Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism

†: These authors contributed equally to this work. 2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(2), 267-270 doi: 10.15171/apb.2016.037 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Reactive oxygen species (ROS), generated by normal cellular metabolism and exogenous agents (e.g.xenobiotics, ionising and nonionsing radiation), may lead to a condition of oxidative stress if its production overwhelms the antioxidant defences. 1,2It has been considered as a significant promoter of cancer, cardiovascular malfunction, aging, and other diseases.Cellular DNA is a particularly sensitive target because of the potential to create cumulative mutations that can disrupt cellular homeostasis.However, the enzymatic repair in living organisms might be inadequate for protecting against permanent DNA mutations.Therefore, for the past few years, it has become a research focus to search for effective and safe antioxidants from natural sources. 3,4ost of natural antioxidants belong to the family of phenolic or polyphenolic compounds, which show their antioxidant activity via a hydrogen atom transfer (HAT) or single electron transfer (SET) mechanism. 5Fisetin (3,3',4',7-tetrahydroxyflavone, Figure 1) is a phenolic flavonoid in various food, such as strawberry, onion and persimmon. 6It has a wide range of pharmacological effects, such as antitumor, antiinflammation, 7 anticoagulant and dissolving thrombus. 8In recent years, its protective effect on the liver in human body (especially patients with diabetes) attracted a wide spread attention.Researches showed that, the fisetin's liver protective effect and promoting glucose homeostasis effect were related to its antioxidant activity. 9However, its antioxidant ability and mechanism remain unknown.Therefore, the present study used DNA protection model to investigate its antioxidant activity then further discuss the possible mechanisms.

Methods
The protective effect of fisetin against •OH-induced DNA damage was determined by our method. 10echanistic analysis experiments included •OH scavenging, •O 2 scavenging, DPPH• scavenging, ABTS• + scavenging, and Cu 2+ -reducing power assays.The •OH scavenging assay was based on the deoxyribose degradation, and improved by our laboratory; 11 in the improved deoxyribose degradation assay, all samples were pre-treated before determination.The •O 2 scavenging assay was based on pyrogallol autoxidation reaction, and also improved by our laboratory; In the improved pyrogallol autoxidation assay, pH was modified as 7.4. 11The other antioxidant assays were described in our previous paper. 10,11In all these assays, BHA and Trolox were used as the positive controls.The inhibition (or protecting DNA, reducing power) percentages were obtained according to the corresponding calculation formulas.The calculation formulas and experimental protocols were detailed in the Suppl.1.

Results and Discussion
ROS can lead to the formation of single and doublestrand breaks, as well as induce chemical and structural modifications to purine and pyrimidine bases, resulting in millions of lesions per cell each day. 4 Deoxythymineglycol (dTG)，8-hydroxy-deoxyguanine (8-OH-dG), malondialdehyde (MDA) and formamidopyrimidine (FAPy) constitute the important markers of DNA oxidative damage. 12,13Among these fragments, MDA is usually used to reflect the DNA protective percentage, because MDA can be easily detected via combining with 2-thiobarbituric acid (TBA). 10s is seen in Figure 2A, the protective percentages of fisetin from •OH-induced DNA damage increased in a dose-dependent fashion.The IC 50 values of fisetin, BHA and Trolox were respectively 1535.00±29.60µM, 2469.00±96.69µM and 1084.67±20.23 µM (Table 1).The results suggested that fisetin could protect DNA against •OH-induced damage.However, its protective effect is inferior to that of Trolox but stronger than that of BHA.  1).This indicated that both •OH and •O 2 -radicals could be eliminated by fisetin at low concentration, and that ROS scavenging may be a possible approach for fisetin to protect DNA.To confirm whether HAT and SET might happen in the ROS scavenging by fisetin, we further measured its radical-scavenging on DPPH•.The previous studies suggested that DPPH• may undergo HAT pathway to be scavenged to yield a stable DPPH-H molecule. 17As illustrated in Figure 2D and Table 1,   Since the formation of ABTS• + from ABTS was previously proven to be via one SET oxidation, 19 ABTS• + scavenging was also reported to be a SET mechanism. 20The effective scavenging ABTS• + by fisetin indicate a SET possibility in its ROS scavenging reaction.The SET possibility was further supported by the Cu 2+ -reducing power assay, in which fisetin efficiently reduced Cu 2+ →Cu + (Figure 2F and Table 1).Cu 2+ -reducing however is well-known as an electron (e) transfer reaction.The IC50 value is defined as the concentration of 50% radical inhibition (or protection percentage, relative reducing percentage).These IC50 values were calculated by linear regression analysis based on the response curves in Figure 2 and converted from µg/mL to µM.Each value in this table is expressed as the mean±SD (n=3).Mean values with different superscripts (a, b, or c) in the same row are significantly different (p < 0.05), while those with same superscripts are not significantly different (p < 0.05).

Conclusion
Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive ROS scavenging approach, which is assumed to be via HAT/SET pathways.In the HAT pathway, the 3',4'-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form.
fisetin scavenged DPPH• radical with high efficiency.It clearly suggests a HAT pathway in the ROS scavenging by fisetin.The possible reaction of fisetin with DPPH• radical can be proposed as Figure 3.In the process, since B ring is regarded as the active sites in the antioxidant process of flavonoids, 18 phenolic-OH in B ring of fisetin is thought to undergo homolysis prior to either the A or C ring, to produce H• and fisetin• radical (Ⅰ).Then DPPH-H molecule may be generated through H• combining with DPPH•.And the fisetin• radical might transform into a semi-quinone form (Ⅲ), which could be further extracted H• by excess DPPH• to form the stable ortho-benzoquinone form.Now it is clear that, the 3',4'-dihydroxyl moiety in B ring of fisetin played an important role, because it could be ultimately oxidized to a stable ortho-benzoquinone form.

Table 1 .
The IC50 values of fisetin and the positive controls (µM)