In Vitro Enzyme Inhibition Potentials and Antioxidant Activity of Synthetic Flavone Derivatives

1Department of Pharmacy, University of Malakand, Chakdara, Dir Lower, Khyber Pakhtunkhwa 18550, Pakistan 2Department of Pharmacology, Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pakhtunkhwa 25000, Pakistan 3Department of Chemistry, University of Malakand, Chakdara, Dir Lower, Khyber Pakhtunkhwa 18550, Pakistan 4Department of Physics, University of Sargodha, Punjab 40100, Pakistan 5Department of Statistics, University of Malakand, Chakdara, Dir Lower, Khyber Pakhtunkhwa 18550, Pakistan


Introduction
Reactive oxygen species (ROS) and the free radicals like superoxide, hydroxyl, alkoxyl, hydroperoxyl, and peroxyl are produced during normal metabolism in humans [1].Free radicals are produced by an important chemical process known as oxidation that in turn initiates chain reactions to damage the cells and originate oxidative stress.This process leads to the development of different disorders like Alzheimer's disease [2,3], Parkinson's disease [4], the pathologies caused by diabetes [5,6], rheumatoid arthritis [7], and neurodegeneration in motor neuron [8].The antioxidants have been used specifically as to stop the chain reactions by the removal of free radical intermediates and slow down other oxidation processes by oxidizing themselves and act as reducing agents like polyphenols or ascorbic acid [9].Naturally, a complex system of enzymes and antioxidant metabolites work in coordination to stop oxidative damage to the cellular components like proteins, DNA, and lipids by preventing the formation or removal of these reactive species before damaging the important components of the cells [10,11].
Lipoxygenases (LOX) are the members of a class of nonheme iron containing dioxygenases that catalyze the first step in the arachidonic acid cascade that lead to formation of lipoxins and leukotrienes involved in the variety of inflammatory responses [12].Alzheimer's disease is the common reason of mental illness (dementia) that shows a progressive loss of cholinergic synapses in the brain regions.Mostly, a decreased level of neurotransmitter acetylcholine (ACh) at neuromuscular junction plays a critical role in this disease.Hence, these types of disorders can be overcome by restoring the adequate level of neurotransmitter to inhibit cholinesterase using agents known as anticholinesterase [13].
In general, the flavonoids are reported to possess potent antioxidant activity [14] by scavenging hydroxyl radicals, superoxide anions, and lipid peroxyradicals [15,16].Based upon the significance of flavonoids, an attempt was made to synthesize the flavones derivatives for their possible enzyme inhibition and antioxidant potentials.Here, the first objective was to report synthesis of flavones derivatives and its evaluation for enzyme inhibition and antioxidant activities.The second objective was to establish the structure activity relationship (SAR) of the flavone derivatives.
1 H-NMR and 13 C NMR spectra were recorded in deuterated chloroform (CDCl3) on Bruker SF spectrometers operating at 300 and 75 megahertz (MHz) frequencies, respectively.Chemical shifts values are expressed in  (ppm) downfield relative to TMS which was used as an internal standard.Infrared spectra were recorded on Thermo Scientific USA (Nicolet 6700) Infrared spectrometer on KBr disk method.All melting points are uncorrected and were taken in open capillary tubes using Electrothermal 9100 apparatus (Barnstead, UK).Reaction extents and final products purities were checked on TLC plates (Merck 60 F254, Darmstadt, Germany) and spots were visualized under UV Lamp (180-365 nm) and with subsequent staining with iodine vapours.

General Procedure for the Synthesis of Flavone Derivatives (F1, F2, F3, F4, and F5).
To an ethanolic solution of 2-hydroxyacetophenone (15 mili mol), sodium hydroxide (10 mL, 40% ethanolic) was added dropwise at room temperature.Then corresponding benzaldehyde derivatives (15 mili mol) were added dropwise to this mixture and stirred for 24 hours at room temperature (25 ± 2 ∘ C).The reaction was monitored by TLC and upon completion of the reaction, it was poured into crushed ice and neutralized with 1N HCl solution resulting in yellow precipitates of corresponding chalcones.The chalcones were filtered and washed with water to remove the impurities.
In the next step, the respective chalcones were cyclized to flavone derivatives in 15 mL DMSO in the presence of iodine (375 mg) at 140 ∘ C for 1 hour separately.Upon completion of reactions, the mixtures were cooled to room temperature and poured into water followed by extraction with ethyl acetate (25 mL × 3), treated with sodium thiosulfate solution (20%) and brine solution, and dried over sodium sulfate.The final products (mixture of flavone and chalcone) were subjected to column chromatography using n-hexane: ethyl acetate (9 : 1) to purify flavones derivatives (Figure 1) [17].[18,19].The antioxidant activity of the synthesized compounds, ascorbic acid, tocopherol, and rutin was measured with the slight modifications using DPPH.2% methanolic solution of DPPH was freshly prepared and 1 mL from this solution was added to each 1 mL of different concentrations of the tested flavone derivatives ranging from 25 to 150 g/mL.

Hydrogen Peroxide Scavenging Activity.
The antioxidant activity of the synthesized compounds, ascorbic acid, tocopherol, and rutin was measured with the slight modifications using hydrogen peroxide.A 2 mM solution of hydrogen peroxide was prepared in phosphate buffer (50 mM, pH 7.4).0.1 mL of flavone derivatives (25-150 g/mL) was transferred into test tubes and their volumes were made up to 0.4 mL with phosphate buffer or solvent.After careful addition of hydrogen peroxide solution (0.6 mL), tubes were then incubated for 10 minutes and were determined against a blank (50 mM phosphate buffer).Ascorbic acid, tocopherol, and rutin were used as a positive control [20].
The scavenging ability was calculated (in triplicate) by the following formula: where  control = absorbance of H

Results and Discussion
The general structure and physical parameters of flavone derivatives are given in Figure 1 and Table 1.The in vitro enzyme inhibition potentials capacity of the flavones derivatives was determined and IC 50 values are given in Table 2.
It is evident that halogenated derived flavones (F3, F4, and F5) showed good activity in comparison with other flavones derivatives (F1 and F2).These results suggest that change in the position or additional moiety may increase or decrease the potency of individual flavones.The antioxidant capacity of the flavones derivatives was estimated with DPPH and H 2 O 2 scavenging systems and results are shown in Tables 3  and 4. The concentration dependent DPPH scavenging effects of flavones derivatives are given.Among the synthesized flavones, the maximum concentration dependent DPPH scavenging effects of 83.16 ( < 0.001) at 120 g/mL were observed by F5 while mild effects of 51.06 ( < 0.05) were produced by F2 at ahigh concentration of 120 g/mL and are comparable with standard ascorbic acid, tocopherol, and rutin.Similar type of findings was observed using H 2 O 2 scavenging system and is given in Table 4.It is evident from the results (Table 1) that nitrogen containing flavone derivative (F2) showed less activity in comparison with halogenated one (F5).Other halogens containing flavones (F3 and F4) also showed antioxidant activity but it was less in comparison to F5, thus suggesting that positioning of halogens may increase or decrease the antioxidant effects as All the values were expressed as mean ± SEM ( = 3).
evident from the findings.Simple flavone (F1) also produced the effects that were almost similar to halogenated ones.These findings may help the future research and open a new window for the synthesis of potent antioxidants for the treatment of wide range of diseases associated with ROS.
In normal situations, the free radicals as by-products are constantly formed by the body's cells from the cellular redox process using oxygen, an essential element of life [23].These are generally called reactive oxygen species (ROS) and have a special attraction for proteins, carbohydrates, lipids, and nucleic acids [24].It has been reported that ROS can be both beneficial and harmful based on the concentration and environment in the biological systems [25,26].The beneficial effects involve a defense against infections, function of cellular signaling, and gene expression.On the other hand, the ROS can mediate injury to cell structures and often referred to as an "oxidative stress" [27].The harmful effects of ROS are counteracted by antioxidants, some of which are enzymes present in the body [28].
Natural antioxidants like -tocopherol, carotenoids, ascorbic acid, flavonoids, and other phenolic compounds might also play a significant role as physiological and dietary antioxidants [29,30].The natural antioxidants are known to possess extensive biological effects that include anticancer, antiviral, antibacterial, anti-inflammatory, antithrombotic, and vasodilatory activities [31].
One method of estimating the antioxidant activity is based on the use of a stable free radical known as DPPH [32][33][34][35] and the electron donation ability of antioxidants can be determined by DPPH purple-colored solution bleaching [36].This method is based on scavenging of DPPH through the addition of an antioxidant that decolorizes the DPPH solution and degree of decolorization is proportional to the free radical scavenging activity indicating its potency [37].Hydrogen peroxide occurs naturally at low concentration in the air, water, food, plants, microorganisms, and human body [38].H 2 O 2 rapidly converts into water and oxygen and may produce hydroxyl radicals ( • OH) that can initiate the lipid peroxidation and cause damage to DNA [39].
LOXs are sensitive to antioxidants as they are involved in inhibition of lipid hydroperoxide formation due to scavenging of lipid oxy-or peroxyradicals and can minimize LOX catalysis by less availability of lipid hydroperoxide substrate [40].Studies have implicated oxygen free radicals in the process of inflammation and phenolic compounds like flavonoids may block the cascade process of arachidonic acid metabolism by inhibiting lipoxygenase activity and may serve as a scavenger of reactive free radicals which are produced during arachidonic acid metabolism [41].
Several studies have reported that natural antioxidants are associated with low rate of heart, cancer, diabetes, and other diseases associated with ageing [52,53].These findings will help the researcher to explore the development of synthetic flavones derivatives for the treatment of wide range of diseases associated with ROS like inflammation and Alzheimer's disease.

Conclusion
In conclusion, the present study confirms the enzyme inhibition and antioxidant activities of flavone derivatives.These findings will open a new channel to synthesize halogenated flavones and explore the development of synthetic flavones derivatives for the treatment of wide range of diseases associated with ROS.
[22]3.In Vitro Lipoxygenase Activity.The lipoxygenase activity of synthesized flavones was determined by spectrophotometric method with slight modification.Inhibition was determined by measuring the loss of soybean 15-LOX activity (5 g) with 0.2 M linoleic acid as the substrate prepared in borate buffer (0.2 M, pH 9.0).The inhibition in triplicate at various concentrations of ∘ C for 15 min using water bath with subsequent addition of substrate solution (5 L).A double beam spectrophotometer (Thermo Electron Corporation, USA) was used to measure the absorbance at 412 nm.IC 50 indicating the concentration of 50% inhibition was also calculated[22].2.4.Statistical Analysis.Data are presented as mean ± SEM.Analysis of variance and Dunnett's test are statistically manipulated with GraphPad Prism 5 version 5.01 software.

Table 1 :
Physical parameters of flavone derivatives.

Table 2 :
In vitro enzyme inhibition potentials of flavone derivatives.

Table 4 :
Hydrogen peroxide radical scavenging activity of flavone derivatives.