Antioxidant Activities and Thin Layer Chromatographic Analysis of Aqueous Extract of Tubers of Drynaria quercifolia (L).J.Sm

Sivaraj, Aashinya, Sripriya, and Arumugam: Antioxidant Activities and Thin Layer Chromatographic Analysis of Aqueous Extract of Tubers of Drynaria quercifolia (L).J.Sm

Authors

INTRODUCTION

Drynaria quercifolia belongs to the family Polypodiceae, which is popularly known as Attukal kizhangu is classified in the botanical division Pteridophyta, order Polypodiales.1 It is commonly found in Bangladesh, India, Pakistan, North America, and Africa. Antioxidants are the substances which inhibit oxidation, and have the ability to remove the potentially damaging oxidizing agents in a living organism. Optimisation of antioxidants from Nelumbo seeds2 and coumarin derivatives as potential antioxidant agents3 manifest the potential of antioxidants having an therapeutic potential in treating various diseases. The presence of phytochemicals in plants are able to prevent the oxidative damage to the human cells which can even cause cancer. It is important to know the antioxidant activities of phytocompounds responsible for inhibiting radicals. In this study, the free radical scavenging activities of the extracts of D. quercifolia is analysed. Free radicals initiate the oxidative stress and damage the healthy cells and DNA along with lipid peroxidation. This damages can contribute to ageing, atherosclerosis, cancer, cardiovascular diseases, and inflammatory diseases.4

MATERIALS AND METHODS

Plant material

The rhizomes of Drynaria quercifolia are creeping and densely covered in brown scales with nested leaves having sterile and fertile fronds. The fertile fronds have a longer stalk growing about 100 cm in length than the sterile frond growing only 20-40 cm in length. The rhizome was associated with water-borne hyphomycetes that may be present as endophytes or epiphytes.5 It has been highly found in Eastern Ghats of India. The tuber extract have potentially high antibacterial activity.6

Extraction

The dried tubers were cut into small pieces and extracted with hot distilled water using a pressure cookware. The hot aqueous extract was cooled and filtered using filter paper. Water was completely removed at 50°C hot plate apparatus. Finally brown gummy residue was obtained and was used for antioxidant activities as well as thin layer chromatographic (TLC) analysis.

Determination of total phenol

Folin-Ciocalteau reagent method was used to estimate total phenolic compounds (Spanos, and Wrosltad, 1990) with slight modifications. One mL of methanol extract (1mg/mL) of aerial parts of C. viscosa was mixed with 1 mL of Folin Ciocalteau reagent (1:10 diluted with distilled water). After 5 min, 1 mL of aqueous solution of Na2CO3 (20%) was added. The mixture was then allowed to stand for 30 min incubation in dark at room temperature. The absorbance was measured by UV-VIS spectrophotometer at 765 nm. The total phenolic content was expressed in terms of Gallic acid.

Determination of total flavonoid

The total flavonoid content of methanol extract of aerial parts of C. viscosa was determined using aluminium chloride colorimetric method with slight modification as described by Liu et al., 2007. One mL of extract (1mg/mL) was mixed with 0.5 mL of 5% sodium nitrite and incubated for 5 min at RT. Then, 0.5 mL 10% aluminium chloride solution was added and after 5 min incubation at RT, 1 mL of NaOH solution (1 M) was added. The total volume was made up to 5 mL with distilled water. Absorbance was measured at 510 nm using spectrophotometer. The result was expressed as quercetin equivalent (μg/mg of dry mass), which is a common reference compound. The phytocompounds like flavonoids and phenolics exhibit various biological activities, the most important being the antioxidant activity.7

Antioxidant activities
Screening of antioxidant activity by dot-blot DPPH staining method

Drops of DPPH (0.4 mM) solution in methanol were loaded onto a 5 cm × 5 cm TLC plate (silica gel 60 F254; Merck) in each column and allowed to dry for 2 min. The first row of TLC plate was considered as control, containing only DPPH. Aqueous extract of tubers of Dynaria quercifolia of various concentrations was carefully loaded onto the DPPH spot in a second row. The third row of TLC plate was considered as standard reference, where ascorbic acid was carefully loaded onto the DPPH spot. The staining of the silica plate was based on the procedure of Soler- Rivas et al.,.8 Stained silica gel layer revealed a purple background with yellow to white spots at the location where radical scavenging capacity observed. The intensity of disappearance of purple colour depends upon the amount and nature of radical scavenger9 present in the tubers of Drynaria quercifolia in aqueous extract

DPPH radical scavenging activity

The DPPH radical scavenging activity was carried out according to the method of Raman et al.,.10 1 mL of 0.1 mM DPPH solution in methanol was mixed with 1mL of various concentrations of aqueous extract in methanol (20-120 μg/mL). Mixer of 1mL and 1mL DPPH solutions were used as control. The reaction was carried out in triplicate and the decrease in absorbance was measured at 517 nm after 30 minutes in dark using UV-Vis spectrophotometer. Ascorbic acid was used as reference standard. The inhibition % was calculated using the following formula.

% of DPPH radical inhibition=ControlSampleControl×100

ABTS•+ radical cation scavenging activity

The antioxidant capacity was estimated in terms of the ABTS•+ radical cation scavenging activity following the procedure described by Delgado-Andrade et al.11 Briefly, 7 mM ABTS stock solution by reacting with 2.45 mM potassium persulfate and the mixture was left to stand in the dark at room temperature for 12-16 h before use. The ABTS solution (stable for 2 days) was diluted with 5 mM phosphate-buffered saline (pH 7.4) to an absorbance at 734 nm of 0.70±0.02. To various concentrations (5-30 μg/mL) of aqueous extract of tubers of D. quercifolia, 1 mL of diluted ABTS+ solution was added and after 10 min, the absorbance was measured at 734 nm. Ascorbic acid was used as reference standard. The ABTS •+ radical cation scavenging activity was expressed as

% of ABTS+ radical inhibition=Control  SampleControl×100

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity was determined using standard protocol.12 Various concentrations (10-60 μg/mL) of the aqueous extract of tubers of D. quercifolia were taken in different test tubes and evaporated to dryness. One milliliter of iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA (0.018%), and 1mL of DMSO (0.85% v/v in 0.1 M phosphate buffer, pH 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 mL of 0.22% ascorbic acid. The test tubes were capped tightly and heated on a water bath at 80- 90°C for about 15mins. The reaction was completed by the addition of 1mL of ice-cold TCA solution (17.5% w/v). One milliliters of Nash reagent (18.75 g of ammonium acetate, 0.75 mL of glacial acetic acid and 0.5 mL of acetyl acetone were mixed and raised to 250 mL with distilled water) was added to all the tubes and left at room temperature for about 15 min for color development. The intensity of the yellow color was measured at 412 nm against reagent blank. Ascorbic acid was used as reference standard. The percentage of hydroxyl radical scavenging was calculated by the following formula

% of OH radical inhibition=Control  SampleControl×100

Nitric oxide radical scavenging activity

Nitric oxide generated from aqueous sodium nitroprusside (SNP) solution interacts with oxygen to produce nitrite ions at physiological pH, which may be quantified and determined according to Griess Illosvoy reaction.13 The reaction mixture contained 1 mL of 10 mM SNP in 0.5 M phosphate buffer (pH 7.4) and 1 mL of various concentrations (10–60 μg/mL) of the aqueous extract of tubers of D. quercifolia. After incubation for 60 min at 37°C, Griess reagent (0.1% ) napthyl ethylenediamine dihydrochloride in water and 1% sulfanilamide in 5% H3PO4) was added. The pink chromophore generated during diazotization of nitrite ions with sulfanilamide and subsequent coupling with napthyl ethylenediamine dihydrochloride were measured spectrophotometrically at 540 nm. Ascorbic acid was used as a positive control. The percentage of nitric oxide radical scavenging activity was calculated by the following formula

% of nitric oxide radical inhibition=Control  SampleControl×100

Reducing power assay

The reducing power of tubers of Drynaria quercifolia in aqueous extract was evaluated according to the method of Ravisankar et al.14 Different concentrations of aqueous extract of tubers of D. quercifolia (20–120 μg/mL) were mixed with 1 mL of 0.2 M phosphate buffer (pH 6.6) and 1 mL of 1% K3Fe(CN)6 . This mixture was incubated at 50°C for 20 min. After, 1 mL of 10% TCA was added and centrifuged at 3000 rpm for 10 min. The upper layer of the solution was mixed with 0.5 mL of FeCl3 (0.1%) solution and the absorbance was measured at 700 nm using a spectrophotometer. Ascorbic acid was used as the standard reference.

Phosphomolybdenum reduction assay

The total antioxidant capacity was measured by spectrophotometeric method of Prieto et al.,.15 At different concentration, aqueous extract of tubers of D. quercifolia (20–120 μg/mL) was combined with 1mL of reagent solution (0.6 M H2SO4, 28 mM sodium phosphate, 4 mM ammonium molybdate). The tubes were incubated at 95°C for 90 min in a water bath. After, the mixture was cooled to room temperature and the absorbance was read at 695 nm. Ascorbic acid was used as the standard reference.

RESULTS AND DISCUSSION

Medicinal plants are an important source of practical and inexpensive new drugs.16 Oxidative stress has been implicated in the pathology of many diseases such as inflammatory conditions, cancer, diabetes and the aging. This study was made to evaluate polyphenolic content and antioxidant activity. Phenolics are composed of one or more aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free radicals by forming stabilized phenoxyl radicals.17

Dot-blot assay for rapid radical scavenging activity

The results of dot-blot assay showed coloured spots where the aliquots of aqueous extract of barks of Drynaria quercifolia were placed in row. The purple zone on the plate indicates no (free radical scavenging) antioxidant activity and the yellow zone indicates antioxidant activity. The more intense the yellow colour, the greater the antioxidant activity (Figure 1). The result indicates that the aqueous extract of tubers of D. quercifolia have significant antioxidant activity when compared to standard ascorbic acid.

Total phenol and flavonoid

The phenolic and flavonoid compounds quantified in the extracts seemed to be responsible for the antioxidant activity. Phenolic acids, flavonoids are the most commonly found polyphenolic compounds in plant extracts.18 The antioxidant activity of phenolics plays an important role in absorption or neutralization of free radicals.19 Polyphenols also enhance the level of cellular antioxidative system and induce the cytochrome P-450 resulting in detoxifying the activity of carcinogens intracellularly.20 Total phenol content was 4.295±1.23 μg/mL of GAE and flavonoid content was 24.564±0.86 μg/mL of QE in the aqueous extract of tubers of D.quercifolia, as shown in Table 1. These investigations provide a comprehensive profile of the antioxidant activity of extracts of plants with respect to their phenols and flavonoids content.

DPPH radical scavenging activity

The ability of aqueous extract of tubers of D. quercifolia to scavenge free radicals formed was assessed using 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH). This was compared with a standard (ascorbic acid). Aqueous extract of tubers of D.quercifolia demonstrated high capacity for scavenging free radicals as shown by the data by reducing the stable DPPH (1,1-diphenyl-2-picrylhydrazyl) radical to the yellow coloured 1,1-diphenyl-2-picrylhydrazine and this capacity increases with increasing concentration as reported earlier by Huang DJ et al.,.21 The maximum percentage of DPPH radical scavenging activity was 55.11±5.27% at 120 μg/mL concentration as shown in Table 2 and Figure 2. It was compared with the standard ascorbic acid (70.95±4.96/120 μg/mL) and the IC50 of DPPH radical scavenging activity was 105.78 μg/mL concentration. The scavenging ability of the aqueous extract of tubers of D.quercifolia may be due to its bio compositions such as phenolic acids and flavonoid. The radical scavenging activities of the extracts were determined by using DPPH a stable free radical at 517 nm. 1,1 -diphenyl-2-picrylhydrazyl is a nitrogen-centred free radical, color of which changes from violet to yellow on reduction by donation of H or e- by the aqueous extract of tubers of D.quercifolia

Figure 1

Dot-blot assay of aqueous extract of D. quercifolia in DPPH Radical scavenging activity. DPPH - 1, 1 Diphenyl-2-picryl hydrazyl STD - Standard (Ascorbic acid).

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Table 1

Total phenol and flavonoid present in aqueous exracts of tubers of Drynaria quercifolia

PhytochemicalsValue (μg/mL)
Phenols4.295±1.23
Flavonoids24.564±0.86

ABTS•+ radical scavenging activity

In the total antioxidant activity, ABTS•+ is a blue chromophore produced by the reaction between ABTS and potassium persulfate and in the presence of the plant extract or trolox, preformed cation radical gets reduced and the remaining radical cation concentration after reaction with antioxidant compound was then quantified.22 The maximum percentage of ABTS•+ radical cation scavenging activity was 54.91±5.55% at 30 μg/mL concentration as shown in Table 3 and Figure 2. It was compared with standard (52.41±3.66/30 μg/mL) ascorbic acid. The IC50 of ABTS•+ radical cation scavenging activity was 27.32 μg/mL concentration.

Hydroxyl (OH•) radical scavenging activity

Scavenging of hydroxyl radical is an important antioxidant activity because of very high reactivity of the OH radical which enables it to react with a wide range of molecules found in living cells, such as sugars, amino acids, lipids and nucleotides. The hydroxyl radical is an extremely reactive free radical formed in biological systems and has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells.23 The maximum percentage of OH radical scavenging activity was 85.91±5.16% at 60 μg/mL concentration as shown in Table 4 and Figure 2. It was compared with standard (97.67±6.83/60 μg/mL) ascorbic acid. The IC50 of OH radical scavenging activity was 26.54 μg/mL concentration.

Nitric oxide (NO•) radical scavenging activity

In this spectrophotometric method, the absorbance of chromophore formed during the diazotization of the nitrile with sulphanilamide and the subsequent coupling with naphthyethylenediamine dihydrochloride was measured. NO, being a potent pleiotropic mediator in physiological processes and a diffusible free radical in the pathological conditions, reacts with superoxide anion and form a potentially cytotoxic molecule, the peroxynitrite (ONOO).24 Its protonated form, peroxynitrous acid (ONOOH), is a very strong oxidant. The main route of damage is the nitration or hydroxylation of aromatic compounds, particularly tyrosine. Under physiological conditions, peroxynitrite also forms anadduct with carbon dioxide dissolved in body fluid and responsible for oxidative damage of proteins in living systems. The maximum NO radical scavenging activity was 52.57±3.67% at 60 μg/mL concentration as shown in Table 4 and Figure 2. It was compared with standard (62.85±4.39/60 μg/mL) ascorbic acid. The IC50 of NO radical scavenging activity was 57.07 μg/mL concentration.

Ferric (Fe3+) reducing power activity

Studies were made on total reduction ability of Fe3+ to Fe2+ transformation in the presence of the tubers of Drynaria quercifolia in aqueous extract and found increasing in showing reduction ability in a dose dependent manner, with increasing concentrations. Increase in absorbance of the reaction mixture indicated increased reducing power. Since the reducing capacity of the aqueous extract of tubers of Drynaria quercifolia serve as a significant indicator of its potential antioxidant activity, the reducing ability was 0.582±0.040 at 120 μg/mL concentration as in Table 2 and Figure 2, which was compared with standard (0.359±0.02/120 μg/mL) ascorbic acid. The antioxidant activity has been reported to be concomitant with development of reducing power.25

Table 2

DPPH, Ferric (Fe3+) reducing power and phosphomolybdenum reduction of aqueous extract of tubers of D. quercifolia

ConcentrationDPPH Assay % of inhibitionFe3+ Reducing power AssayPhosphomolybdenum Reducing Assay
2013.07±0.910.038±0.0020.135±0.009
4016.12±1.120.15±0.0100.187±0.013
6028.10±1.960.197±0.0130.267±0.018
8033.55±2.340.383±0.0260.344±0.024
10047.27±3.300.481±0.0330.394±0.027
12055.11±5.270.582±0.0400.441±0.030
Table 3

ABTS.+ radical cation scavenging activity of aqueous extract of tubers of D. quercifolia

Concentration (μg/mL)% of inhibition
511.60±0.81
1015.17±1.06
1523.66±1.65
2037.50±2.62
2542.85±2.99
3054.91±5.55
Table 4

Nitric oxide and hydroxy radical scavenging activity of aqueous extract of tubers of Drynaria quercifolia

Concentration (μg/mL)Nitric oxide scavenginghydroxy radical
1010.94±0.7619.82±1.38
2014.59±1.0239.13±2.73
3030.81±2.1556.52±3.95
4034.86±2.4475.65±5.29
5043.10±3.0183.65±5.85
6052.57±3.6785.91±5.16
Figure 2

Graphical representation of antioxidant activities of aqueous extract of tubers of Drynaria quercifolia

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Figure 3

Thin layer chromatography analysis of aqueous extract of tubers of D. quercifolia

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Phosphomolybdenum reduction activity

The total antioxidant activity of aqueous extract of tubers of D. quercifolia was measured spectrophotometrically by the phophomolybdenum method, which is based on the reduction of Mo (VI) by the petroleum ether fraction and the subsequent formation of green phosphate/Mo (V) complex at acidic pH, with a maximum absorption 695 nm. It evaluates both water-soluble and fat-soluble antioxidants with a high absorbance value of the petroleum ether fraction indicated its strong antioxidant acitivity.26 The maximum absorbance was 0.441±0.030 at 120 μg/mL concentration, as in Table 2 and Figure 2, which was compared with standard (0.359±0.02/120 μg/mL) ascorbic acid.

Thin layer chromatography

TLC analysis was carried out for aqueous extract of tubers of D. quercifolia by using Hexane:Ethyl acetate (0.2:1.8) solvent system. The separated bands were visualized by UV light at 254 nm. The Rf values of the separated compounds were measured (Figure 3).

CONCLUSION

The replacement of synthetic with natural antioxidants may be advantageous. In the present study, aqueous extract of tubers of D. quercifolia tested with respect to their total phenolic and flavonoid content, antioxidant capacity and oxidative stability. Extractions were performed using the conventional method reflux and methanol as solvent. The existence of phenolic and flavonoid compounds was confirmed by the Folin-Ciocalteu and AlCl3 methods. The antioxidant capacity was measured by DPPH free radical scavenging method was proven to be high. Finally, the results in this study indicate that the examined extract contains significant sources of antioxidants.

GRAPHICAL ABSTRACT

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SUMMARY

  • The aqueous extract of tubers of Drynaria quercifolia was prepared and analyzed for antioxidant activities such as, DPPH, OH,NO radical scavenging assays and Fe3+ reducing power as well as phosphomolybdenum reduction assay methods. The results revealed that the aqueous extract of D. quercifolia showed significant antioxidant activities. The radical scavenging activity was further confirmed by Dot-blot assay method where, the purple colour of DPPH changed to yellow upon reduction.

ABOUT AUTHORS

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Perumal Arumugam: Director, Armats Biotek Training & Research Institute, Guindy, Chennai-600032.

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Chandrasekaran Sivaraj: Postdoctoral Fellow, Armats Biotek Training & Research Institute, Guindy, Chennai-600032.

ACKNOWLEDGEMENT

We are grateful to the support provided by ARMATS Biotek Training and Research Institute, and Department of Biotechnology, Vel Tech High Tech Dr.Rangarajan Dr.Sakunthala engineering college, Tamil Nadu, India.

Notes

[1] Conflicts of interest CONFLICT OF INTEREST There is no conflict of interest associated to the work done.

ABBREVIATIONS USED

DPPH•

2,2-diphenyl-1-picrylhydrazyl radical

NO•

Nitric oxide radical

•OH

Hydroxyl radical

O2•-

Superoxide anion radical

Fe2+

Ferrousion

IC50

Inhibitory concentration 50

REFERENCES

1. 

Smith AR, Pryer KM , authors. A Classification for Extant Ferns. 2006;Taxon. 55(3)

2. 

Nawaz H, Muhammad AS , authors. Extraction Optimisation of Phenolic Antioxidants from microwave treated Nelumbo nuclear Seed Flour. Free Radicals and Antioxidants. 2017;7(1):63–73

3. 

Amiery AA, Saour KY, Duhaidahawi DL, Al- Majedy YK, Kadhum AA, Mohamad AB , authors. Comparative Molecular Modelling Studies of Coumarin Derivatives as Potential Antioxidant Agents. Free Radicals and Antioxidants. 2017;7(1):31–5

4. 

Kumar V, Lemos M, Sharma M, Shriram V , authors. Antioxidant and DNA damage protecting activities of Eulophia nuda Lindl. Free Radicals Antioxidants. 2013;3(2):55–60

5. 

Karamchand KS, Sridhar KR , authors. Association of water-borne conidial fungi with epiphytic tree fern (Drynaria quercifolia). Acta Mycologica. 2009;44:19–27

6. 

Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P, Vidal N , authors. Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compound. Food Chemistry. 2006;97:54–60

7. 

Adil FW, Ahlam M, Muneeb UR, Seema A, Mubashir HM , authors. In vitro antioxidant and antimicrobial activities of propolis from Kashmir Himalaya region. Free Radicals and Antioxidants. 2015;6(1):1–7

8. 

Soler-Rivas C, Espin JC, Wichers HJ , authors. An easy and fast test to compare total free radical scavenger capacity of foodstuffs. Phytochem. Analysis. 2000;11:1–9

9. 

Huang DJ, Lin CD, Chen HJ, Lin YH , authors. Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas [L.] Lam Tainong 57) constituents, Bot. Bull Acad Sin. 2004;45:179–186

10. 

Raaman N, Sivaraj C, Tenzing , authors. Antioxidant activities and Phytochemical analysis of methanol extract of leaves of Artocarpus Heterophyllus lam. Int J Pharm Science. 2014;6:289–293

11. 

Delgado-Andrade C, Rufian-Henares JA, Morales FJ , authors. Assessing the antioxidant activity of melanoidins from coffee brews by different antioxidant methods. J Agric Food Chem. 2005;53:7832–7836

12. 

Klein SM, Cohen G, Cederbaum AI , authors. Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical generating systems. Biochemistry. 1981;20(21):6006–6012

13. 

Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR , authors. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry. 1982;131–8

14. 

Ravisankar N, Sivaraj C, Seeni S, Joseph J, Raaman N , authors. Antioxidant activites and phytochemical analysis of methanol extract of leaves of Hypericum hookerianum. Int J Pharm Pharm Sci. 2014;6:456–460

15. 

Prieto p, Pineda M, Anguilar M , authors. Spectrophotometric quantitation of antioxidant capacity through the formation of a Phosphomolybdenum Complex. Specific application to the determination of Vitamin E. Anal Biochem. 1999;269(2):337–41

16. 

Anonymous, 1948-1976. The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products. Vol. I-XI. Publication and Information Directorate, CSIR; New Delhi, India:

17. 

Kang MH, Lee MS, Choi MK, Min KS, Shibamoto T , authors. Hypoglycemic Activity of Gymnema Sylvestre Extracts on Oxidative Stress and Antioxidant Status in Diabetic Rats. J Agric Food Chem. 2012;60(10):2517–24

18. 

Wolfe K, Wu X, Lin RH , authors. Antioxidant activity of apple peels. J Agric Food Chem. 2003;51:609–14

19. 

Basile A, Giordano S, Lopez-Sacz JA, Cobianchi RC , authors. Antibacterial activity of pure flavonoids isolated from mosses. Phytochem. 1999;52:8:1479–82

20. 

De Flora S , author. Problems and prospects in antimutagenesis and anticarcinogenesis. Mutat Res. 1988;202(2):279–83

21. 

Huang DJ, Ou BX, Prior RL , authors. The chemistry behind antioxidant capacity assays. J Agric Food Chem. 2005;53(6):1841–56

22. 

Johnston JW, Dussert S, Gale S, Nadarajan J, Harding K, Benson EE , authors. Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm. Plant Physiol Biochem. 2006;44(4):193–201

23. 

Yasuda T, Inaba A, Ohmori M, Endo T, Kubo S, Ohsawa K , authors. Urinary metabolites of gallic acid in rats and their radical scavenging effect on DPPH. J Nat Prod. 2000;63(10):1444–6

24. 

Malinski T , author. Nitric oxide and nitroxidative stress in Alzheimer’s disease. J Alzheimers Dis. 2007;11(2):207–18

25. 

Yang JH, Lin HC, Mau JL , authors. Antioxidant properties of several commercial mushrooms. Food Chem. 2002;77(2):229–35

26. 

Abbasi Am, Saleem H, Rehman A, Riaz T , authors. Determination of antioxidant activity and phytoconstituent screening of Euphorbia heterophylla Linn. British J of Pharmaceutical Research. 2013;3(2):202–16