ascorbic acid , not that of 2-deoxy - d - ribose .
2-deoxy - d - ribose most probably competes with ascorbic acid as ligand for iron .
this affects the portion of the iron ions that are complexed by the 2-deoxy - d - ribose molecules ( 12 ) . if this is the case , then the possibility that formed hydroxyl radicals directly attack 2-deoxy - d - ribose increases ( 13 ) .
juglone boosted the pro - oxidative effect of ascorbic acid in both variants of the assay .
the amplification of the activity was stronger in the variant without addition of edta ( figure 3e ) .
the effect increased in concentrations 63125 m and slightly decreased in concentrations from 250 to 500 m .
the redox properties of the ironjuglone complex and the reduction activity of the hydroxyl group of juglone and the potentially formed trihydroxynaphthalene contribute most probably to the pro - oxidative effect that is evident in this system of the assay .
trihydroxynaphthalene may arise following reduction by ascorbic acid , superoxide anion radical or hydrogen peroxide .
juglone and its reduced derivative , trihydroxynaphthalene , participate in the redox cycling of iron(iii)/iron(ii ) besides of ascorbic acid ( figures 4 and 5 ) .
this effect was visible in both of the variants with and without addition of edta .
these results suggest that the most important contribution was the more easy reduction of the ironjuglone complex compared to that of the feedta complex .
this effect , however , is not fully visible in the above - mentioned systems because of the presence of hydrogen peroxide that may undergo similar reactions from the start of the assay . in this system ,
hydrogen peroxide is generated by concomitant activities of juglone , ascorbic acid and iron ions .
by contrast , this is not the case in the classic system of the 2-deoxy - d - ribose degradation assay
. there exists , however , an assay system that uses phospholipid liposomes for detection of hydroxyl radicals ( 34,35 ) ; it uses a similar setup as in this modified system of the 2-deoxy - d - ribose degradation assay . in the variant
without addition of edta , the potential shift in the redox properties may cause the strong pro - oxidative effects observed in the 63 and 125 m concentrations where iron might be better available for fenton reaction .
the decrease of the pro - oxidative effect in the higher concentrations may be caused by changing concentrations of reactants and reaction products .
quercetin inhibited the pro - oxidative activity of ascorbic acid more or less acting as an antioxidant .
in contrast to juglone , this effect was more apparent in the variant without addition of edta ( figure 3f ) .
it is possible that both mechanisms , scavenging of ros and chelation of iron ions , contributed to the observed effect .
the results from the variant without addition of edta indicate that the contribution of complexation of iron ions may be higher ; ironquercetin complexes decrease the activity of iron ions in the fenton reaction as their reduction seems to be more difficult ( 36 ) .
further studies are required to explore if quercetin protects the iron ions against reduction by ascorbic acid by acting as more efficient competitor in their chelation .
the coupling reactions between semiquinones and monodehydroascorbic acid also may influence the activities of both juglone and quercetin ( 37 ) .
the principle is similar to the system where ascorbic acid generates ros , but here the test compound replaces ascorbic acid ( reactions 911).alternatively , the following reaction is also possible : iron(ii ) ions can reduce molecular oxygen into superoxide anion radicals ( reaction 8) .
complex formation between the tested substance and iron ions plays a key role ; in the various complexes , iron ions possess variable redox properties that affect their participation in oxidation or reduction reactions ( reactions 12 and 13 ) .
similar to the last system described , scoring was performed after 16 h because the system also depends on the diffusion of molecular oxygen from the air into the reaction mixture .
juglone showed notable pro - oxidant activity that was more pronounced in the variant without edta addition .
the efficient concentrations were 63500 m for the variant without edta addition and 125500 m for variant with edta addition .
the results suggest that juglone reduced iron(iii ) to iron(ii ) ions , either directly by its hydroxyl group or indirectly by superoxide anion radicals that were formed by reduction of molecular oxygen ( figures 4 and 5 ) .
the reduction of molecular oxygen to the superoxide anion radical and its subsequent dismutation generates hydrogen peroxide which initiates the fenton reaction ( figure 4 ) .
the reduction activity of juglone explains the pro - oxidative effect in both variants of this assay system .
the higher pro - oxidative effect in the variant without addition of edta suggests that iron ions are more easily reduced in the complex with juglone than in the complex with edta .
the pro - oxidative effect of quercetin was more evident in the variant with addition of edta than in the variant without addition of edta ( figure 3h ) .
in the variant without addition of edta , the pro - oxidative effect was only apparent in the lower concentrations ( 463 m ) .
the results suggest that quercetin affects the degradation of 2-deoxy - d - ribose in this system assay by chelation of iron ions and strong redox properties . in the variant without the addition of edta , iron was chelated by quercetin and this prevented the reduction of iron(iii ) to iron(ii ) . in the variant with addition of edta
quercetin may also interact with molecular oxygen by transfer of either one or two electrons ( figures 7 and 8) ; this generates hydrogen peroxide .
it is suggested that hydrogen peroxide may arise mainly through the superoxide anion radical intermediate ( 28,38 ) .
reduction of molecular oxygen and iron(iii ) started the fenton reaction in the variant without the addition of edta .
all modified systems of the 2-deoxy - d - ribose degradation assay demonstrated that the chosen test compounds , juglone and quercetin , enter complex redox reactions depending on the presence and absence of other components in the assay system . in the author s view , this set of assays provides easy and cost - effective characterization of the potential pro- and antioxidative effects of the test compound in changing milieus , a highly critical issue in the characterization of the redox chemistry of a test compound .
electron transfer reactions determined by fenton chemistry are highly milieu - dependent and this especially merits attention in the classification of antioxidative properties of tested compounds or compound mixtures .
edta , ethylenediaminetetraacetic acid ; feedta , chelate of edta with iron ions ; mda , malonyldialdehyde ; ros , reactive oxygen species .
 interest in the redox properties of natural products has led to the development of various assays for the detection of antioxidant activities and ros - scavenging properties . here , additional modifications of the 2-deoxy - d - ribose degradation assay are introduced that specifically allow the determination of interactions of the test compound with the autoxidation of ascorbic acid and the autoxidation of the test compound itself . to illustrate this , 
 juglone and quercetin were used as examples . 
 the modified assay systems provide insights into their specific antioxidative and pro - oxidative properties . in additional , 
 an extensive characterization of the redox properties of their complex with iron is possible , if iron ions are added in the free form or complexed with edta . 
 the jugloneiron complex proved to be pro - oxidative in a wider range of milieus than the quercetiniron complex . 
Introduction
Materials and Methods
Results and Discussion
Abbreviation Used