Antioxidant activity and mechanism of commercial Rama Forte persimmon fruits (Diospyros kaki).

This study aimed to characterize the antioxidant properties of Rama Forte persimmon, a tannin-rich fruit variety produced in Brazil. Extracts prepared with lyophilized pulps from fruits obtained in local markets were analyzed individually to evaluate the extent of antioxidant protection and investigate the antioxidant mechanism. Iron-mediated hydroxylation of 5,5-dimethyl-1-pirrolidine-N-oxide, determined by electron paramagnetic resonance (EPR), and oxidative degradation of 2-deoxyribose (2-DR) were inhibited by fruit extracts in a dose-dependent manner. There was a considerable individual variability in inhibition of 2-DR degradation by individual fruits. Higher protection of 2-DR degradation (by the extracts) was observed in Fe(III)-citrate/ascorbate in comparison with Fe(III)-EDTA/ascorbate system; however, antioxidant effectiveness of fruit extracts was not diminished by increasing EDTA concentration by 10-fold. Other competition experiments using the 2-DR assay (varying pre-incubation time and 2-DR concentration) indicated that protection comes mainly from free radical scavenging, rather that metal chelation antioxidant activity. Persimmon extracts prevented iron-mediated lipid peroxidation in rat liver homogenates, which correlated significantly with the inhibition of 2-DR oxidation. Finally, sugar content of individual fruits correlated inversely with inhibition of 2-DR degradation, which could indicate that maturation decreases soluble antioxidant concentration or efficiency. In conclusion, lipid peroxidation, 2-DR and EPR experiments indicated that extracts from commercial fruits showed mainly radical-scavenger activity and relevant antioxidant activity.

Ellagic acid inhibits iron-mediated free radical formation.

Polyphenols are reported to have some health benefits, which are link to their antioxidant properties. In the case of ellagic acid (EA), there is evidence that it has free radical scavenger properties and that it is able to form complexes with metal ions. However, information on a possible link between the formation of iron-EA complexes and their interference in Haber-Weiss/Fenton reactions was not yet determined. Thus, the present study investigated the in vitro antioxidant mechanism of EA in a system containing ascorbate, Fe(III) and different iron ligands (EDTA, citrate and NTA). Iron-mediated oxidative degradation of 2-deoxyribose was poorly inhibited (by 12%) in the presence of EA (50µM) and EDTA. When citrate or NTA - which form weak iron complexes - were used, the 2-deoxyribose protection increased to 89-97% and 45%, respectively. EA also presented equivalent inhibitory effects on iron-mediated oxygen uptake and ascorbyl radical formation. Spectral analyses of iron-EA complexes show that EA removes Fe(III) from EDTA within hours, and from citrate within 1min. This difference in the rate of iron-EA complex formation may explain the antioxidant effects of EA. Furthermore, the EA antioxidant effectiveness was inversely proportional to the Fe(III) concentration, suggesting a competition with EDTA. In conclusion, the results indicate that EA may prevent in vitro free radical formation when it forms a complex with iron ions.

Blog construction as an effective tool in biochemistry active learning.

To boost active learning in undergraduate students, they were given the task of preparing blogs on topics of clinical biochemistry. This "experiment" lasted for 12 teaching-semesters (from 2008 to 2013), and included a survey on the blogs' usefulness at the end of each semester. The survey (applied in the 2008-2010 period) used a Likert-like questionnaire with eight questions and a 1-to-6 scale, from "totally disagree" to "fully agree." Answers of 428 students were analyzed and indicated overall approval of the blog activity: 86% and 35% of the responses scored 4-to-6 and 6, respectively. Considering the survey results, the high grades obtained by students on their blogs (averaging 8.3 in 2008-2010), and the significant increase in average grades of the clinical biochemistry exam after the beginning of the blog system (from 5.5 in 2007 to 6.4 in 2008-2010), we concluded that blogging activity on biochemistry is a promising tool for boosting active learning. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):205-215, 2017.

Reevaluation of the 2-deoxyribose assay for determination of free radical formation.

BACKGROUND: The 2-deoxyribose (2-DR) degradation assay is a widely used test for determining anti/pro-oxidant properties of molecules and plant extracts. Most reports use reaction blanks omitting 2-DR or thiobarbituric acid (TBA). However, when studying Fe(II)-mediated reactions, we verified that these blanks are not appropriate. Fe(III)--a product of these reactions--causes a relevant artifact in the assay, where 2-DR is oxidized by Fe(III). METHOD: 2-DR degradation was determined at 532 nm as TBA-reactive substances. RESULTS AND CONCLUSION: HPLC determinations indicated that Fe(III) added after or before TBA generates considerable amounts of malondialdehyde (2-DR degradation product) in comparison with assays employing Fenton reagents or Fe(II) autoxidation. Addition of catalase and thiourea has no effect on Fe(III)-induced 2-DR degradation indicating lack of ROS involvement. This Fe(III)-mediated 2-DR damage is dependent on iron and 2-DR concentrations, but not on H2O2, buffer composition or iron-chelators. Depending on the assay conditions Fe(III)-interference accounts for 20% to 90% of 2-DR degradation mediated by Fe(II). SIGNIFICANCE: A new reaction blank is proposed herein-based on the use of Fe(III)-for the assay. The lack of such correction has caused the underestimation of antioxidant capacity of various compounds in many studies in the last 2 decades.

The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals.

Tannic acid (TA) has well-described antimutagenic and antioxidant activities. The antioxidant activity of TA has been previously attributed to its capacity to form a complex with iron ions, interfering with the Fenton reaction [Biochim. Biophys. Acta 1472, 1999, 142]. In this work, we observed that TA inhibits, in the micromolar range, in vitro Cu(II) plus ascorbate-mediated hydroxyl radical (*OH) formation (determined as 2-deoxyribose degradation) and oxygen uptake, as well as copper-mediated ascorbate oxidation and ascorbate radical formation (quantified in EPR studies). The effect of TA against 2-deoxyribose degradation was three orders of magnitude higher than classic *OH scavengers, but was similar to several other metal chelators. Moreover, the inhibitory effectiveness of TA, by the four techniques used herein, was inversely proportional to the Cu(II) concentration in the media. These results and the observation of copper-induced changes in the UV spectra of TA are indications that the antioxidant activity of TA relates to its copper chelating ability. Thus, copper ions complexed to TA are less capable of inducing ascorbate oxidation, inhibiting the sequence of reactions that lead to 2-deoxyribose degradation. On the other hand, the efficiency of TA against 2-deoxyribose degradation declined considerably with increasing concentrations of the *OH detector molecule, 2-deoxyribose, suggesting that the copper-TA complex also possesses an *OH trapping activity.