J. Chil. Chem.

Soc, 52, N 4 (2007), pgs: 1326-1329

ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM HYPERICUM TERNUM

A.P.M. BERNARDI1, C. LPEZ-ALARCN2, A. ASPEE3, S. RECH1, G.L. VON POSER1*, R. BRIDE1 AND E. LISSP3 1 Programa de Pos Graduacao em Ciencias Farmacuticas, UFRGS. Av. Ipiranga, 2752, 90610-000 Porto Alegre, RS, Brazil, Fax (+55) 51 3308 5437 2 Departamento de Farmacia, Facultad de Qumica, Pontificia Universidad Catlica de Chile. Av. Vicua Mackenna n4860, Santiago, Chile. 3 Facultad de Qumica y Biologa, Universidad de Santiago de Chile, 307 Casilla, Correo 2, Santiago, Chile.

ABSTRACT In the present work we have studied the scavenging activity of several flavonoids isolated fromHypericum ternum A. St. Hil. The evaluation of the free radical scavenging capacity was based on the 2,2-diphenyl-l-picrylhydrazyl radical (DPPH) consumption elicited by their addition. Also, we have attempted to evaluate their capacity to delay pyrogallol red consumption promoted by peroxyl radicals. The compounds isolated and characterized were 13,118-biapigenin and five quercetin derivatives (quercetin 3-methyl ether, quercetin 3,7-dimethyl ether, hyperoside, isoquercitrin and guaijaverin). All compounds were able to scavenge DPPH radicals. The order in scavenging capacity (from highest to lowest) was: guaijaverin > hyperoside isoquercitrin > quercetin-3-methyl-ether. No protection of pyrogallol red was evidenced for all flavonoids derivatives at relatively high (100 M) concentrations. This lack of protection contrasts with the efficient protection afforded by 10 M quercetin, indicating that substitution at the 3-position in quercetin strongly reduces the capacity of the molecule to scavenge peroxyl radicals. Key words: Hypericum ternum, antioxidant activity, flavonoids.

INTRODUCTION
Reactive oxygen species (ROS) are involved in hMan diseases such as atherosclerosis, cardiovascular and neurodegenerative diseases.1,2 The ability of some natural antioxidants, such as flavonoids and other polyphenols, to scavenge several oxygen and nitrogen free radicals has been associated to the health benefits of diets rich in fruits and vegetables.3 Moreover, the effect of some medicinal plants has been related with their polyphenolic and flavonoids components." For this reason, in the last years, many works have been aimed to find active phytochemicals compounds in a variety of different plants.5-10

The genus Hypericum (Guttiferae) includes approximately 400 species, with twenty of them native from southern Brazil.11,12 Hypericum species, contain several chemically active compounds such as naphthodianthrones, flavonoids, xanthones, and phloroglucinol derivatives. In addition, in BrazilianHypericum species, benzophenones and benzopyrans have been identified. In particular, Hypericum ternum A St. Hil. grows only in South Brazil.11 The only work about this plant reports a preliminary analysis of flavonoids by thin layer chromatography and tannins

quantification.13 No study on the antioxidant potential was found. Therefore, in this work we isolated and identified flavonoids from the aerial parts of Hypericum ternum and evaluated their antioxidant potential using methodologies based on the consumption of the stable free radical DPPH (2,2-diphenyl-l-picrylhydrazyl) and on the measurement of oxygen radical absorbance capacities employing pyrogalol red as target molecule. DPPH bleaching is elicited by a great variety of compounds of widely different free radical scavenging capacity,14 while the proposed ORAcumethodology is suitable only for compounds of high reactivity.15

Materials and Methods.

Plant Material Aerial parts of H. ternum were collected in the Rio Grande do Sul state, in January 2003. Plants were identified and deposited in the herbariM of the Universidade Federal do Rio Grande do Sul (ICN). Extraction and Isolation of flavonoids The methanolic extract was obtained from the air dried and powdered plant material (aerial parts) by staticumaceration (3 x 24 hours) at room temperature. The extract was evaporated under reduced pressure and the residue partitioned between water and ethyl acetate. The organic solvent extract was evaporated to dryness under reduced pressure and the main compounds isolated by colMn chromatography on silica gel using a CH2Cl2-MeOH gradient system. Hyperoside, isoquercitrin and guaijaverin were obtained in high amounts. Quercetin 3-methyl ether, quercetin 3,7-dimethyl ether, and 13,118-biapigenin were purified by preparative TLC on silica gel using CH2Cl2-MeOH (9:1) as mobile phase. Only few milligrams were obtained of

quercetin3,7-dimethyl ether and 13,118-biapigenin, precluding a quantitative evaluation of their antioxidant capacities. The compounds were identified by NMR spectroscopy. Copies of the original spectra are obtainable from the author of correspondence. Antioxidant activity Studies using DPPH free radicals To obtain a preliminary evaluation of the antioxidant capacity, a DPPH bleaching assay was used as a rapid TLC screening method (DPPH-TLC). TLC was performed with an acetyl acetate (AcOEt) extract ofH. ternum solution 20 mg/mL (20 L) on silica gel GF254 using as solvent AcOEt: MeOH:H20 (100:13.5:10). After developing and drying, TLC plates were sprayed with a 0.2% DPPH solution inumeOH and examined 10 min after spraying.16 Compounds with capacity to reduce DPPH appear as yellow spots against a purple background. After the preliminary studies, the interaction of active compounds with DPPH was quantitatively measured by UV-visible spectroscopy.17 Briefly, ethanol solutions of flavonoids (12.5 or 100 M), were added to a DPPH ethanolic solution (60 M). The change in sample absorbance was recorded in a Hewlett Packard 8453 (Palo Alto, CA, USA) UV-visible spectrophotometer. The absorbance was measured at 517 nm (DPPH molar absorptivity = 11500 M-1cm-1) at 25C. Measurements started immediately after mixing the solutions. The absorbance decrease of DPPH was evaluated after 50 or 600 seconds. For the evaluation of the antioxidant capacity, experimental data (kinetics profiles of DPPH decay) were adjusted to a bi-exponential equation

In absence of flavonoids DPPH absorbance was not changed in the time of the experiments. DPPH and flavonoids solutions were freshly prepared daily.

The activity of the tested compounds was characterized by: (A2 + A2) = total bleaching capacity. (Abs0 - Abs50s) = consumption of DPPH at a short reaction time (50 sec). It takes into account only fast processes. (Abs0 - Abs600s) = consumption of DPPH at long reaction time (600 sec). It takes into account fast and slow processes. Oxygen Radicals Absorbance Capacities (ORAC) assay We used the ORAC (Oxygen Radical Absorbance Capacity) methodology employing pyrogallol red (PGR) as target molecule.15 This method (ORAC-PGR) is based on the ability of the antioxidant compounds to delay the consumption of PGR elicited by AAPH. Stock solutions of flavonoids were prepared inumethanol immediately before their use. Stock solutions of AAPH (0.6 M) and PGR (1x10-4M) were prepared daily in phosphate buffer (75 mM pH 7.4):methanol (60:40). A reactionumixture containing AAPH (10 mM), PGR (5 M) and the additives (100 M) was incubated at 37C in the thermostatized cuvette of a UV-visible spectrophotometer Hewlett Packard 8453 (Palo Alto, CA, USA). Runs carried out in the absence of additives were employed as controls. All experiments were carried out in triplicate. The PGR consumption during its incubation in the presence of AAPH, was evaluated from the progressive absorbance decrease measured at 540 nm. Values of (A/ A0) were plotted as a function of time. Integration of the area under the curve (AUC) was employed to obtain ORAC-PGR values. Statistical analysis Experiments were performed in triplicate and results were expressed as means standard deviation. Statistical analysis was performed by oneway analysis of variance (ANOVA) followed by Tukey test for multiple comparisons. The SPSS (Statistical Package for the Social Sciences) software. Differences were considered significant at P<0.05.

RESULTS AND DISCUSSION

Six flavonoids have been identified in H. ternum Two flavonoids (quercetin 3-methyl ether and quercetin 3,7-dimethyl ether) have not been previously isolated from Hypericum species. On the other hand, the otherflavonoids (13,118-biapigenin, hyperoside, isoquercitrin and guaijaverin) are compounds frequently found in Hypericum species.18-22 All compounds were identified by 1H and 13 C NMR analysis. The data are in accordance with that reported in the literature.23,24 Since only a few milligrams were obtained of quercetin 3,7-dimethyl ether and 13,II8-biapigenin, the free radical scavenging capacity of these compounds was only qualitatively assessed by a DPPH-TLC method.

Bleaching of DPPH mediated by flavonoids DPPH is a stable free radical with a visible band at 517 nm whose bleaching has been widely employed for the evaluation of the antioxidant capacity of pure compounds (AH) and complex

mixtures.25 The methodology is based on the bleaching of a methanolic DPPH solution26 elicited by the added antioxidant, according to reaction [2]

Before performing quantitative studies of DPPH bleaching by the isolated flavonoids, a preliminary test was carried out. For this purpose, we used a DPPH-TLcumethod (see materials and methods section). When the plates (with flavonoids) were sprayed with a DPPH solution (0.2%), all flavonoids showed a significant DPPH bleaching capacity. Therefore, all flavonoids can be considered as potential free radical scavengers. The antioxidant capacity of quercetin 3-methyl ether, hyperoside, isoquercitrin, and guaijaverin was quantitatively evaluated following the DPPH absorbance bleaching at 517 nm. When flavonoids (12.5-100 M) were added to a DPPH solution (60 M), a fast bleaching of DPPH was observed .

As can be seen in this figure, all tested compounds were able to bleach DPPH in a concentration dependent way. Guaijaverin and quercetin 3-methyl ether were the flavonoids with the highest and lowest antioxidant activity, respectively. Hyperoside and isoquercitrin showed similar (intermediate) antioxidant capacities.

The kinetic profiles of DPPH bleaching induced by flavonoids showed a biphasic decay. In the first seconds it is observed a fast absorbance diminution followed, at longer reaction times, by a considerably slower process. These kinetic profiles preclude a simple treatment of the data and could be related to the presence of two or more reactive centers of different reactivity.26 This multiplicity of reactive centers can be taken into account by fitting the decay to a bi-exponential function. Therefore, the kinetics profiles were adjusted to a bi-exponential equation in order to estimate the total bleaching capacity. Also, in order to have some insight on the reactivity of the

tested compounds, we have arbitrarily defined two parameters: the decay at 50 sec. and the decay at 600 sec.

Shows the extent of DPPH consumption induced by flavonoids (25 M) after 50 and 600 sec. of reaction. In this figure are also included total consumptions obtained by extrapolating to infinite time the bi-exponential decays. Guaijaverin was the flavonoid with the highest capacity to remove DPPH radicals. In particular, it is interesting to note that its scavenging capacity measured at short reaction times is similar to that of quercetin, with almost one DPPH radical bleached per added flavonoid molecule. On the other hand, it is observed that DPPH consumption by quercetin, measured at long reaction times, is considerably larger than that of all tested 3-substituted compounds. This would suggest that the OH at this position contributes to the free radical scaveging capacity of quercetin. These results agree with previous work, that have shown that the DPPH consumption was two or three times larger with quercetin than hyperoside and rutin respectively.27-30

Significant differences on the DPPH radical removal capacity was observed for the four 3substituted compounds tested in the present work. This implies that the substituent present at the 3-positionumodulates the reactivity and/or secondary reactions of the products generated in the first step (reaction (2)). Quercetin 3-methyl-ether was the flavonoid with the lower antioxidant capacity, in particular at long reaction times. 3.2 Oxygen radical absorbance capacity (ORAC-PGR) assay The results given in

show that substitution at the 3-position decreases the stoichiometry of the free radical removal process by the flavonoids. However, it does not provide clear evidence about their reactivity towards free radicals. In order to compare the reactivity of the flavonoids towards peroxyl radicals, we used an ORAcumethod based on the bleaching of pyrogallol red (ORAC-PGR).

shows the effect of isoquercitrin (100 M) or quercetin (10 M) on the consumption of PGR (5 M) mediated by AAPH derived peroxyl radicals. It is observed that quercetin (10 M) delays PGR consumption, a result attributable to its capacity to remove peroxyl radicals. On the other hand, it is observed that the addition of isoquercitrin (100 M) does not afford any protection to the PGR molecule. Similar results were obtained when the other 3-substituted compounds were employed. This lack of activity of the flavonoids studied indicate that quercetin high reactivity towards peroxyl radicals is due to the presence of a reactive hydrogen atom at the 3-position. Blockage of this position renders a molecule that is not able to compete with the highly reactive PGR.152831 It can be speculated that, if the 3-position group is cleaved in the organism prior and/or after absorption, the scavenging capacity of these compounds could be considerably increased.

CONCLUSIONS
Our results show that Hypericum ternum is a source of 5,7,3',4'-hydroxy-substituted flavonoids. These compounds showed a significant DPPH bleaching capacity. On the other hand, and contrary to quercetin, they were unable to protect PGR from its bleaching by peroxyl radicals. The present results show that the magnitude of the effect of blockage of the 3-position upon the free radical scavenging capacity of quercetin depends on the methodology employed in its evaluation. In particular, the lack of protection of PGR indicates that 3-substituion notable reduces the capacity of quercetin to remove peroxyl radicals.

ACKNOWLEDGEMENTS
This work was supported by CAPES, CNPq, FAPERGS, PROPESQ-UFRGS, LARC-IBRO and FONDECYT (n 11060323). Also, the support of Vicerrectora Adjunta de Investigacin y Doctorado (VRAID), Pontificia Universidad Catlica de Chile (DIPUC n2006/28) is acknowledged.

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