INTRODUCTION:

In the human body, free radicals are continuously generated as a consequence of normal metabolic processes and interaction with environmental stimuli¹. An atom or molecule which has an unpaired electron is a free radical and is therefore unstable. This unstable radical appears to become stable in healthy human cells by electron pairing with biological macromolecules such as proteins, lipids, and DNA, causing damage to proteins and DNA². The production of free radicals in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a normal healthy human body is effectively regulated by various levels of antioxidant protection mechanisms such as superoxide dismutase (SOD), catalase, glutathione, glutathione peroxidases and reductase, vitamin E (tocopherols and tocotrienols) and vitamin C³.

When the generation of Reactive oxygen species exceeds the levels of antioxidant mechanism, it leads to oxidative damage of tissues and bio molecules, eventually leading to many degenerative human diseases like diabetes mellitus, atherosclerosis, cancer, neurodegenerative disorders, Alzheimer’s disease, Parkinson’s disease, and other free radical mediated diseases⁴. By taking appropriate quantities of exogenous antioxidants, defense against free radicals can be improved. A stable molecule that donates an electron to a rampaging free radical and terminates the chain reaction before destroying vital molecules is an antioxidant. Cellular damage may be delayed or inhibited by free radical scavenging potential of antioxidants⁵. Antioxidants serve as reducing agents, free radical scavengers, quenchers of singlet oxygen molecules, and antioxidant enzyme activators to regulate the harm caused by free radicals in living systems⁶.

Many antioxidant compounds, naturally occurring from plant sources, have been identified as free radical or active oxygen scavengers. Much attention has recently been invested in the development of ethnomedicinal products with potent antioxidant properties but low cytotoxicity to replace synthetic antioxidants, which are being restricted due to their side effects such as carcinogenicity. Natural antioxidants may protect the human body from free radical damage and therefore delay the progress of many chronic diseases and lipid oxidative rancidity in foods⁷. Many plants often contain substantial amounts of antioxidants including vitamin C and E, carotenoids, flavanoids and tannins and can be utilized to scavenge the excess free radicals from human body⁸. Epidemiological evidence also suggests an inverse correlation between the consumption of phenolic compound-rich foods and the reduction of some chronic diseases⁹. In addition, the antioxidant effect of plant products has been determined to be primarily due to the radical scavenging action of phenolic compounds such as flavonoids, polyphenols, tannins and phenolic terpenes¹⁰. Tabebuia roseo-alba (Ridl) Sand, known as the White Trump pet tree, belongs to the Bignoniaceae family and, due to its ornamental use and its ethano pharmacological components, is considered to be an economically significant species. Previous research has shown that the leaves and stem bark of this plant have demonstrated antimicrobial activity against microorganisms of nosocomial infection. In addition, it has been shown that this plant species has powerful cytotoxic activity¹¹. The present study was therefore aimed at evaluating the free radical scavenging efficiency of different Tabebuia roseo-alba (T. roseo-alba) extracts.

For the evaluation of antioxidant activities, there are different models available. Depending on the test used, the chemical complexity of different extracts and the mixture of compounds present may lead to dispersion of the results¹². Therefore, it would be more informative and even appropriate to use an approach with many assays to determine the antioxidant capacity of extracts. Free radical scavenging activities of various extracts were assessed in this study and all findings were compared with standard findings.

MATERIALS AND METHODS:

Collection and authentication of the plant sample:

Fresh young leaves of T. roseo-alba were collected in P.S.G college of Arts and Science campus, Coimbatore district and authenticated at Botanical Survey, Tamil Nadu Agriculture University (TNAU) Coimbatore, India (BSI/SRC/5/23/2019/Tech/3236).

Preparation of leaf extract:

The leaves were washed with distilled water several times to remove dust particles and shade dried for 2 to 3 weeks at room temperature. Dried leaves were powdered and extracted with various solvents of increasing polarities like petroleum ether, chloroform, ethanol and water using Soxhlet apparatus. The extracts were then filtered through muslin cloth, evaporated under reduced pressure, stored in air tight containers separately at 4°C and used for further experiments.

DPPH radical scavenging assay:

The free-radical scavenging activities of various extracts were tested by their ability to reduce the stable radical DPPH. The antioxidant activity using DPPH (1, 1-diphenyl-2- picrylhydrazyl) assay was evaluated according to the method of Blois¹³. The reaction mixture contained 100μM DPPH in methanol and various concentrations (20-100μg/ml) of plant extract. Absorbance at 517nm was documented after 30 min at room temperature and the scavenging activity were calculated as a percentage of the radical scavenging. The experiment was performed in triplicates. Ascorbic acid was taken as reference standard. The percentage inhibition vs. concentration was plotted and the concentration required for 50% inhibition of radicals was expressed as IC₅₀value.

Total antioxidant activity assay by radical cation (ABTS⁺):

ABTS radical scavenging assay was performed by the method of Re et al¹⁴. ABTS radical cation (ABTS⁺) was produced by reacting 2.45mM potassium persulfate (final concentration) with ABTS stock solution and allowing the mixture to stand in the dark for 12-16 h before use at room temperature. The solution was diluted in methanol (approx. 1:89 v/v) prior to the assay and adjusted to 30°C to give an absorbance of 0.700±0.02 in a 1cm cuvette at 734nm. Varying concentrations of the plant extracts (20-100μg/ml) was allowed to react with 1mL of the ABTS^•+ solution and the absorbance was taken at 734nm between 3-7 min using the spectrophotometer. The ABTS^•+ scavenging capacity of the extract was compared with that of the standard and the percentage inhibition was calculated using the following formula,

ABTS⁺radical scavenging activity (%) = [(Abs_control-Abs_sample)/Abs_control)]x100

Abs_control - absorbance of ABTS radical and methanol: Abs_sample - absorbance of ABTS radical + sample /standard.

Hydroxyl radical scavenging activity:

The scavenging activity of sample extracts on hydroxyl radical was measured according to the method of Klein et al¹⁵. Various concentrations (20-100μg/ml) of the extracts were added with 1.0mL of iron-EDTA solution, 0.5mL of EDTA solution (0.018%), and 1.0mL of DMSO (0.85% DMSO (v/v) in 0.1M phosphate buffer, pH 7.4) sequentially. By adding 0.5mL of ascorbic acid (0.22 %), the reaction was initiated and incubated in a water bath at 80-90°C for 15 minutes. The reaction was terminated by the addition of 1.0mL of ice-cold TCA (17.5 % w/v) after incubation. Three mL of Nash reagent was added and left at room temperature for 15 min. The intensity of the colour produced against the reagent blank was measured spectrophotometrically at 412nm. The % hydroxyl radical scavenging activity was calculated using the formula as given above.

Hydrogen peroxide radical scavenging assay:

The capacity of the extracts to scavenge hydrogen peroxide was calculated by Ruch et al¹⁶. In a phosphate buffer (pH 7.4), a solution of hydrogen peroxide (2mmol /l) was prepared. Extracts (20-100μg/ml) were added to hydrogen peroxide solution (0.6ml). After 10 min, the absorption of hydrogen peroxide at 230nm against a blank (solution containing phosphate buffer without hydrogen peroxide) was calculated and compared with the reference compound, ascorbic acid, The % hydrogen peroxide radical scavenging activity was calculated using the formula as given above.

Superoxide radical scavenging assay:

The measurement of superoxide scavenging activity is based on the method as described by Liu et al¹⁷ and is assayed by the reduction of nitroblue tetrazolium (NBT). Tris HCl buffer (3ml, 16mM, pH 8.0) containing 1 ml NBT (50μM) solution, 1ml NADH (78μM) solution and a sample solution of extracts (20-100μg/ml) in water were mixed. The reaction was initiated by the addition of 1ml of phenazine methosulfate (PMS) solution (10μM) to the mixture. The reaction mixture was incubated at 25 °C for 5 min, and the absorbance was read at 560nm against the corresponding blank samples. Increased superoxide anion scavenging activity was measured by decreased absorption of the reaction mixture. Ascorbic acid was used as the standard and the % superoxide radical scavenging activity was calculated using the formula as given above.

Nitric oxide scavenging assay:

The interaction of various extracts of T. roseo-alba with nitric oxide (NO) was assessed by the nitrite detection method¹⁸. Various concentrations of the extracts (20-100 μg/ml) were mixed with 2.5ml of sodium nitroprusside and made upto 3.0ml with PBS. Then the mixture was incubated for 15 minutes at 25˚C. After incubation, for 6 min at 37°C, 0.5ml of the reaction mixture was removed and 0.5ml of Griess reagent (a-napthyl-ethylenediamine 0.1% in water and sulphanilic acid 1% in H₃PO₄) was added. And the absorbance was measured at 546nm. The same reaction mixture without the extract of sample but with equivalent amount of distilled water served as control. Ascorbic acid was used as positive control. By comparing the test results with those of controls not treated with the extract, the percent inhibition was determined.

Reducing power assay:

The reducing power capacity of the plant was assessed by the modified method of Oyaizu¹⁹. Various concentrations (20-100ug/mL) of the extracts (0.5mL) were mixed with 0.5mL phosphate buffer (0.2 M, pH 6.6) and 0.5ml potassium hexacyanoferrate (0.1%), following 50 incubation in a water bath for 20 minutes. After incubation, 0.5mL of TCA (10%) was added to end the reaction. The upper portion of the solution (1ml) was mixed with 1ml distilled water, and 0.1mL ferric chloride solution (0.01%) was added. At room temperature, the reaction mixture was left for 10 min and the absorbance was measured at 700nm against a suitable blank solution. Greater reducing power was indicated by the higher absorbance of the reaction mixture. Ascorbic acid was used as a positive control.

STATISTICAL ANALYSIS:

All experiments were performed in triplicates and the data obtained were statistically analyzed and results were reported as mean+SD.

RESULTS:

DPPH radical scavenging assay:

The assay was performed using varying concentrations of T. roseo-alba extracts ranging from 20-100μg along with the standard Ascorbic acid. The scavenging abilities of different solvent extracts of T. roseo-alba were concentration-dependent and expressed in Figure 1. Concentration of the sample necessary to decrease the initial concentration of DPPH by 50% (EC₅₀) was calculated.

Figure 1. DPPH radical scavenging assay:

From the results it is evident that, all the extracts were able to scavenge DPPH radicals in a dose dependant manner. The percentage of scavenging potential on the DPPH radical was concurrently increased with increase in the concentration of all the extracts. However, the maximum activity was observed at 100μg which confirms the dose dependent activity of the extracts of T. roseo-alba. Among the different extracts used, the ethanolic extract of T. roseo-alba was efficient in scavenging DPPH radicals with an EC₅₀ value of 61.13μg/ml, while others exhibited moderate activity. DPPH free radical scavenging effect of different T. roseo-alba extracts and standards was in this order: Ascorbic acid>Ethanol >Aqueous >P.ether>Chloroform. The results confirmed that all extracts of T. roseo-alba were able to scavenge the DPPH radical indicating the antioxidant potential of T. roseo-alba which may be due to the presence of mixture of phytochemicals. Hydrogen may be donated to free radicals by antioxidants with DPPH radical scavenging activity, in particular to lipid peroxides or hydroperoxide radicals, which are the key propagators of lipid chain autoxidation, and to form non-radical species, resulting in the inhibition of the propagating phase²⁰.

ABTS⁺ radical scavenging Assay:

The total antioxidant potential of the extract through ABTS⁺ radical scavenging was calculated from the decolorization of ABTS⁺ radical which was measured spectrophotometrically at 734nm. Figure 2 indicates the ABTS⁺ radical scavenging potential of the various extracts of T. roseo-alba at different concentrations. The results showed that different extracts of T. roseo-alba showed varying degree of scavenging potential for ABTS⁺ radicals in concentration dependent manner. From the results, it may be postulated that, the ethanolic extract of T. roseo-alba exhibited a higher inhibitory potential compared to other extracts with a maximum percentage inhibition of 70.41 ± 0.12 at 100μg/mL with an EC₅₀value of 55.4μg/mL. The results were compared with the standard Vitamin C and the values evidenced the antioxidant potential of the extract.

Figure 2. ABTS⁺ radical scavenging assay

Hydroxyl radical scavenging assay:

The results of hydroxyl radical scavenging potential of the plant extract and ascorbic acid were depicted in Figure 3. Hydroxyl radicals were formed from the reaction of H₂O₂ and the ferric compound that would react with 2-deoxyribose. Hydroxyl radical scavenging potential of an extract or compound is directly proportional to its antioxidant property that is evident from the low intensity of red colour. Hydroxyl radicals were efficiently scavenged and degradation of 2-deoxyribose was prevented by all the extracts of T. roseo-alba when added to the mixture.

Figure 3. Hydroxy radical scavenging assay:

Among the various extracts tested, ethanolic extract exhibited greater antiradical activity with the maximum inhibition of 71.24 at 100μg/ml with an EC₅₀value of 44.11μg/ml while standard ascorbate was found to possess 76.94% scavenging activity at 100μg/ml, which was higher than that of all the extracts tested. The extracts with lower EC₅₀ value shows more potential of free radical scavenging ability than the extracts with higher EC₅₀.

Hydrogen peroxide radical scavenging Assay:

The scavenging of hydrogen peroxide by the extract in a dose dependent manner is illustrated in (Figure 4). In the present study, the various extracts of T. roseo-alba were subjected to oxidative stress by the addition of H₂O₂. Though all the extracts tested were able to scavenge H₂O₂ to a considerable extent, ethanolic extract was found to exhibit greater antiradical activity when compared to other extracts. Maximum scavenging activity was observed at 500μg/ml with an EC₅₀ value of 76.88μg/ml. However, with a consistent increase in the concentration of all the extracts of T. roseo-alba, increased Hydrogen peroxide radical scavenging activity was observed.

Figure 4. Hydrogen peroxide radical scavenging assay

Nitric oxide radical scavenging assay:

In the present study, various extracts of T. roseo-alba leaves were assessed for their inhibitory potential against NO generation in vitro. All the extracts were able to inhibit nitric oxide generation in a concentration dependent manner and the results are shown in Figure 5. On comparison with other extracts, ethanolic extract was found to be effective in nitric oxide radical scavenging activity. The ethanolic extract showed maximum inhibition of 73.73±0.23 at a concentration of 100μg/mL with an EC₅₀ value of 35.62μg/mL. The study proved the antioxidant potential of the plant by inhibiting nitric oxide generation and thus can be used as valuable source of natural antioxidants that can protect against free radicals mediated damages.

Figure 5. Nitric oxide radical scavenging assay

Superoxide radical scavenging assay:

The ability to inhibit the generation of superoxide by different extracts of T. roseo-alba was assessed and the results are presented in Figure 6. The decrease in absorbance at 560nm represents the free radical scavenging potential of the plant extracts against the superoxide anion in the reaction mixture. Although all the extracts were able to exhibit inhibitory activity against superoxide generation in a concentration dependent manner, the ethanolic extract of T. roseo-alba leaves exhibited maximum superoxide radical scavenging activity with an EC₅₀ value of 55.4μg/mL. From the results, it may be suggested that the ethanolic extract of T. roseo-alba leaves acquire antioxidant activity by inhibiting superoxide radical generation which may be due to the presence of secondary metabolites in the plant.

Figure 6. Superoxide radical scavenging assay

Reducing power Assay:

The presence of reductants in the antioxidant sample reduces the Fe³⁺/ferricyanide complex to the form of Fe²⁺/ferrous in this assay. By measuring the formation of Perl's Prussian blue at 700nm, the reduction power of the sample can thus be monitored.

Figure 7. Reducing power assay

The reduction potential of various T. roseo-alba extracts and standards at different concentrations was measured by their ability to transform Fe³⁺ to Fe²⁺ and the results are depicted in the Figure 7. On the basis of the results, it is documented that there is a concentration dependent increase in reducing power of all the extracts. Among the different extracts tested, ethanolic extract of T. roseo-alba was found to have higher reduction potential. Maximum reducing power of ethanolic extract was found at 100μg/ml signifying their dose dependent activity which was comparable with the standard ascorbic acid. Hydrogen donation from phenolic compounds, which is also related to the presence of a reducing agent, may be attributed to the ability to reduce Fe(III). Furthermore, the number and position of hydroxyl group in the phenolic compounds also contribute to their antioxidant activity²¹.

DISCUSSION:

DPPH is a nitrogen-centered radical with a characteristic absorption at 517nm, commonly used for assessing the radical scavenging activity of plant extracts and to test the ability of compounds as free-radical scavengers or hydrogen donors. The capacity of natural products to donate electron can be determined by 2,2- diphenyl-1- picrylhydrazyl radical (DPPH), which is based on the bleaching of purple coloured solution²². Due to its hydrogen accepting capability, the antioxidants react with DPPH and convert it to 1,1-diphenyl-2-picryl hydrazine at a very rapid rate. The degree of discoloration is dependent on the strength and concentration of the antioxidants and indicates the scavenging potential of the antioxidants¹². In the present study, the antioxidant activity was confirmed by a decrease in absorbance band upon increasing concentrations of various extracts of T. roseo-alba leaves.

The ABTS assay is an excellent method for evaluating the antioxidant activity of natural compounds that donate hydrogen and chain breaking antioxidants²³. A protonated radical, ABTS⁺ radical, has a characteristic absorption maximum at 734nm, which decreases with proton radical scavenging, which is known as an excellent peroxidase substrate frequently used to study the antioxidant properties of natural compounds²⁴. In present study the scavenging potential of T. roseo-alba for ABTS radical was recorded in a concentration dependent manner

The hydroxyl radical can cause oxidative damage to DNA, lipids and proteins. The hydroxyl radical in the cells can simply cross the cell membrane at specific sites, react with the majority of biomolecules and furthermore can cause tissue damage and finally lead to cell death. Thus, removing hydroxyl radical from the body is very important for the protection of living systems²⁵. In the presence of decreased transition metals such as Fe²⁺ and H₂O₂, hydroxyl radical can be generated by the Fenton reaction, which is considered to be the most reactive of all reduced forms of dioxygen and is thought to induce in vivo cell damage²⁰. The hydroxyl radical scavenging activity was evident at all the concentrations of T. roseo-alba leaves and correlated well with increasing concentrations.

Hydrogen peroxide is a weak oxidising agent which, typically through the oxidation of essential thiol (-SH) groups, directly inactivates a few enzymes. In order to form hydroxyl radicals, it can probably react with Fe²⁺ and possibly Cu²⁺ ions and this may be the cause of many of its toxic effects. Scavenging of hydrogen peroxide relies on the phenolic content present in the plant extract that can donate electrons to H₂O₂, thereby neutralizing it into water¹. In our study, various extracts of T. roseo-alba scavenged H₂O₂, thus proving its antioxidant potential which may be due to the presence of phenolics, which could donate electrons to H₂O₂, thereby neutralizing it into water.

Nitric oxide is a free radical generated in mammalian cells that is involved in the control of neurotransmission, vascular homeostasis, antimicrobial and antitumor activities of various physiological processes. Since excess production of NO is associated with many illnesses, development of potent and selective inhibitors of NO for future therapeutic use would be interesting²⁶. In the present study, T. roseo-alba exhibited potent nitric oxide radical scavenging activity, which competes with oxygen to react with nitric oxide and thus inhibits the generation of nitrite.

Directly or indirectly, superoxide radicals destroy biomolecules by forming H₂O₂, OH, peroxyl nitrate or singlet oxygen during ageing and pathological events such as injury and ischemic repurfusion. It has also been found that superoxide initiates lipid peroxidation directly²⁷. Superoxide anion is a relatively weak oxidant, but it can produce more hazardous species, like singlet oxygen and hydroxyl radicals, potentially causing tissue damage²⁸. Superoxide radical reduces NBT to a blue coloured formazan that is measured at 560nm in the present analysis. Higher concentrations of the extracts were more efficient in scavenging free radicals. Our findings suggest that the phenolic components and other secondary metabolites present in the plants extracts might be responsible for superoxide radical scavenging effect.

The reducing power of the plant extract act as a indicator of its potential antioxidant activity. The decreasing properties are typically related to the presence of reductones that demonstrate antioxidant activity by breaking the chain reactions by contributing hydrogen atoms²⁹. In the present study with T. roseo-alba leaves, all the extracts demonstrated reducing power that increased linearly with increasing concentration.

In an attempt to analyse free radical scavenging and antioxidant potential of various extracts of T. roseo-alba, all the radicals tested in the present study are competently scavenged to a greater extent by the ethanolic extract of T. roseo-alba which was comparable with the standard. Based on the various in vitro assays, it can be concluded that the ethanol extract of T. roseo-alba, possesses strong antioxidant activity due to the presence of phenolic components and other active metabolites in the extract, which was confirmed by the free radical scavenging property. The results obtained in this study indicate that ethanolic extract of T. roseo-alba scavenges free radicals efficiently and there by diminishing lipid peroxidation, ameliorating the damage caused by oxidative stress in various pathological conditions and serve as easily accessible potential source of natural antioxidant.

CONFLICT OF INTEREST:

The Authors declare no conflict of interest.

REFERENCES:

1. Pattabiraman K., Muthukumaran P. Antidiabetic and Antioxidant Activity of Morinda tinctoria roxb Fruits Extract in Streptozotocin-Induced Diabetic Rats. Asian J. Pharm. Tech. 2011; 1(2): 34-39

2. Rahman Md.M, Islam Md.B, Biswas M and Alam AHMK. In vitro antioxidant and free radical scavenging activity of different parts of Tabebuia pallida growing in Bangladesh. BMC Res Notes. 2015; 30(8): 621.

3. N. Naidu, G. Sudheer Kumar, K. Sivakrishna, K. Anjinaik, L. Praveen Kumar, G. Sneha. Antimicrobial and antioxidant evolution of aqueous extract of Terminalia chebula using disc diffusion, H2O2 scavenging methods. Asian J. Res. Pharm. Sci. 2017; 7(2): 112-114.

4. Tiwari P, Rakesh K. Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of Ashwagandharishta Prepared by Traditional and Modern Methods. Asian J. Pharm. Ana. 2013; 3(4): 147-152.

5. Yadav M, Yadav A, Yadav JP. In vitro antioxidant activity and total phenolic content of endophytic fungi isolated from Eugenia jambolana Lam. Asian Pac J Trop Med. 2014; 7(Suppl 1): S256-S261.

6. Okoro I. Comparative study of the in vitro antioxidant properties of different extracts of Ethulia conyzoides. Nigerian Journal of Science and Environment. 2017; 15(1): 37-43

7. P. Muthukumaran P, Salomi S , Umamaheshwari R. In vitro Antioxidant Activity of Premna serratifolia Linn. Asian J. Res. Pharm. Sci. 2013; 3(1): 15-18.

8. Valli G, Jeyalakshmi M.. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. Asian J. Pharm. Res. 2012; 2(4): 153-155.

9. Tiwari P. Phenolics and Flavonoids and Antioxidant Potential of Balarishta Prepared by Traditional and Modern Methods. Asian J. Pharm. Ana. 2014; 4(1): 5-10.

10. Ghosh S, Derle A, Ahire M, More P, Jagtap S, Phadatare SD, Patil AB, Jabgunde AM, Sharma GK, Shinde VS, Pardesi K, Dhavale DD, Chopade BA. Phytochemical Analysis and Free Radical Scavenging Activity of Medicinal Plants Gnidia glauca and Dioscorea bulbifera. PLoS One. 2013; 8: e82529.

11. Silva JC, Santos WBD, Araujo MGS, Oliveira JDS, Verissimo RCSS, Bernardo T et al. Evaluation of the Cytotoxic, Antimicrobial and Antioxidant Activity of the Plant species Tabebuia roseo-alba (Ridl) Sand. Journal of Chemical and Pharmaceutical Research. 2017; 9(4): 148-153.

12. Sannigrahia S, Mazuder UK, Pal DK, Parida S, Jain S. Antioxidant Potential of Crude Extract and Different Fractions of Enhydra fluctuans Lour. Iranian Journal of Pharmaceutical Research. 2010; 9(1): 75-82.

13. Blois MS. Antioxidant determinations by the use of stable free radical. Nature.1958; 1: 1199-2000.

14. Re R, Pelligrini N, Proteggeenate M, Yang C, Evans R. Antioxidants activity of applying an improved ABTS radical cation decolorisation assay. Free Radical Biology and Medicine. 1999; 26(9-10): 1231-1237.

15. Klein SM, Cohen G, Cederbaum AI. Production of formaldehyde during metabolism of dimethyl sulphoxide by hydroxyl radical generating system. Biochemistry. 1991; 20: 6006-6012.

16. Ruch RJ, Cheng SJ, Klaunig JE. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis. 1989; 10(6): 1003-1008.

17. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and nitrate in biological fluids. Analytical Biochemistry. 1982; 126: 131– 138.

18. Liu F, Ooi VEC, Chang ST. Free radical scavenging activity of mushroom polysaccarides extract. Life Science. 1997; 60.

19. Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Japan Journal of Nutrition. 1986; 7: 307-315.

20. Sowndhararajan K, Kang SC. Free radical scavenging activity from different extracts of leaves of Bauhinia vahlii Wight and Arn. Saudi Journal of Biological Sciences. 2013; 20(4): 319–325.

21. Chanda S, Dave R, Kaneria M. Invitro antioxidant property of some Indian Medicinal plants. Research Journal of Medicinal Plants. 2011; 5(2): 169.

22. Nunes PX, Silva SF, Guedes RJ, Almeida S. Biological Oxidations and Antioxidant activity of natural Products. Croatia: Tech Publisher. 2012; pp.1-20.

23. Priyanga S, Hemmalakshmi S, Sowmya S, Vidya B, Chellaperumal P, Gopalakrishnan VK, Devaki K. In vitro Enzyme Inhibitory Evaluation and Free Radical Scavenging Potential of Ethanolic Leaf Extract of Macrotyloma uniflorum (L.). International Journal of Current Pharmaceutical Review and Research. 2015; 6(3): 169-177.

24. Sanmugapriya K, Saravana PS, Payal H, Peer Mohammed S, Binnie W. Antioxidant activity, total phenolic and flavanoid contents of Artocarpus heterophyllus and Manilkara zapota seeds and its reduction potential. International Journal of Pharmacy and Pharmaceutical Science. 2011; 3(5): 256-260.

25. Yang JX, Guo J, Yuan JF. In vitro antioxidant properties of rutin. LWT Food Science and Technology. 2008; 41(6): 1060-1066.

26. 26.Hepsibha BT, Sathiya S, Babu CS, Premalakshmi V, Sekar T. In vitro studies of antioxidant and free radical scavenging activities of Azima tetracantha. Lam leaf extract. Indian J Sci Technol. 2010; 3(5): 571-577.

27. Gautham SA, Onkarappa R. In Vitro Antioxidant activity of Metabolite from Streptomyces fradiae strain GOS1, International Journal of Drug Development and Research. 2013; 5(1): 235-244.

28. Kalaiselvi M, Ravikumar G, Gomathi D, Uma C. Invitro free radical scavenging activity of Ananas comosus (L.) Merrill peel. International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4: 604.

29. Venkatachalam U, Muthukrishnan S. Free radical scavenging activity of ethanolic extract of Desmodium gangeticum. Journal of Acute Medicine. 2012; 2(2): 36-42.

Received on 22.10.2020 Modified on 30.11.2020

Research J. Pharm. and Tech. 2021; 14(9):4801-4807.

DOI: 10.52711/0974-360X.2021.00835

Total antioxidant activity assay by radical cation (ABTS +):

Hydroxyl radical scavenging activity:

Total antioxidant activity assay by radical cation (ABTS⁺):