1. INTRODUCTION:

Free radicals are highly reactive and unstable molecules containing one or more unpaired electrons, behaves like oxidants or reductants.

Because of this property these free radicals attaches very quickly on the defective cell or that area causes a micro crack in the mitochondrial cell, causes cell damage¹. In modern life we are frequently exposed to free radicals, which can damage any cell in the body with which they come into contact and cause hazardous effects. The excess free radicals can cause a condition called oxidative stress which is mainly responsible for development of many oxidative diseases such as cardiovascular diseases, atherosclerosis, diabetes, rheumatoid arthritis, aging, and skin lesions etc, mainly due to harm done to the body by free radicals². Though the body has its own mechanism to combat oxidative stress, when exposure is more than body’s capacity, the problem aggravates^3.There comes the importance of exogenous antioxidants which alleviates the harmful effects of free radicals^4,5. Antioxidants are a group of compounds that neutralize or prevent free radicals or reactive species and avoid cell or tissue damage caused by oxidative potential of free radicals^6,7. Many natural substances have been explored for their antioxidant activities^8,9. There is still need to evaluate new substances for their antioxidant potential to find better alternative sources for treatment of conditions related to oxidative stress caused by free radicals.

Flavonoids are polyphenolic compounds widely distributed and found in plant materials including naturally occurring plant based food products. These are also considered as natural antioxidants as they are known to be the integral part of human and animal diet ¹⁰. Majority of the flavonoids studied, selectively react with free radicals to act as antioxidants^11-12. Depending on the available information of synthetic flavonoids, the present study was undertaken to evaluate and confirm the radical scavenging antioxidant effects of synthetic flavonoids (dibenzopyrone as a basic nucleus with thiophen substitution) such as VMF41, VMF 43, VMF 45 and VMF 46, using in-vitro radical scavenging activity methods. Nitric oxide, hydroxyl and superoxide anion free radical scavenging methods were used for evaluating antioxidant activities.

2. MATERIAL AND METHODS:

2.1 Materials:

The synthetic flavonoids (SFs) VMF 41, VMF 43, VMF 45 and VMF 46, collected from Dr Vijayakumar D Ptofessor and Principal at Cauavery college of pharmacy Mysore Karnataka India, to which synthesis and characterization details have already been reported elsewhere¹³. 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol, disodium hydrogen phosphate (Na₂HPO₄), sodium nitroprusside, sulphanilamide, phosphoric acid, nicotinamide adenine dinucloetide (NADH), Ethylene Diamine Tetra Acetate (EDTA), butylated hydroxy anisole (BHA), nitrobluetetrazolium (NBT), thiobarbituric acid, 2-deoxy-2- ribose, trichloroacetic acid, phenazine methosulphate, potassium ferricyanide and standard ascorbic acid, were obtained from SD fine chemicals, Sigma loba chemicals. All other chemicals used were of AR grade and used as received.

2.2. Evaluation of In Vitro Antioxidant Activity:

2.2(a). Nitric oxide radical scavenging assay:

The nitric oxide scavenging activity was estimated according to the earlier described method.¹⁴ Various concentrations (10μg, 50μg and 100μg) of SFs compounds dissolved in DMSO (1mg/ml), as well as Butylatedhydroxy anisole (BHA) a standard compound were taken in separate tubes and the volume was uniformly made up to 3ml with 0.1M phosphate buffer (pH 7.2). Then 1ml of sodium nitroprusside (5mM) at phosphate buffer saline (pH7.2) was added to each tube. At room temperature the reaction mixture was incubated for 30 min. Without the test compound a control with an equivalent amount of methanol was used. After the incubation, 1.5ml of above solution including control was mixed with 1.5ml of Griess reagent (2% phosphoric acid, 1% Sulphanilamide and 0.1% N-1- Naphthylethylene diamine dihydrochloride). By using UV-visible spectrometer the absorbance of chromophore formed was measured at 546nm. The IC₅₀ values for each test compounds as well as standard preparation were calculated. Percentage nitric oxide radical scavenging activity was calculated using the following formula:

% NO radical scavenging activity = (Control OD - Sample OD) ×100/ Control OD

The IC₅₀ values for each test compounds as well as standard preparation were calculated.

2.3 (b). Hydroxyl radical Scavenging activity (HRS):

The hydroxyl free radical scavenging activity of synthetic flavonoids(SFs) were assayed according to the method¹⁵described earlier. This assay was used to determine the scavenging activity of test samples at different concentrations in the presence of free hydroxyl radicals which damage the body cells.

The ascorbic acid-iron-EDTA model was used as hydroxyl free radical generating system. In this system, totally aqueous system in which iron - EDTA and ascorbic acid was used that reacts with each other to form hydroxyl radicals. Various concentration of the test SFs (10μg, 50μg and 100μg) in DMSO(1mg/ml), were separately taken in test tubes and made up to 250μl with 0.1M phosphate buffer. The above sample solutions were mixed with 1ml of iron EDTA solution, 0.5ml of EDTA solution, 1ml of DMSO, and then 0.5ml of ascorbic acid was added to start the reaction. The reaction mixture after 15 min of incubation in a boiling water bath, ice-cold TCA was added to stop the reaction. Then 3ml of Nash reagent was added and again the mixture was kept incubation for 15min at room temperature to develop the colour. Then absorbance of the yellow colour formed was measured spectrophotometrically at 412nm against reagent blank. Ascorbic acid (AA) was used as reference standard and percentage hydroxyl radical scavenging activity (% HRSA) was determined by

% HRSA = (Control OD - Sample OD) ×100/ Control OD.

The IC₅₀ values for each test compounds as well as standard preparation were calculated using the Microsoft excel.

2.3 (c). Superoxide Anion Radical (O2^*-) Scavenging Activity:

Scavenging of the superoxide (O2•-) anion radical was measured by the reduction of NBT, according to method which was previously reported¹⁶. The reaction mixture of 1ml contained phosphate buffer (20mM, pH 7.4), NBT (50μM), PMS (15μM), NADH (73μM), and various concentrations (10, 50 and 100μg/ml) of test samples were incubated for 5 min at room temperature. The blank consisted of pure DMSO instead of alkaline DMSO. The absorbance was measured at 562nm using a UV–VIS spectrophotometer. against blank to determine the quantity of formazan generated. All tests were repeated three times. As positive control ascorbic acid was used. Super oxide radical scavenging activity in percentage was calculated using the following formula:

% SO radical scavenging activity = (Control OD - Sample OD) ×100/Control OD.

The test compounds and standard drug IC₅₀ value were calculated by using the Microsoft excel.

3. RESULTS:

Different concentrations ranging from 10, 50 and 100 μg/ml of test novel synthetic flavonoids (SFs) VMF 41, VMF 43, VMF 45 and VMF 46 in DMSO were tested for their free radical scavenging activity with different in vitro methods. It was observed that the test compounds showed a promising free radical scavenging activity in a dose dependent manner. The maximum percentage inhibition with different concentrations of novel synthetic flavonoids for different radical scavenging models is given in the following Tables.

Nitric Oxide radical scavenging:

Nitric oxide scavenging activity was performed with four test novel synthetic flavonoid (SFs) compounds and BHA as standard drug. the reductive potential of these compounds exhibited in a dose dependent manner as shown in Table- 1. From these results, it is clear that the IC₅₀ value calculated for SF compounds were found significant as compared to BHA. The IC₅₀ of compounds VMF 41, VMF 43, VMF 45 and VMF 46 were 5.72 μg/ml, 5.65μg/ml, 3.65μg/ml and 3.99μg/ml respectively which were found higher than IC₅₀ of BHA standard drug. At 100µg/ml concentration the percentage inhibition of VMF 45 and VMF 46 compounds on nitric oxide radicals showed 42.61% which is almost equivalent to that of standard BHA.

Table. 1. Percentage Nitric oxide Radical Scavenging Activity of Synthetic Flavonoids (SFs) compared with Standard

Test Samples and their percentage scavenging activity
Concentration	VMF 41	VMF 43	VMF 45	VMF 46	BHA
10µg	5.65	10.00	11.30	5.65	14.35
50µg	15.65	22.61	18.70	13.04	34.78
100µg	24.35	26.96	42.61	42.61	47.83
IC₅₀	5.72± 5.12	5.56± 0.28	3.65± 1.32	3.99± 0.13	3.06± 0.24

Nitric oxide radical scavenging assay. The nitric oxide radical scavenging activity of novel synthetic flavonoid (SFs) and the standard BHA. The data represent the percentage nitric oxide inhibition.

Hydroxyl radical scavenging:

Results were tabulated in Table-3. Here the SFs VMF 41, VMF 43, VMF 45 and VMF 46 at different concentrations (10μg/ml - 100μg/ml) were tested, where they found to exhibit a dose dependent hydroxyl radical scavenging activity.

The SF compounds VMF 43and VMF 46 at 100µg/ml concentration the percentage inhibition of hydroxyl radical were 41.67% and 46.83% respectively. And their IC₅₀values were 3.66μg/ml and 2.06μg/ml respectively where as that of standard ascorbic acid percentage inhibition on hydroxyl radical was 94.84% with IC₅₀ value 1.1 suggesting that test flavonoids have less potent than standard drug. The test flavonoids VMF 41 and VMF 45 in this study showed negligible scavenging effect on hydroxyl radicals as shown in the table 2.

Table. 3. Percentage Hydroxyl Radical Scavenging Activity of Synthetic flavonoids (SFs) compared with Standard

Test Samples and their percentage scavenging activity
Concentration	VMF 41	VMF 43	VMF 45	VMF 46	Ascorbic acid
10µg	8.33	5	4	9	48.01
50µg	16.17	12.17	19.00	24.67	69.84
100µg	33.33	41.67	29.50	46.83	94.84
IC₅₀	4.46 ± 3.27	3.66 ± 0.97	4.54± 1.02	2.06± 2.81	01.11± 0.13

Hydroxyl radical scavenging assay. The hydroxyl radical scavenging activity of novel synthetic flavonoid (SFs) and the standard ascorbic acid. The data represent the percentage inhibition of deoxyribose degradation.

Superoxide radical scavenging:

The superoxide radicals generated from dissolved oxygen by PMS-NADH coupling can be measured by their ability to reduce NBT. The decrease in absorbance at 562nm with the test synthetic flavonoids (SFs) VMF 41, VMF 43, VMF 45 and VMF 46 and the reference compound ascorbic acid indicates their abilities to quench superoxide radicals in the reaction mixture. As shown in Table 1, IC₅₀ values of test synthetic flavonoids (SFs) VMF 41, VMF 43, and the standard ascorbic acid on superoxide radical scavenging activity were 3.44±0.36, 4.51±0.36 and 02.2±0.62 respectively. The IC₅₀ value of the synthetic flavonoids were more than that of the standard drug. It suggests that the only test flavnoids VMF 41 and VMF 43exhbited more superoxide radical scavenging activity than other test flavonoids but lesser than ascorbic acid. At 50 μg/ml, the percentage inhibition of the synthetic flavonoids (VMF 41 and VMF 43) were 24%, 18.4%, whereas that of ascorbic acid was 46.88%.

Table. 4 Percentage Super oxide Anion Radical Scavenging Activity of Synthetic Flavonoids (SFs) compared with Standard

Test Samples and their percentage scavenging activity
Concentration	VMF 41	VMF 43	VMF 45	VMF 46	Ascorbic acid
10µg	0.8	0.8	1.6	0	37.36
50µg	24	18.4	8	09.6	46.88
100µg	40	28.0	16	15.2	59.12
IC₅₀	3.44± 0.36	4.51± 0.36	7.75± 0.22	7.491± 0.31	02.2± 0.62

Super oxide radical scavenging assay. The Super oxide radical scavenging activity of novel synthetic flavonoid (SFs) and the standard ascorbic acid.

4. DISCUSSION:

Free radicals are highly reactive molecules, associated with oxidative damage where as antioxidants are reducing agents, since they donate electrons to free radicals and preventing them from oxidative damage to biological structures. Otherwise these excess free radicals may leads to various disease conditions, especially degenerative diseases, and extensive lysis¹⁷.

Antioxidants are molecules that prevent the oxidation from other compounds through hydrogen or electron donors as well neutralize free radicals. Recently, many natural antioxidants have been isolated from different plant materials. According to a Afnan E. Abd-Almonuim et al,. flavonoids and phenolic compounds possess the antioxidant and radical scavenging properties. Flavonoids are polyphenolic compounds widely distributed and found in plant materials including naturally occurring plant based food products¹⁸. Majority of the flavonoids studied, selectively react with free radicals to act as antioxidants. Eventhough, natural flavonoids are highly potent, they show certain limitations as far as their stability, solubility characteristics and kinetics are concerned¹⁹.

Therefore, the present study was undertaken to study the free-radical scavenging ability of synthetic flavonoids through invitro assay methods.

Nitric oxide is an important chemical mediator generated by endothelial cells, macrophages, neurons and involved in the regulation of various physiological functions such as vascular homeostasis, neurotransmission, antitumor, and antimicrobial activities etc. However, many reports suggest that, excess concentration of nitric oxide due to unstable nature under aerobic environment, is implicated in the cytotoxic effects observed in various disorders such as AIDS, cancer, Alzheimer’s and arthritis etc^20-21. Drugs which scavenge the nitric oxide compete with oxygen, leading to less formation of nitrite ions. It would be interesting to develop potent and selective inhibitors of NO* for potential therapeutic use²².

From results shown in Table1, it is clear that synthetic flavonoids VMF 41, VMF 43, VMF 45 and VMF 46 have shown as antioxidants compared to the BHA but lesser than the standard. These compounds can compete with oxygen to react with nitric oxide radical and thus reduce the generation of the nitrite and peroxy nitrite anions. This property of these compounds may be due to the electron donating nature of the substituent’s OH and -CH₃ groups of benzopyran nucleus²³.

Hydroxy radical is a highly reactive in biological systems and has been implicated as highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells and is involved in many pathophysiological processes including DNA strand breakage and K+ loss from the cell membrane^24-25.If any drug scavenges the hydroxyl radical, they may either scavenge the radical or may chelate the Fe2+ ion making them unavailable for the Fentons reaction. Results tabulated in table-3 suggests that the synthetic flavonoids found to exhibit a dose dependent hydroxyl radical scavenging activity when tested at different concentrations (10μg/ml, 50μg/ml and 100μg/ml). Among these test flavonoids only VMF 43 and VMF 46 based on their IC₅₀ values, makes clear that of having good hydroxyl radical scavenging activity but lesser than that of standard ascorbic acid which is having 01.11±0.13 as IC₅₀ value .

The radical scavenging power of the test flavonoid and standard drug can be explained by the presence of OH groups and methyl groups in their chemical strictures, as they quench the oxygen-derived free radicals by donating a hydrogen atom or an electron to the free radicalor neutralize free radicals or by their chelating ability due to their high nucleophilic character of the aromatic ring²⁶.

Superoxide anion is also very harmful to cellular components^27,28. Robak and Glyglewski²⁹ reported that flavonoids are effective antioxidants mainly because they scavenge superoxide anions.

From results, it was found that the compounds (SFs) VMF 41, VMF 43, VMF 45 and VMF 46 showed potent free radical scavenging activity compared to the ascorbic acid (standard). However the compounds VMF 41 and VMF 43 exhibited the highest radical scavenging power 40% and 28% respectively than the other test synthetic flavonoids in this study. The flavonoids are often considered as antioxidants as they have property of donating electrons due to the presence of electron donating substituent groups like –OH, -CL and –CH₃ in their chemical benzopyran nucleus³⁰. Therefore these compounds donated their electrons to the superoxide and scavenge them to prevent their further interaction with NBT followed by inhibition of formation of blue colour formazan product.

5. CONCLUSION:

From the results obtained in this study, it is concluded that the synthetic flavonoids (SFs) VMF 41, VMF 43, VMF 45 and VMF 46 exhibited the radical scavenging activity. For nitric oxide scavenging activity, VMF 45 and VMF 46 test flavonoids (SFs) have showed better antioxidant activity than VMF 41, VMF 43. In case of hydroxyl radical scavenging assay, the compounds VMF 43 and VMF 46, exhibited the highest scavenging power than the other synthetic test flavonoids. Lastly, the compounds VMF 41and VMF 43 synthetic flavonoids represented the good antioxidant than the VMF 45 and VMF 46 compounds in scavenging the superoxide radicals generated in the assay reactions. To conclude based on results in this study; it is clear that these synthetic flavonoids can be considered as a potential antioxidant agents which need to be further explored for proper design of lead antioxidant drug candidates. Furthermore, the in-vivo antioxidant activity of these synthetic flavonoids needs to be assessed prior to clinical use.

6. ACKNOWLEDGEMENT:

The authors are grateful to the authorities of BLDE’s B.M.Patil Medical College and B.V.V.S’s S.N.Medical Bagalkot, for the facilities.

7. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.02.2021 Modified on 29.09.2021

Research J. Pharm. and Tech 2022; 15(9):3947-3951.

DOI: 10.52711/0974-360X.2022.00661