Synthesis and Evaluation of Radical Scavenging Potencies and DNA Binding of Metal-Flavonoid Complexes for their Versatile Applications

Abstract

In the present work Cu (II) and Fe (III) complexes of three flavonoids (Fls); morin (mo), quercetin (quer) and primuletin (prim), were synthesized and characterized through Density Functional Theory (DFT), UV-Vis spectroscopy, cyclic voltammetry (cv), FTIR, thermo-gravimetric analysis (TGA), differential scanning calorimetry (DSC) and elemental analysis (EA). The flavonoids (Fls) and their metal complexes (M-Fls) were subjected to evaluate their; (i) antioxidant activity ̶ both invitro and in-vivo (Sprague-dawley rat models), (ii) anti-diabetic study, (iii) human plasma proteins (HPPs) interaction and its effect on antioxidant activity ̶ also restoration of the antioxidant activity using (2-hydroxypropyl)-β-cyclodextrin (HP- βCD), (iv) OH• sensing ability, (v) DNA-binding in the presence and absence of HP- βCD, (vi) in-vitro DNA interaction study using cancer cell lines (MCF-7 and Hela). In the first step, the metal complexes of Fls (Cu-mo, Fe-mo, Cu-quer, Fe-quer Cu-prim and Fe-prim) were synthesized and scrutinized through structure analysis. The UV-Vis spectroscopy and cv data revealed that all the complexes exhibited negative ΔG indicating spontaneous complex formation. Cu-mo and Fe-quer were found to be most stable among all synthesized complexes. The stoichiometry of Cu:Fls and Fe:Fls was found to be 1:2 and 1:3 respectively. DFT calculations predicted 3-OH and 4C=O link as preferred chelation site for mo and quer and 5-OH and 4C=O group as chelation site for prim through stability gain of HOMO orbital. This was confirmed through FTIR data, where the appearance of Fe-O and Cu-O bonds along with more shifts in C-OH bands at predicted sites. The TGA and DSC results also supported the information obtained from FTIR analysis. The radical scavenging activity (RSA) was studied for DPPH• (1,1-diphenyl-2- picryhydrazyl), TEMPO• (tetramethyl-pipyridinyl), hydroxyl (OH•) and superoxide (O2•‐) radicals in terms of IC50 values (the antioxidant quantity for 50% radicals capture) at 310 K and pH-7.4. ΔEHOMO and ionization potential (IP) values were used for computation of RSA which were in agreement with experimental IC50 trend. The computed bond dissociation energy predicted that all three ways of bond dissociation; hydrogen atom transfer (HAT), sequential proton loss electron transfer (SPLET) & single electron transfer followed by proton transfer (SET-PT) are equally probable in iv ABSTRACT case of mo, quer and their complexes while HAT was dominant mechanism for prim and its complexes. The data revealed that each M-Fl exhibited lower IC50 value than respective Fl clearly indicating that metal complexation has increased the antioxidant potential of the flavonoids. In-vivo ROS (reactive oxygen species) controlling potential of Fls and M-Fls was evaluated in terms of hepatoprotective activity leading to diabetes control by injecting Fls and M-Fls to normal and diabetic rats. The glucose level was significantly reduced in diabetic rat indicating that the injected flavonoids and metalflavonoids have anti-diabetic potential. In-vivo RSA was also found to be higher for M-Fls than for Fls in diabetic rats. In a separate in-vitro study, it was found that Fls and M-Fls show significant interactions (Pseudo formation constant Kfp ≈ 103-104 (M-1)) towards HPPs which in turn cause a decrease in their RSA. The co-administrated HP-βCD raised the RSA of the compounds (Fls and M-Fls) in both molecular level interactions and in-vivo trials. In-vivo hepato-protective activity was studied by monitoring all the parameters which are expected to rise in hepatic damage. All these parameters were significantly reduced in the presence of the compounds depicting their hepato-protective activity as well. For some physical applications, Fls and M-Fls were deposited over APTESFTO (3-aminopropyl triethoxy saline ̶ fluorine doped TiO2) to fabricate Fl/M-Fl- APTES-FTO electrode where the successive addition of OH• at nano-molar level caused a decrease in anodic square wave signal leading to OH• detection at very low concentration. Significant current decay was induced by OH• as compare to higher concentration of any other ROS suggesting that Fl/M-Fl-APTES-FTO is reasonably sensitive as well as selective towards OH•. In ds.DNA (double strand DNA) interaction study, the binding constants were found to be in the range of 102-104 (M-1) and ΔG was negative. The absorbance and current variation patterns were used to assign the modes of binding (intercalation, groove binding, electrostatic binding or mixed ones). The relative strength of binding was found to be higher for M-Fls than that for corresponding Fls except in case of Fequer. v ABSTRACT In in-vitro study, the Cu2+ and Fe3+ complexes of prim and mo exhibited significant cytostatic activity against human breast cancer (MCF-7) cell line while Cu-quer revealed significant cytotoxicity against it. Similarly, for human uterine cancer cell line (Hela) cancer cell lines, Cu-mo, Cu-prim and Fe-quer were cytostatic while Cu-quer and prim were cytotoxic against it. Thus, Cu-quer having cytotoxicity comparable to that of doxorubicin can have anti-cancerous potential. In the presence of HP-βCD; Fe mo, Fe-quer and Fe-prim as well as Cu-prim showed electrostatic mode of binding as dominant way of interaction with ds.DNA as the binding constants were found to be of the order of 102 (M-1). Whereas Cu mo and Cu-quer can bind with DNA in the presence of HP-βCD through groove binding as their binding constants are found to be of the order of 103-104 (M-1). Three methods were used for the removal of metal ions from solution through their chelation by flavonoid bound to ds.DNA immobilized over APTES. Immobilization of M-Fl-DNA over APTES removed 60% of metal ions from solution. M-Fl adsorbed/intercalate into DNA-APTES films up to 80% from the solution. The metal ion chelation over Fl-DNA-APTES was successful in ion removal from the solution up to 99%. Comparing ion removing by Fl through ds.DNA binding; prim was found to be better metal ion chelator towards all the ions followed by morin. This method can successfully remove hazardous ions from the solution up to safer limits.