ORIGINAL ARTICLES
3.5. Determination of the half-wave potentials
3. Experimental
Electrochemical measurements were performed in a two-compartment Pyr-
ex cell. The working electrode was a Pt wire. Before each measurement
the Pt wire electrode was cleaned by heating it up to the red on a burner
flame. The counter electrode was a platinum foil of large area (approxi-
mately 2 cm2). The reference electrode was an aqueous saturated calomel
electrode (SCE). Solutions were deaerated by bubbling pure nitrogen. All
measurements were performed at 25 ꢅ 0.5 ꢄC, employing H2O/NaOH
10 mM as solvent.
3.1. Chemicals
7-Hydroxyflavonol, 7,30-dihydroxyflavonol and 30-hydroxyflavonol were
synthetized, purified and identified as described below (Tanaka and Sujino,
2001). Quercetin, Kaempferol and Rutine were naturally obtained from
Organic Chemistry Area, San Luis National University. Hypoxanthine and
xanthine oxidase were purchased from Sigma Chem. Co.
The convolution of faradaic current was calculated by applying the algo-
rithm proposed by Oldham (1986) to the background corrected voltammo-
grams. The measuring system for linear scan voltammetry (LSV) was con-
structed from a EG & G PARC Model 273 potentiostat run with model
PAR270 electrochemical analysis software.
3.2. Synthesis of the flavonoids
3.2.1. Synthesis of 7-hydroxyflavonol (1)
Crystals of 20,40-dihydoxychalcone (0.250 g 100% pure), an aq. NaOH so-
lution (2.5 mL 8 M) and a 30% hydrogen peroxide solution (2.15 mL)
were stirred at room temperature for 2 h. The reagent mixture was neutra-
lized with HCl (6 M) in an ice bath. The precipitate was suction filtered,
water washed, dried and recrystallized from a methanol-water mixture to
give a beige crystalline solid yielding 95% of the pure product. The struc-
ture of 1 was determined by the chromatographic and spectroscopic data:
Rf (TLC): 0.22; UV lmax (MeOH) nm: 330 (18162.96); 253 (16672.64);
226 (20416.64). EI-MS m/z (rel. int.) 270 [Mþ] (100); 137 (11.56).
Acknowledgements: Financial support from Secretar´ıa de Ciencia y Te´cni-
ca de la Universidad Nacional de San Luis (SECyT UNSL), Consejo Na-
cional de Investigaciones Cient´ıficas y Te´cnicas (CONICET), Agencia Na-
cional de Promocio´n Cient´ıfica y Tecnolo´gica (ANPCyT) and Secretar´ıa de
Ciencia y Te´cnica de la Universidad Nacional de R´ıo Cuarto (SECyT
UNRC) is grateful acknowledged.
3.2.2. Synthesis of 7,30-dihydroxyflavonol (2):
References
Crystals of 20,40,3-trihydroxychalcone (0.500 g 100% pure), an aq. NaOH
solution (5 mL 8 M) and a 30% hydrogen peroxide solution (5 mL) was
stirred at room temperature for 2 h. The reagent mixture was neutralized
with HCl (6 M) in an ice bath. The precipitate was suction filtered, water
washed, dried and recrystallized from methanol-water mixture. The product
was purified by chromatoghaphy using a Sephadex LH 20 column and
methanol as eluent. The structure of 2 was determined by the chromato-
graphic and spectroscopic data: Rf (TLC): 0.14; UV lmax (MeOH) nm:
318.6 (16052.30); 244.8 (15447.56). EI-MS m/z (rel.int.) 270 [Mþ] (100);
137 (52.01); 121 (30.89); 120 (15.08).
Bielski HJ, Cabelli DE, Arudi RL, Ross AB (1985) Reactivity of HO2/
O.2–– radicals in aqueous solution. J Phys Chem Ref Data 14: 1041–
1100.
Blasiak J, Arabski M, Pertynski T, Malecka-Panas E, Wozniak K Drze-
woski J (2002) DNA damage in human colonic mucosa cells evoked by
nickel and protective action of quercetin-involvement of free radicals?
Cell Biol Toxicol 18: 279–288.
Carlsson DJ, Suprunchuk T, Miles DM (1976) Photooxidation of unsatu-
rated oils: effects of singlet oxygen quenchers. J Oil Chem Soc 53:
656–660.
Choi JS, Chung HY, Kang SS, Jung MJ, Kim JW, No JK, Jung HA
(2002) The structure-activity relationship of flavonoids as scavengers of
peroxynitrite. Phytother Res 16: 232–235.
3.2.3. Synthesis of 30-hydroxyflavonol (4):
Crystals of 20,3-dihydroxychalcone (0.500 g 100% pure), an aq. NaOH
solution (5 mL 8 M) and a 30% hydrogen peroxide solution (4.15 mL) was
stirred at room temperature for 2 h. The reagent mixture was neutralized
with HCl (6 M) in an ice bath. The precipitate was suction filtered, water
washed, dried and recrystallized from methanol-water mixture. A beige
crystalline solid was obtained yielded 95% of the pure product. The struc-
ture of 3 was determined by the chromatographic and spectroscopic data:
Rf (TLC): 0.32; UV lmax (MeOH) nm: 344.4 (20665.47); 304.4
(12807.99); 243.8 (24782.82). EI-MS m/z (rel. int.) 254 [Mþ] (100); 121
(53.83).
Cotelle N, Bernier JL, Catteau JP, Pommery J, Wallet JC, Gaydou EM
(1996) Anfioxidant properties of hydroxyflavones. Free Rad Biol Med
20: 35–43.
´
Criado S, Gutie´rrez MI, Avila V, Bertolotti SG, Garc´ıa NA (1996) Medium
and substitution pattern effects on the action of hydroxyflavones as
photoprotectors against singlet molecular oxygen-mediated photooxida-
tion of fats. Fett/lipid 98: 172–175.
Csokay B, Prajda N, Weber G, Olah E (1997) Molecular mechanisms in
the antiproliferative action of quercetin. Life Sci 60: 2157–2163.
Das NP, Pereira TA (1990) Effects of flavonoids on thermal autoxidation
of palm oil: structure-activity relationships. J Am Chem Oil Soc 67:
255–258.
3.3. Generation and inhibition of superoxide radical anion
Superoxide radical anion was generated enzymatically by a hypoxanthine-
xanthine oxidase system and was detected by the nitrite method (Hu et al.
1995). The test solutions were prepared by mixing 2 mL of stock solution
Duarte J, Jimenez R, O’Valle F, Galisteo M, Perez-Palencia R, Vargas F,
Perez-Vizcaino F, Zarzuelo A, Tamargo J (2002) Protective effects of the
flavonoid quercetin in chronic nitric oxide deficient rats. J Hypertens 20:
1843–1854.
El-Sukkary MMA, Speier G (1981) Oxygenation of 3-hydroxyflavones by
superoxide anion. J Chem Soc Chem Comm 745.
Fang X, Jin F, Jin H, Von Sonntad C (1998) Reaction of superoxide
radical with N-centered radical derived from N.acetyltryptophan methyl
ester. J Chem Soc Perkin Trans 2: 259–263.
(1.2 ꢂ 10ꢃ3 M hypoxanthine, 1.2 ꢂ 10ꢃ3 M hydroxylamine and 6 ꢂ 10ꢃ4
M
EDTA), 1.4 mL buffer (0.0624 M KH2PO4 and 0.0498 M Na2B4O7) with
flavonoids (or without flavonoids as controls). Flavonoids were prepared in
concentrated methanolic solutions and 25 to 200 mL were added (complet-
ing to 200 mL with MeOH) in the test tubes. The reaction was started by
adding 10 mL xanthine oxidase (final concentration: 2 ꢂ 10ꢃ3 U/mL). After
incubation (30 min, 37 ꢄC), 0.3 mL HCl (1 M) and 0.1 mL dye reagent
(sulfanilic acid 0.019 M and N-1-naphtylethylenediamine 2.60 ꢂ 10ꢃ3 M)
were added. The mixtures were allowed to stand for 45 min at room tem-
perature and absorbances at 530 nm were determined in a spectrophot-
ometer Shimadzu UV-160A. The scavenging activities were determined
with different concentrations of flavonoids.
Garc´ıa NA (1994) Singlet molecular oxygen-mediated photodegradation of
aquatic phenolic pollutants.
A kinetic and mechanistic overview. J
Photochem Photobiol B: Biology 22: 185–196.
Grossman L, Moldave K (1967) Methods in Enzymology. Vol XII, Nucleic
Acid Part A. In: Academic Press (ed), New York, p. 5.
Hu JP, Calomme M, Lasure A, de Bruyne T, Pieters L, Vlietinck AJ, Van
den Berghe D (1995) Structure-activity relationship of flavonoids with
superoxide scavenging activity. Biol Trace Elem Res 47: 327–331.
Janssen K, Mensink RP, Cox FJ, Harryvan JL, Hovenier R, Hollman PC
(1998) Effects of the flavonoids quercetin and apigenin on hemostasis in
healthy volunteers: results from an in vitro and a dietary supplement
study. Am J Clin Nutr 67: 255–262.
Jorgensen LV, Skibsted LH (1998) Flavonoid deactivation of ferrylmyoblo-
din in relation to ease of oxidation as determined by cyclic voltametry.
Free Rad Res 28: 335–351.
Kim DO, Lee KW, Lee HJ, Lee CY (2002) Vitamin C equivalent anti-
oxidant capacity (VCEAC) of phenolic phytochemicals. J Agric Food
Chem 50: 3713–3717.
Knekt P, Kumpulainen J, Jarvinen R, Rissanen H, Heliovaara M, Reuna-
nen A, Hakulinen T, Aromaa A (2002) Flavonoid intake and risk of
chronic diseases. Am J Clin Nutr 76: 560–568.
3.4. Inhibition assay of xanthine oxidase by flavonols
Xanthine oxidase (XO) enzyme activity was determined as described by Kup-
pusamy et al. (1990). A XO solution (final concentration 2 ꢂ 10ꢃ3 U/mL)
was preincubated with 0.2 mL of methanolic flavonoid solution and
1.4 mL pH 7 buffer for 15 min at 37 ꢄC. The volume was made up to
3.2 mL with distilled water. Control tubes contained the enzyme, methanol,
buffer and water in the same proportion as assay tubes were prepared.
After preincubation, assay was started by adding 0.4 mL of a stock solu-
tion containing hypoxanthine (6 ꢂ 10ꢃ3 M), hydroxilamine (6 ꢂ 10ꢃ3 M)
and EDTA (3 ꢂ 10ꢃ3 M) to each tube and incubated for 30 min at 37 ꢄC.
Reactions were stopped by addition of 0.3 mL HCl (1 M) and the colora-
tion reaction was carried out in the same way as described above.
On the basis of data obtained in the enzymatic inhibition assay, absorbance
of nitrite (proportional to the XO activity) was plotted vs. flavonoid con-
centration. Values were fitted by regression and the 25% inhibition concen-
tration (IC 25X) was graphically evaluated.
Kuppusamy UR, Khoo HE, Das NP (1990) Structure-activity studies of
flavonoids as inhibitors of hyaluronidase. Biochem Pharmacol 40: 397–
401.
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