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. Flavones have got special position in research field of natural and synthetic organic chemistry due to their biological capabilities as antioxidant. The antioxidants are known to possess extensive biological effects that include antiviral, antibacterial, anti-inflammatory, antithrombotic, and vasodilatory activities. The simple flavone (
Reactive oxygen species (ROS) and the free radicals like superoxide, hydroxyl, alkoxyl, hydroperoxyl, and peroxyl are produced during normal metabolism in humans [
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 [
In general, the flavonoids are reported to possess potent antioxidant activity [
Ketone and benzaldehyde derivatives, DPPH, ascorbic acid, quercetin, rutin, silica, DTNB 5,5-dithiobisnitrobenzoic acid, enzymes including 15-lipoxigenase (soybean), AChE
1H-NMR and 13C 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
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 (
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
Structure of flavone derivatives.
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
After 30 minutes, the absorbance was measured at 517 nm. Ascorbic acid, tocopherol, and rutin were used as a positive control. The scavenging activity of tested samples was calculated by the following formula in triplicate [
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
The scavenging ability was calculated (in triplicate) by the following formula:
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
The
Data are presented as mean ± SEM. Analysis of variance and Dunnett’s test are statistically manipulated with GraphPad Prism 5 version 5.01 software.
The general structure and physical parameters of flavone derivatives are given in Figure
Physical parameters of flavone derivatives.
Flavone | R1 | R2 | R3 | Yield | Appearance |
|
M.P. (°C) |
---|---|---|---|---|---|---|---|
F1 | –H | –H | –H | 68.7% | Creamy white solid | 0.58 | 96–98 |
F2 | –H | –H | –N(CH3)2 | 73.6% | Brick red solid | 0.67 | 107–109 |
F3 | –Cl | –H | –Cl | 87.0% | White solid | 0.57 | 90.5 |
F4 | –Cl | –Cl | –H | 81.0% | White solid | 0.64 | 88.3 |
F5 | –H | –Cl | –Cl | 79.3% | White solid | 0.61 | 195–197 |
Sample test | LOX |
AChE |
---|---|---|
F1 | 77.29 ± 1.32 | 187.23 ± 1.65 |
F2 | 98.63 ± 1.91 | ≥250 |
F3 | 65.75 ± 1.45 | 131.33 ± 1.05 |
F4 | 61.62 ± 1.15 | 126.29 ± 1.39 |
F5 | 58.33 ± 1.69 | 112.33 ± 1.68 |
Indomethacin | 53.66 ± 1.38 | — |
Quercetin | 38.50 ± 1.72 | — |
Galantamine | — | 25.66 ± 1.09 |
All the values were expressed as mean ± SEM (
DPPH radical scavenging activity of flavone derivatives.
% Scavenging activity (DPPH) | ||||||
---|---|---|---|---|---|---|
25 |
50 |
75 |
100 |
125 |
150 | |
Ascorbic acid | 65.23 ± 0.41 | 71.56 ± 0.61 | 75.26 ± 0.68 | 81.09 ± 0.42 | 86.11 ± 0.87 | 90.68 ± 0.58 |
Tocopherol | 72.32 ± 0.36 | 79.16 ± 0.57 | 84.41 ± 0.61 | 90.18 ± 0.53 | 92.24 ± 0.75 | 94.33 ± 0.49 |
Rutin | 79.03 ± 0.76 | 83.11 ± 0.43 | 87.63 ± 0.81 | 91.25 ± 0.61 | 95.35 ± 0.66 | 97.67 ± 0.51 |
F1 | 32.12 ± 0.61 | 38.46 ± 1.17 | 46.48 ± 0.57 |
55.81 ± 0.81 |
61.65 ± 1.06 |
63.44 ± 0.75 |
F2 | 16.65 ± 1.04 | 21.62 ± 0.67 | 30.94 ± 0.63 | 37.63 ± 0.39 | 44.74 ± 0.86 |
47.76 ± 0.82 |
F3 | 42.47 ± 0.84 |
51.62 ± 1.21 |
59.74 ± 0.75 |
63.18 ± 0.54 |
68.21 ± 0.61 |
70.16 ± 0.69 |
F4 | 35.31 ± 0.92 | 38.32 ± 0.62 |
55.27 ± 0.43 |
61.65 ± 0.49 |
65.36 ± 0.72 |
68.14 ± 0.55 |
F5 | 41.96 ± 0.54 |
53.78 ± 1.31 |
61.21 ± 0.69 |
65.23 ± 0.71 |
71.96 ± 0.47 |
73.12 ± 0.48 |
All the values were expressed as mean ± SEM.
Hydrogen peroxide radical scavenging activity of flavone derivatives.
% Scavenging activity (H2O2) | ||||||
---|---|---|---|---|---|---|
25 |
50 |
75 |
100 |
125 |
150 | |
Ascorbic acid | 71.23 ± 0.65 | 76.57 ± 0.49 | 84.06 ± 0.81 | 88.19 ± 0.56 | 92.21 ± 0.71 | 96.63 ± 0.63 |
Tocopherol | 73.02 ± 0.43 | 76.26 ± 0.66 | 82.43 ± 0.73 | 90.38 ± 0.87 | 93.65 ± 0.63 | 95.47 ± 0.48 |
Rutin | 77.13 ± 0.69 | 82.15 ± 0.87 | 88.43 ± 0.34 | 92.35 ± 0.61 | 97.16 ± 0.46 | 98.84 ± 0.74 |
F1 | 28.21 ± 0.84 | 32.52 ± 0.77 | 38.33 ± 0.87 |
47.41 ± 0.36 |
55.34 ± 0.67 |
57.84 ± 0.69 |
F2 | 13.75 ± 0.60 | 17.12 ± 0.97 | 25.77 ± 0.68 | 32.35 ± 0.79 | 36.47 ± 0.68 | 42.19 ± 0.48 |
F3 | 37.43 ± 0.48 | 48.35 ± 0.91 |
53.19 ± 0.57 |
56.78 ± 0.45 |
60.46 ± 0.46 |
63.16 ± 0.84 |
F4 | 31.62 ± 0.54 | 35.13 ± 0.77 | 46.91 ± 0.74 |
51.85 ± 0.78 |
57.74 ± 0.56 |
61.39 ± 0.43 |
F5 | 43.07 ± 0.48 |
51.72 ± 0.76 |
57.68 ± 0.49 |
62.86 ± 0.65 |
70.21 ± 0.71 |
72.26 ± 0.78 |
All the values were expressed as mean ± SEM.
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 [
Natural antioxidants like
One method of estimating the antioxidant activity is based on the use of a stable free radical known as DPPH [
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 [
Flavonoids are phenolic compounds that are mostly present in fruits, vegetables, and tea; they are an integral part of the human diet [
Several studies have reported that natural antioxidants are associated with low rate of heart, cancer, diabetes, and other diseases associated with ageing [
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.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors are thankful to Department of Pharmacy, University of Malakand, Khyber Pakhtunkhwa, Pakistan, for provision of laboratory facilities.