Jamun fruits used in present investigations were procured from the local market of Hisar. All the chemicals used in this investigation were of high-quality analytical grade and were purchased from M/s Sigma Chemicals Co., USA, Sisco Research Laboratory, Hi-Media, and E. Merck, Mumbai.

Seeds were separated from the pulp, chopped, shade dried in the dark, and ground to powder. Total phenolic compounds were extracted from the powder under the pre-optimized conditions using 25 ml/g volume of solvent (50% acetone), 193 rpm agitation rate, and 150 min extraction time (unpublished data). The extract was evaluated for its in-vitro antioxidant potential using an array of oxidants.

A dose-dependent increase in the quenching of free radicals was observed for JSE. In the present study, the IC₅₀ value of JSE was calculated as 0.24 μg/ml (Table 1), indicating it as a good scavenger of DPPH radical. The JSE showed 64.21% DPPH scavenging at 0.25 µg/ml while BHA exhibited only 14.69% activity at the same concentration, thus proving JSE a better scavenger of free radicals than BHA, a well-known synthetic antioxidant.

Table 1
DPPH radical scavenging activity of JSE.

A decrease in concentration-dependent color formation in the presence of the extract indicates iron-chelating potential. The metal chelating activity of JSE (IC₅₀ value, 48.39 μg/ml) was calculated (Table 2) and compared with standard antioxidant – EDTA (IC₅₀ value, 37.99 μg/ml) which was used as a positive control.

Results show that JSE possesses remarkable ABTS scavenging activity, and it increases as the concentration rises. In the present study, the IC₅₀ value of JSE was 0.31 μg/ml (Table 3), indicating it a good scavenger of ABTS radicals.

Table 2
Metal chelating activity of JSE.

Table 3
ABTSradical scavenging activity of JSE.

When compared with BHA (IC₅₀ 21.77 μg/ml) at equivalent concentrations, JSE exhibited better activity (IC₅₀ 5.67 μg/ml), as depicted in Table 4.

Table 4
Hydrogen peroxide scavenging activity of JSE.

The IC₅₀ value from Table 5 depicts that the plant extract (5.75 μg/ml) is a better hydroxyl radical scavenger than the standard BHA (18.01 μg/ml). In contrast, Pal et al. (2011) showed that gallic acid had better hydroxyl radical scavenging activity than ashwagandha root extracts.

The extract was found to possess substantial TAA with AO_0.5AU value of 5.37 µg/ml (Table 6) as compared with gallic acid (AO_0.5AU 9.62 µg/ml).

A rise in absorbance of the reaction mixture depicts an increase in reducing capacity due to the increased formation of the complex. The RP_{0.5 AU} was found to be 3.10 µg/ml (Table 7), which was relatively more pronounced than that of gallic acid (5.47 µg/ml).

Table 5
Hydroxyl radical scavenging activity of JSE.

Table 6
Total antioxidant activity of JSE.

The nitric oxide radical scavenging activity of JSE (IC₅₀ value, 37.01 μg/ml), as shown in Table 8, was compared with standard antioxidant – gallic acid (IC₅₀ value, 33.2 μg/ml).

The decrease in MDA level by JSE illustrates its role as an antioxidant. The present study shows that JSE (IC₅₀, 5.38 μg/ml) (Table 9) has more influential lipid peroxidation inhibitory activity as compared to BHA (IC₅₀, 18.97 μg/ml).

The DPPH scavenging activity of the Jamun Seed Extract (JSE) was taken as a parameter to estimate its antioxidant potential. DPPH is a relatively stable radical, and bleaching of its absorption (515 nm) by a test compound is representative of its capacity to scavenge free radicals by donating a hydrogen atom to become a stable diamagnetic molecule. DPPH radical has the undeniable advantage of being unconcerned by the side reactions, i.e., enzyme inhibition and metal chelation [22Pal J, Ganguly S, Tahsin KS, Acharya K. In vitro free radical scavenging activity of wild edible mushroom, Pleurotus squarrosulus Mont: Singer 2010.]. Our results indicate distinct scavenging activity of the extract towards DPPH radicals in comparison with BHA. A high DPPH scavenging activity has also been reported in the acetonic extract of S. cumini leaves [23Kaneria M, Chanda S. Evaluation of antioxidant and antimicrobial capacity of Syzygium cumini L. leaves extracted sequentially in different solvents. J Food Biochem 2013; 37(2): 168-76.
[http://dx.doi.org/10.1111/j.1745-4514.2011.00614.x] ].

Table 7
Total reducing power of JSE.

Table 8
Nitric oxide radical scavenging activity of JSE.

Table 9
Lipid peroxidation inhibitory activity of JSE

Iron an acutely reactive metal and causes oxidative damage to lipids, proteins, and other cellular components. Metal chelating capacity is desirable to decrease the concentration of transition metals, causing lipid peroxidation. In this assay, EDTA had slightly higher chelating activity as compared to JSE, and similar results were found by Bholah et al. [24Bholah K, Ramful Baboolall D, Neergheen Bhujun VS. Antioxidant Activity of Polyphenolic Rich Moringa oleifera Lam. Extracts in Food Systems. J Food Biochem 2015; 39(6): 733-41.
[http://dx.doi.org/10.1111/jfbc.12181] ] in Moringa oleifera Lam. extracts and Pal et al. [12Pal A, Bhushan B, Narwal RK, Saharan V. Extraction and evaluation of antioxidant and free radical scavenging potential correlated with biochemical components of red rose petals. Iran J Sci Technol A 2018; 42(3): 1027-36.
[http://dx.doi.org/10.1007/s40995-016-0071-2] ] in red rose petal extracts.

The ABTS scavenging activity is taken as a parameter for desolvation of chain-breaking antioxidants in case of lipid peroxidation and antioxidant activity of hydrogen donating antioxidants [25Singh JP, Kaur A, Shevkani K, Singh N. Influence of jambolan (Syzygium cumini) and xanthan gum incorporation on the physicochemical, antioxidant and sensory properties of gluten‐free eggless rice muffins. Int J Food Sci Technol 2015; 50(5): 1190-7.
[http://dx.doi.org/10.1111/ijfs.12764] ]. The ABTS method is established as a rapid method for the determination of antioxidant activity and could be a convenient tool to screen samples and cultivars to obtain great content of natural antioxidants in foods. A high ABTS scavenging activity has also been found in Jamun fruit peel extract than standard compounds such as gallic acid, caffeic acid, sinapic acid, and quercetin [26Singh JP, Kaur A, Singh N, et al. In vitro antioxidant and antimicrobial properties of jambolan (Syzygium cumini) fruit polyphenols. Lebensm Wiss Technol 2016; 65: 1025-30.
[http://dx.doi.org/10.1016/j.lwt.2015.09.038] ].

The generation of even low levels of H₂O₂ in biological systems may be pernicious since naturally occurring iron complexes react with H₂O₂ to produce highly reactive hydroxyl radicals in a superoxide-driven ‘Fenton’ reaction [27Namiki M. Antioxidants/antimutagens in food. Crit Rev Food Sci Nutr 1990; 29(4): 273-300.
[http://dx.doi.org/10.1080/10408399009527528] [PMID: 2257080] ]. Pal et al. [11Pal A, Naika M, Khanum F, Bawa AS. In-vitro studies on the antioxidant assay profiling of Withania somnifera L. (Ashwagandha) Dunal root: Part 1. J Pharmacogn 2011; 3(20): 47-55.
[http://dx.doi.org/10.5530/pj.2011.20.10] ] showed that ascorbic acid had higher hydrogen peroxide scavenging activity than ashwagandha root extracts. Tchimene et al. [28Tchimene MK, Nwaehujor CO, Ezenwali M, Okoli CC, Iwu MM. Free radical scavenging activity of lupeol isolated from the methanol leaf extract of Crateva adansonii Oliv.(Capparidaceae). Int J Pharmacogn Phytochem Res 2016; 8: 419-26.] reported that leaf extract of Crateva adansonii had better hydrogen peroxide scavenging activity than ascorbic acid.

This radical can cause strand damages in DNA, causing carcinogenesis, mutagenesis, and cytotoxicity. Moreover, hydroxyl radicals are capable of quick inception of the lipid peroxidation process by abstracting hydrogen atoms from unsaturated fatty acids [29Kappus H. Lipid peroxidation-mechanism and biological relevance. Free Radicals Food Addit 1991; 59-75., 30Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc 1998; 75(2): 199-212.
[http://dx.doi.org/10.1007/s11746-998-0032-9] [PMID: 32287334] ]. The hydroxyl radicals were produced in the present study by incubating ferric-EDTA with ascorbic acid and H₂O₂ at pH 7.4 and allowed to react with 2-deoxy-2-ribose to produce malondialdehyde (MDA)-like product. This compound then formed a pink chromogen upon heating with TBA at low pH.

Since the assay is simple and unaffected by other antioxidant measurements commonly employed, its application has been expanded to plant extracts [18Prieto P, Pineda M, Aguilar M. 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.
[http://dx.doi.org/10.1006/abio.1999.4019] [PMID: 10222007] ]. The assay is successfully used in the quantification of vitamin E in seeds. Pal et al. [11Pal A, Naika M, Khanum F, Bawa AS. In-vitro studies on the antioxidant assay profiling of Withania somnifera L. (Ashwagandha) Dunal root: Part 1. J Pharmacogn 2011; 3(20): 47-55.
[http://dx.doi.org/10.5530/pj.2011.20.10] , 12Pal A, Bhushan B, Narwal RK, Saharan V. Extraction and evaluation of antioxidant and free radical scavenging potential correlated with biochemical components of red rose petals. Iran J Sci Technol A 2018; 42(3): 1027-36.
[http://dx.doi.org/10.1007/s40995-016-0071-2] ], differently, stated that gallic acid had higher TAA than ashwagandha root and red rose petal extracts.

The reducing capacity of a compound can be estimated by the direct reduction of Fe³⁺ to Fe²⁺. The addition of free Fe³⁺ to the reduced product leads to the formation of intense Perl’s Prussian blue complex, which possesses strong absorbance at 700 nm. This method is often used as an indicator of electron-donating activity, which is an important mechanism for testing the antioxidant activity of plant extracts [31Yildirim A, Mavi A, Kara AA. Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. J Agric Food Chem 2001; 49(8): 4083-9.
[http://dx.doi.org/10.1021/jf0103572] [PMID: 11513714] ]. Many reports have affirmed that there is a direct correlation between antioxidant activities and reducing the power of certain plant extracts [31Yildirim A, Mavi A, Kara AA. Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. J Agric Food Chem 2001; 49(8): 4083-9.
[http://dx.doi.org/10.1021/jf0103572] [PMID: 11513714] , 32Oktay M, Gülçin İ, Küfrevioğlu Öİ. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. Lebensm Wiss Technol 2003; 36(2): 263-71.
[http://dx.doi.org/10.1016/S0023-6438(02)00226-8] ]. Our present results imply that JSE has the potential to donate electrons to reactive free radicals, hence, converting them to more stable non-reactive species and terminating the free radical chain reaction.

Nitric oxide is a dynamic pleiotropic inhibitor of physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation, and regulation of cell-mediated toxicity [33Hagerman AE, Riedl KM, Jones GA, et al. High molecular weight plant polyphenolics (tannins) as biological antioxidants. J Agric Food Chem 1998; 46(5): 1887-92.
[http://dx.doi.org/10.1021/jf970975b] [PMID: 29072454] ]. The excessive production of NO is associated with various diseases like adjuvant arthritis, cancer, etc. Our results are following those reported by Pal et al. [11Pal A, Naika M, Khanum F, Bawa AS. In-vitro studies on the antioxidant assay profiling of Withania somnifera L. (Ashwagandha) Dunal root: Part 1. J Pharmacogn 2011; 3(20): 47-55.
[http://dx.doi.org/10.5530/pj.2011.20.10] ], where ascorbic acid showed higher nitric oxide radical scavenging activity than different extracts of ashwagandha root. The mechanism involved in the scavenging activity of the extract may be associated with its phenolic compounds, as indicated in earlier studies [34Kumar S, Mishra A, Pandey AK. Antioxidant mediated protective effect of Parthenium hysterophorus against oxidative damage using in vitro models. BMC Complement Altern Med 2013; 13(1): 120.
[http://dx.doi.org/10.1186/1472-6882-13-120] [PMID: 23721571] ].

Lipid peroxidation is a crucial biological consequence of oxidative cellular damage and aging. The polyunsaturated fatty acyl side chains, due to their susceptibility to oxidative damage in membrane phospholipids, pose a consistent threat to cellular integrity and functions [35Jain SK. Evidence for membrane lipid peroxidation during the in vivo aging of human erythrocytes. Biomembranes 1988; 937(2): 205-10.
[http://dx.doi.org/10.1016/0005-2736(88)90242-8] [PMID: 3337801] ]. In the process, cyclic peroxides, lipid peroxides, and cyclic end peroxides are produced, which ultimately get fragmented into aldehydes like malondialdehyde (MDA). MDA forms a pink chromogen with TBA that has a strong absorbance at 532 nm. JSE inhibited the amount of MDA generated in the egg homogenate model. Anjum et al. [36Anjum S, Gani A, Ahmad M, et al. Antioxidant and antiproliferative activity of walnut extract (Juglans regia L.) processed by different methods and identification of compounds using GC/MS and LC/MS technique. J Food Process Preserv 2017; 41(1): e12756.
[http://dx.doi.org/10.1111/jfpp.12756] ] also showed that walnut extracts could prevent oxidative deterioration of linoleic acid. It may be due to their polyphenolic compounds which protect the fatty acids from getting rancid.