1108 Biol. Pharm. Bull. 26(8) 1108—1114 (2003) Vol. 26, No. 8

Oxidation of Flavonoids which Promote DNA Degradation Induced by Bleomycin–Fe Complex

Narumi SUGIHARA,* Arisa KANEKO, and Koji FURUNO Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; Sanzou, Gakuen-cho, Fukuyama, Hiroshima 729–0292, Japan. Received January 28, 2003; accepted May 15, 2003

Sixteen flavonoids including and and their relatives were examined for their ability to promote DNA degradation induced by the bleomycin (BLM)–Fe complex. Three hydroxyl groups in the flavonoidal nucleus were proposed as a crucial structural requirement for effectively promoting DNA degrada- tion: 1) the C7-hydroxyl substitution in the A-ring; 2) the C4؅-hydroxyl substitution in the B-ring; and 3) the C3- hydroxyl substitution in the C-ring. Flavonoids, which lack even one of these hydroxyl substitutions, showed re- pϽ0.001) between activity to promote ,0.920؍markably diminished activity. There was a good correlation (r DNA degradation and oxidizability, which was measured following the Fe(III)-induced oxidation of flavonoids themselves, among the 16 flavonoids. The oxidizability of flavonoids which have the crucial hydroxyl substitu- tions, was remarkably enhanced in the presence compared with the absence of BLM. On the other hand, the ex- tent of oxidation of flavonoids lacking these substitutions was enhanced little or not at all by BLM. No correla- tion between the Fe(III)-reducing activity and DNA degradation-promoting activity was found among flavonoids satisfying the crucial structural requirements. Furthermore, the correlation between the extent of oxidation of flavonoids and the Fe(III)-reducing activity was not confirmed among these flavonoids. Therefore, it was sug- gested that Fe(III)-reducing activity was not the only factor determining DNA degradation-promoting activity in flavonoids having the three hydroxyl groups necessary for effectively promoting DNA degradation induced by BLM–Fe complex. Key words flavonoid; bleomycin; DNA degradation; Fe(III)-reducing activity; pro-oxidant

The dietary intake of flavonoids, which are widely distrib- their Fe(III)-reducing activity. uted in fruits and vegetables, is currently believed to have preventive effects on a variety of diseases associated with MATERIALS AND METHODS free radical-mediated damage in pathologically generating processes.1—4) The antioxidant activity of flavonoids is con- Materials Materials and chemical reagents were ob- sidered to be responsible for some of the pharmacological ef- tained from the following companies: flavonoids from Fu- fects. However, flavonoids are known to behave as pro-oxi- nakoshi Co. (Tokyo, Japan); bleomycin hydrochloride (BLM) dants.5—9) One such example was the finding that quercetin and deoxyribonucleic acid sodium salt from salmon sper- or accelerated DNA degradation which was in- mary, and Ferric chloride from Wako Pure Chemical Indus- duced by bleomycin (BLM)–Fe complex.10) tries, Ltd. (Osaka, Japan). All chemicals used were of the BLM exerts antitumor activity by causing the oxidative highest purity available. cleavage of DNA strands. Fe ion, oxygen and a suitable re- BLM–Fe Complex-Induced DNA Degradation The re- ducing agent are essential cofactors for BLM-mediated DNA action mixtures consisted of flavonoids at concentrations 11—13) degradation. BLM possesses functional domains for ranging from 5 to 100 m M: 0.5 mg of DNA, 10 mM MgCl2, binding DNA and Fe ion in its structure. The BLM–Fe(II) 5 mg of bleomycin, and 50 m M FeCl3 in a final volume of complex with dioxygen undergoes a transformation to gener- 1.0 ml of 50 mM Tris–HCl buffer, pH 7.4. Flavonoids were ate so-called “activated BLM” by the addition of an electron dissolved in dimethyl sulfoxide. The final concentration of provided by another BLM–Fe(II) complex or by a reducing dimethyl sulfoxide was less than 1%, which had no de- agent. Activated BLM decomposes and releases an active tectable effect on DNA degradation. Incubations were started radical, which is regarded as the substance causing the oxida- with the addition of FeCl3 to reaction mixtures and continued tive cleavage of DNA strands. BLM–Fe(II) is transformed to for 1 h at 37 °C. The DNA degradation-promoting activity of BLM–Fe(III) via these processes.14,15) Some flavonoids have flavonoids was evaluated as malondialdehyde (MDA) equiva- the capability to reduce metal ions such as Fe(III) and Cu(II) lents. Thiobarbituric acid reactive substances (TBARS) ions. Thus, flavonoids are considered to accelerate the rate of which arose from deoxyribose degradation of DNA were as- redox-cycling of the BLM–Fe complex owing to their metal- sessed by a modification of the Uchiyama and Mihara reducing ability.10,16—18) However, no systematic study on the method.19) Briefly, to 1.0 ml of the reaction mixture in a correlation of the DNA degradation-promoting activity of 12 ml glass tube, 2.5 ml of 1% phosphoric acid and 1 ml of flavonoids with their Fe(III)-reducing activity has been re- 0.67% thiobarbituric acid were added. The tube was capped ported until now. with a screw cap and heated at 100 °C for 35 min. After cool- In the present study, we have characterized in detail the ing in ice water, 3 ml of n-butanol was added. The mixture structure–activity relationships governing the promoting ef- was then shaken and centrifuged to separate the organic fect of flavonoids on DNA degradation induced by the layer. The fluorescence intensities in the butanol layer were BLM–Fe complex. We also studied whether the ability of measured at excitation and emission wavelengths of 515 and flavonoids to promote DNA degradation was correlated with 553 nm, respectively.

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2003 Pharmaceutical Society of Japan August 2003 1109

UV–Visible Spectral Measurement of Flavonoids duced DNA degradation was examined at concentrations UV–visible spectra were measured with a UV–visible spec- ranging from 5 to 100 m M. As shown in Fig. 2A, myricetin, trophotometer (Jasco UV/VIS V-550). Flavonoids were quercetin, fisetin, and kaempferol effectively promoted added at a final concentration of 20 m M to 2.0 ml of 50 mM the DNA degradation. The DNA degradation promoted by

Tris–HCl buffer at pH 7.4 containing 10 mM MgCl2 with and myricetin, quercetin, fisetin or kaempferol sharply increased without 10 mg of bleomycin hydrochloride. The spectral trac- in a dose-dependent manner up to about 20 to 50 m M and de- ing was started by the addition of 50 m M FeCl3. After the ad- creased at higher concentrations. The concentration that pro- dition of Fe(III), spectra were recorded every 6 min for 1 h duced the maximum enhancement of DNA degradation was (trace 1 to 11 in Fig. 3). The change of spectral absorption at 20 m M for myricetin, quercetin and fisetin, and 50 m M for the peak in the longest wavelength area was calculated from kaempferol, respectively. Morin promoted the degradation the difference of trace 1 and 11. as its concentration increased up to 100 m M. In terms of the Fe(III) Reducing Activity Fe(III) reduced by flavonoids efficiency of their promoting activity at 20 m M, the flavo- was evaluated by colorimetric determination of Fe(II) nols ranked as follows: kaempferolӷquercetinϾmyricetinϾ chelated with bathophenanthroline disulfonic acid.20) fisetinϾmorin. Flavonoid at 20 m M was added to 2.0 ml of 25 mM citrate Figure 2B shows the DNA degradation-promoting activity buffer, pH 7.0, containing 50 m M FeCl3. Bathophenanthroline of quercetin-related flavonols including , isorham- disulfonic acid was added at a final concentration of 0.5 mM netin and geraldol. Rhamnetin, in which the C7 hydroxyl to the mixture. The absorbance of the bathophenanthroline substitution in the A-ring of quercetin is methylated, had disulfonic acid–Fe(II) complex was read at 540 nm after 31% of the activity of quercetin at 20 m M. In contrast, 30 min. For the total bathophenanthroline disulfonic , CЈ3 hydroxyl-methylated quercetin, promoted acid–Fe(II) complex concentration, a 0.25% (w/v) hydroxyl- DNA degradation 2.6 times as much as quercetin. Geraldol, ammonium chloride solution was used instead of the lacking the C5-hydroxyl substitution in the A-ring of flavonoid solution. The percentage of Fe(III) reduced by the isorhamnetin, promoted DNA degradation remarkably. How- flavonoid was calculated using the following equation: ever, the degradation promoted by geraldol was less exten- sive than that by isorhamnetin. percentage of Fe(III)-reductionϭ(C /C )ϫ100 FL HA Figure 2C shows the activity of kaempferol-related where CHA is the concentration of Fe(II), reduced by the ad- flavonols including kaempferide, and 5-deoxy- dition of 0.25% (w/v) hydroxylammonium chloride solution, kaempferol. Both kaempferide and galangin, in which the Ј and CFL is the concentration of Fe(II), reduced by 20 m M 4 -hydroxyl substitution in the B-ring is methylated and flavonoid. absent, respectively, were scarcely effective. 5-Deoxy- Statistical Analysis The data in the figures are given as kaempferol, lacking the C5-hydroxyl substitution in the the meanϮS.D. of three to five experiments. Differences be- A-ring of kaempferol, had 58% of the promoting activity of tween treatment groups and a control group were determined with Dunnett’s test using Stat-100 (BIOSOFT, U.K.). A p value of less than 0.05 was considered significant.

RESULTS

The Activity of Flavonoids to Promote DNA Degrada- tion Induced by BLM–Fe Complex The ability of 16 flavonoids (Fig. 1, Table 1) to promote BLM–Fe complex-in- Fig. 1. Basic Structures of Flavonoids

Table 1. Variation in Number and Arrangment of the Hydroxyl Substitutions among Flavonoids

Substitutions Compound C3 C5 C6 C7 C2Ј C3Ј C4Ј C5Ј

Myricetin OH OH H OH H OH OH OH Quercetin OH OH H OH H OH OH H OH H H OH H OH OH H Morin OH OH H OH OH H OH H Kaempferol OH OH H OH H H OH H

Rhamnetin OH OH H OCH3 HOHOHH Isorhamnetin OH OH H OH H OCH3 OH H Geraldol OH H H OH H OCH3 OH H 5-Deoxykaempferol OH H H OH H H OH H

Kaempferide OH OH H OH H H OCH3 H Galangin OH OH H OH H H H H Luteolin H OH H OH H OH OH H

Diosmetin H OH H OH H OH OCH3 H Apigenin H OH H OH H H OH H Chrysin H OH H OH H H H H Baicalein H OH OH OH H H H H 1110 Vol. 26, No. 8

Fig. 2. Effects of (A), Quercetin-Related Flavonols (B), Kaempferol-Related Flavonols (C) and Flavones (D) on DNA Degradation Induced by BLM–Fe complex

The reaction mixtures containing 0.5 mg of DNA, 10 mM MgCl2, 5 mg of BLM and 50 m M FeCl3 were incubated with flavonoids at concentrations ranging from 5 to 100 m M in a final volume of 1.0 ml of 50 mM Tris–HCl buffer at pH 7.4. After the incubation for 1 h at 37 °C, the DNA degradation was evaluated based on the amount of MDA produced by de- oxyribose degradation. The amount of MDA produced by BLM–Fe without flavonoids was about 0.5 nmol. The values represent the meanϮS.D. of at least three separate experi- ments. Flavonoids: myricetin (᭺), quercetin (᭹), fisetin (᭝), morin (᭜) and kaempferol (᭡) in A; quercetin (᭹), rhamnetin (᭺), isorhamnetin (᭿) and geraldol (ᮀ) in B; kaempferol (᭡), 5-deoxykaempferol (᭝), kaempferide (᭢) and galangin (᭞) in C; luteolin (᭹), diosmetin (᭺), apigenin (᭿), chrysin (ᮀ) and baicalein (᭝) in D. kaempferol at 20 m M. or the absence of BLM (data not shown). The results for the Figure 2D shows the promoting activity of flavones that flavonoids tested are summarized in Table 2. In the absence lack the C3-hydroxyl substitution in the C-ring such as lute- of BLM, myricetin, quercetin and fisetin, which possessed a olin, diosmetin, apigenin, chrysin and baicalein. Luteolin and catechol or pyrogallol moiety, exhibited a large decrease in baicalein were barely effective. Apigenin, chrysin and dios- absorption. metin rather inhibited the DNA degradation. The extent of the oxidation of flavonoids satisfying the These results indicate the following three hydroxyl substi- structural requirements for promoting DNA degradation was tutions in the flavonoidal nucleus are essential for effectively remarkably enhanced in the presence compared with the ab- promoting DNA degradation induced by the BLM–Fe com- sence of BLM. In particular, isorhamnetin, geraldol and plex: 1) the C7-hydroxyl substitution in the A-ring, 2) the kaempferol, which were very active in promoting DNA C4Ј-hydroxyl substitution in the B-ring and 3) the C3-hy- degradation in the presence of BLM, exhibited a remarkable droxyl substitution in the C-ring. Furthermore, the 5-hy- increase in oxidation by the addition of BLM. On the other droxyl substitution in the A ring is likely to be necessary for hand, flavonoids lacking these structural requirements more effectively promoting the DNA degradation. showed little change in their absorption spectrum in both the Fe(III)-Induced Oxidation of Flavonoids in the Pres- presence and absence of BLM. As shown in Fig. 4, there was ence and Absence of BLM The oxidation of flavonoids a good correlation between the extent of oxidation of was evaluated as the decrease in spectral absorption in the flavonoids and the activity to promote DNA degradation in long wavelength area (330—380 nm) of flavonoids induced the presence of BLM (rϭ0.920, pϽ0.001) among the sixteen by the addition of Fe(III) in the presence and absence of flavonoids. BLM. On addition of Fe(III) to the solution containing Fe(III)-Reducing Activity of Flavonoids The Fe(III)- flavonoids, the absorption peak in the long wavelength area reducing activity of flavonoids was measured by the colori- rapidly migrated to a longer wavelength and decreased with metric determination of reduced Fe(II) which was chelated time. The change in spectral absorption of kaempferol is with bathophenanthroline disulfonic acid. Table 3 shows the shown as an example in Fig. 3. The spectral tracing was re- Fe(III)-reducing activity of the 16 flavonoids at a concentra- peated every 6 min for 1 h from traces 1 to 11 after the addi- tion of 20 m M. Flavonoids having good metal-reducing abili- tion of Fe(III). The decrease in absorption was calculated ties such as isorhamnetin or geraldol exhibited strong activity from the difference of traces between 1 and 11 at the peak in to promote DNA degradation. Morin, which had the weakest the long wavelength area. The oxidation of flavonoids was DNA degradation-promoting activity at 20 m M, had the low- not influenced by the addition of DNA in either the presence est level of Fe(III)-reducing activity among flavonoids having August 2003 1111

Fig. 3. Fe(III)-Induced Changes of Spectral Absorption of Kaempferol in the Presence (A) and Absence (B) of BLM

The reaction mixture containing 20 m M flavonoid and 10 mM MgCl2 in 2.0 ml of 50 mM Tris–HCl buffer, pH 7.4 with and without 10 mg of bleomycin was kept at 25 °C. The spectral tracing was started with the addition of 50 m M FeCl3 and spectra of the flavonoids were recorded every 6 min for 1 h (trace 1 to 11).

Table 2. Oxidation of Flavonoids in the Presence and Absence of BLM

Difference Difference Flavonoids (20 m M) Flavonoids (20 m M) BLM (ϩ) BLM (Ϫ) BLM (ϩ) BLM (Ϫ)

Myricetin 0.066Ϯ0.007 0.052Ϯ0.008 Rhamnetin 0.021Ϯ0.007 0.008 Quercetin 0.066Ϯ0.005 0.032Ϯ0.007 Kaempferide 0.014Ϯ0.001 0.011 Fisetin 0.052Ϯ0.008 0.029Ϯ0.006 Galangin 0.019Ϯ0.003 0.004 Morin 0.088Ϯ0.013 0.012Ϯ0.001 Luteolin 0.009Ϯ0.005 0.005 Kaempferol 0.109Ϯ0.024 0.016Ϯ0.006 Diosmetin n.d. n.d. Isorhamnetin 0.131Ϯ0.009 0.023Ϯ0.005 Apigenin 0.002 0.002 5-Deoxykaempferol 0.062Ϯ0.018 0.010Ϯ0.002 Chrysin n.d. n.d. Geraldol 0.095Ϯ0.009 0.025Ϯ0.001 Baicalein 0.009Ϯ0.003 0.009

The difference was calculated from traces of 1 and 11 at the peak in the longest wavelength area. n.d. not detected. The values are meansϮS.D. of at least three separate exper- iments.

Fig. 4. Correlation between the Oxidation of Flavonoids in the Presence of BLM and Their Activity to Promote the Degradation of DNA Induced by BLM–Fe Complex for All Flavonoids Tested (A) and Flavonoids Which Have Essential Structures (B)

The values are for the oxidation in the presence of BLM and DNA degradation-promoting activity of flavonoids at a concentration of 20 m M. Flavonoids: flavonoids which have essential structures (᭹) and flavonoids which lack these structures (᭡). myricetin (Myr), quercetin (Que), fisetin (Fis), morin (Mor), kaempferol (Kaem), isorhamnetin (IsoRham), 5-deoxykaempferol (5-deoxy Kaem), geraldol (Ger), rhamnetin (Rham), kaempferide (Kaemd), galangin (Gal), luteolin (Lut), diosmetin (Dios), apigenin (Api), chrysin (Chr) and baicalein (Bai). rϭ0.920, pϽ0.001 in A and rϭ0.844, pϽ0.01 in B. 1112 Vol. 26, No. 8 these hydroxyl substitutions. Furthermore, apigenin, chrysin Therefore, a correlation (rϭ0.320) between the Fe(III)-re- and deosmetin, which inhibited DNA degradation induced by ducing activity and DNA degradation-promoting activity was the BLM–Fe complex, were inactive. Thus, there was a sig- not confirmed among flavonoids satisfying the structural re- nificant correlation between the Fe(III)-reducing activity and quirements for promoting DNA degradation (Fig. 5B). the DNA degradation-promoting activity among the 16 As shown in Fig. 6B, the extent of oxidation of flavonoids flavonoids (rϭ0.706, pϽ0.01, Fig. 5A). However, myricetin satisfying the structural requirements correlated well with the which had the strongest Fe(III)-reducing activity was less ac- Fe(III)-reducing activity in the absence of BLM (rϭ0.854, tive than isorhamnetin and geraldol. Despite having weak pϽ0.01). In contrast, no correlation (rϭ0.014) was found be- low Fe(III)-reducing activity, kaempferol promoted remark- tween the oxidation of flavonoids and their Fe(III)-reducing ably DNA degradation induced by the BLM–Fe complex. activity in the presence of BLM (Fig. 6A).

Table 3. Fe(III)-Reducing Activity of Flavonoids DISCUSSION

Flavonoids Fe(III)-reducing Flavonoids Fe(III)-reducing The ability of flavonoids to promote the degradation of (20 m M) activity (%) (20 m M) activity (%) DNA induced by the BLM–Fe complex was examined partic- Myricetin 95.8Ϯ2.9 Rhamnetin 62.5Ϯ0.7 ularly in relation to the number and pattern of hydroxyl sub- Quercetin 73.9Ϯ2.8 Kaempferide 9.3Ϯ1.4 stitutions in the flavonoidal nucleus. The DNA degradation- Fisetin 68.1Ϯ1.1 Galangin 6.6Ϯ0.5 promoting activity was vastly different among the flavonoids. Morin 34.9Ϯ1.1 Luteolin 34.0Ϯ1.4 Kaempferol 53.3Ϯ5.1 Diosmetin 0.2Ϯ0.1 Unexpectedly, isorhamnetin and kaempferol which have only Isorhamnetin 81.6Ϯ2.5 Apigenin 0.2Ϯ0.1 one free hydroxyl substitution in the B-ring were more active 5-Deoxykaempferol 41.0Ϯ1.6 Chrysin 0.6Ϯ0.2 than myricetin and quercetin which are strong reducers due Geraldol 82.6Ϯ2.7 Baicalein 44.8Ϯ6.3 to their pyrogallol and catechol moiety, respectively, in the B- ring. Quercetin lost its DNA degradation-promoting activity The values are meansϮS.D. for at least three separate experiments. with methoxylation at position C7 in the A-ring as shown in

Fig. 5. Correlation between the Fe(III)-Reducing Activity and Activity to Promote DNA Degradation Induced by BLM–Fe Complex for All Flavonoids Tested (A) and Flavoniods Which Have Essential Structures (B)

The values are for Fe(III)-reducing activity and DNA degradation-promoting activity of flavonoids at a concentration of 20 m M. Flavonoids: flavonoids which have essential struc- tures (᭹) and flavonoids which lack these structures (᭡). myricetin (Myr), quercetin (Que), fisetin (Fis), morin (Mor), kaempferol (Kaem), isorhamnetin (IsoRham), 5-de- oxykaempferol (5-deoxy Kaem), geraldol (Ger), rhamnetin (Rham), kaempferide (Kaemd), galangin (Gal), luteolin (Lut), diosmetin (Dios), apigenin (Api), chrysin (Chr) and baicalein (Bai). rϭ0.706, pϽ0.01 in A and rϭ0.320 in B.

Fig. 6. Correlation between the Fe(III)-Reducing Activity and the Oxidation of Flavonoids Which Have Essential Structures in the Presence (A) and Ab- sence (B) of BLM

The values are for Fe(III)-reducing activity and the oxidation of flavonoids at 20 m M. Flavonoids: myricetin (Myr), quercetin (Que), fisetin (Fis), morin (Mor), kaempferol (Kaem), isorhamnetin (IsoRham), 5-deoxykaempferol (5-deoxy Kaem), geraldol (Ger). rϭ0.014 in A and rϭ0.854, pϽ0.01 in B. August 2003 1113 rhamnetin, whereas methoxylation at position C3Ј in the B- an important role in the donation of the two electrons since ring rendered the promoting activity more effective as shown flavones hardly display any decrease in absorption at this in isorhamnetin. Kaempferol remarkably lost its promoting wavelength. A good correlation (rϭ0.920, pϽ0.001) be- activity when the C4Ј-hydroxyl substitution in the B-ring was tween the extent of oxidation of flavonoids and their DNA blocked by methoxylation, as shown in kaempferide. Another degradation-promoting activity was recognized among the 16 flavonol like galangin which lacks a hydroxyl substitution in flavonoids as shown in Fig. 4. The oxidizability of the B-ring had little promoting activity. Dehydroxylation at flavonoids, which have the three hydroxyl substitutions re- position C5 in the A-ring of quercetin, isorhamnetin and quired for effectively promoting DNA degradation, was re- kaempherol slightly attenuated the activity as shown in markably enhanced in the presence compared with the ab- fisetin, gelaldol and 5-deoxykaempherol, respectively. In sence of BLM. On the other hand, the extent of oxidation of contrast, dehydroxylation at the C3 position in the C-ring of flavonoids lacking these hydroxyl substitutions was enhanced quercetin rendered the activity much less effective as shown little or not at all by BLM (Table 2). In the absence of BLM, in luteolin. All other flavones tested in the present study also flavonoids possessing pyrogallol or catechol moieties in the exhibited very weak or no promoting activity. These findings B-ring such as myricetin, quercetin and fisetin, exhibited indicated that the following three hydroxyl substitutions in extensive oxidation. The order among flavonoids having the flavonoidal nucleus are essential for effectively promot- crucial hydroxyl substitutions was as follows: myricetinϾ ing DNA degradation induced by the BLM–Fe complex: 1) quercetinϾfisetinϾgeraldolϾisorhamnetinϾkaempferolϾ the C7-hydroxyl substitution in the A-ring; 2) the C4Ј-hy- morinϾ5-deoxykaempferol. Of note, the flavonoids with droxyl substitution in the B-ring; and 3) the C3-hydroxyl strong DNA degradation-promoting activity such as isorham- substitution in the C-ring. Flavonoids, which lack even one netin, kaempferol and geraldol exhibited a remarkable in- of these hydroxyl substitutions, had remarkably diminished crease in oxidation on the addition of BLM. The order in promoting activity. terms of the extent of their oxidation was as follows: The promoting effect of flavonoids on DNA degradation isorhamnetinϾkaempferolϾmorinϾgeraldolϾmyricetinϭ increased with the concentration up to about 20 m M, but de- quercetinϾ5-deoxykampferolϾfisetin. creased at higher concentrations. The pyrogallol and catechol Flavonoids possessing catechol or pyrogallol have been re- Ϫ moieties in the B-ring are the most important for radical ported to produce O2· and H2O2 through their oxida- scavenging, and are also involved in metal chelating activ- tion.31,32) Therefore, active oxygen species generated by the ity.21,22) Mycetin and quercetin which have a pyrogallol and a oxidation of flavonoids might promote DNA degradation in- catechol moiety in the B-ring, respectively, were less active duced by the BLM–Fe complex. However, the degradation than isorhamnetin and kaempferol which have only one hy- promoted by flavonoids was not inhibited by addition of cata- droxyl substitution. The DNA degradation promoted by lase (tested up to 2000 units) or of superoxide dismutase (up myricetin and quercetin might be restrained due to their to 800 units) to the reaction mixture (data not shown). strong antioxidant properties compared to isorhamnetin and Laughton et al. also indicated that these enzymes were inef- kaempferol. Thus, the decrease of the ability of flaovnoids to fective in promoting-activity of myricetin and quercetin.16) promote DNA degradation at higher concentration might be Hence, these active oxygen species are not likely to be in- ascribed to their anti-oxidative property. volved in the DNA degradation-promoting activity of BLM-induced DNA degradation requires Fe ion, molecu- flavonoids. lar oxygen and a suitable reducer.11) BLM possesses func- The mechanism underlying the degradation of DNA pro- tional domains for the binding of DNA and Fe ion.12) By moted by quercetin or myricetin has been proposed to be an bringing Fe to DNA strands directly, BLM mediates the sec- acceleration in the reduction of the Fe(III)–BLM–DNA to the tioning of DNA strands predominantly at G–C and G–T se- Fe(II) form because these flavonoids have the capability of quences. Activated BLM, namely DNA–BLM–Fe(III)–OOH, reducing metal ions such as Fe(III) and Cu(II).10,16) The pre- is the last detectable intermediate before DNA degradation. sent study also showed that flavonoids having good metal-re- The formation of activated BLM is carried out via reduction ducing abilities such as myricetin and quercetin effectively processes including the reduction of Fe(III) which forms a promoted the DNA degradation induced by the Fe–BLM complex with BLM.23,24) Flavonoids have a conjugation be- complex, whereas flavonoids which are inefficient in promot- tween the B- and C-rings through the C2,3-double bond ing the degradation such as keampferaid and galandin, had which is extended to the C4 carbonyl group in the C ring on little or no Fe(III)-reducing activity. Furthermore, flavones their structures. Several electrons are donated due to the such as chrysin, apigenin and diosmetin, which lack Fe(III)- availability of free hydroxyl substitutions in conjugated phe- reducing activity, actually inhibited the DNA degradation in- nolic rings.25—27) Hence, the activity of flavonoids to promote duced by the BLM–Fe complex. The correlation between DNA degradation has been assumed to arise from the reduc- Fe(III)-reducing activity and the DNA degradation-promot- tive ability. We examined whether the DNA degradation-pro- ing activity was significant among the sixteen flavonoids, as moting activity of flavonoids correlates with their oxidizabil- shown in Fig. 5A (rϭ0.706, pϽ0.01). Therefore, the activity ity. The oxidation of flavonoids was evaluated by measuring of flavonoids to promote DNA degradation seems to be in- the decrease in spectral absorption in the long wavelength volved in the reduction of Fe(III) which forms a complex area related to the B-ring which is regarded as the major de- with BLM. However, no correlation between Fe(III)-reducing terminant for donating hydrogen atoms from available free activity and DNA degradation-promoting activity was found hydroxyls.28—30) The donation of more than two electrons re- among the flavonoids having crucial hydroxyl substitutions sults in the loss of the conjugation between the B- and C- as shown in Fig. 5B, although there was a significant correla- rings. The C3 hydroxyl substitutions are considered to play tion among flavonoids which lack these substitutions 1114 Vol. 26, No. 8

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