Antioxidants are compounds that prevent the oxidation of essential biological macromolecules by inhibiting the propagation of the oxidizing chain reaction. Keeping in mind the adverse effects of synthetic antioxidants, researchers have channelled their interest in isolating natural antioxidants  which are very effective to control the oxidative stress and hence prevent the initiation of disease propagation. Interestingly, quite a few studies on the antioxidant properties of the three plant materials, viz., T. chebula [22, 23], T. belerica [24, 25] and E. officinalis [26, 27] have been done earlier. However, this study provides a definitive report about the free radical scavenging capacity of T. chebula, T. belerica and E. officinalis, since the antioxidant activity of a drug may depend on the free radical scavenging activity .
is a blue colored chromophore which is reduced to ABTS on a concentration dependant manner upon addition of the fruit extract. The results are compared with trolox and the TEAC value demonstrates the extracts as a potent antioxidant, with their TEAC values following the order T. chebula > E. officinalis > T. belerica. The effect of the fruit extract in the scavenging assay of DPPH radical furthermore assured the fact that the extracts smoothly act as antioxidants, since the study on TEAC and DPPH scavenging can be observed as complementary to each other , although it followed the order T. belerica > E. officinalis > T. chebula.
The most detrimental of the free radicals formed in biological systems is the hydroxyl radical that causes enormous damage on biomolecules of the living cells . As the extracts or standard mannitol is added to the Fenton reaction mixture the hydroxyl radicals are scavenged and thereby sugar damage can be blocked. The results, as can be found from Figure 3 and Table 2, indicate that the fruit extracts are better hydroxyl radical scavengers than standard mannitol, with T. chebula being the best in comparison to T. belerica and E. officinalis.
Superoxide anion is also another harmful reactive oxygen species as it damages cellular components in biological systems . The ability of the fruit extracts and the reference compound quercetin to quench superoxide radicals from reaction mixture is reflected in the decrease of the absorbance at λ = 560 nm. From the results (Figure 4 & Table 2), it can be put forward that the fruit extracts are more potent scavenger of superoxide radical than the standard quercetin with a decreasing order of T. chebula > E. officinalis > T. belerica.
Nitric oxide radicals play important roles in various types of inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis and ulcerative colitis [32, 33]. The nitric oxide generated from sodium nitroprusside reacts with oxygen to form nitrite anion that is well restrained by the extracts. The scavenging activities of the extracts and curcumin proved that the nitric oxide scavenging activity of the former is better than the latter, with T. chebula and E. officinalis showing nearly the same activity, which is better that that of T. belerica.
Furthermore, the lethal consequence of NO increases significantly upon reaction with superoxide radical resulting in the formation of highly reactive peroxynitrite anion (ONOO-), especially its protonated form, peroxynitrous acid (ONOOH). ONOO- has added to the pathogenesis of diseases such as heart disease, Alzheimer's disease, and atherosclerosis [34, 35]. However, as revealed in Figure 6, highly considerable results were obtained for the scavenging effects of the studied extracts, which illustrated similar result to the standard gallic acid in the order E. officinalis > T. belerica > T. chebula.
Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. It can cross cell membrane rapidly, once inside the cell, H2O2 can probably react with Fe2+ and possibly Cu2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects . From the results, it appeared that H2O2 scavenging activity of the fruit extracts is very negligible compared to standard sodium pyruvate.
A high energy form of oxygen, singlet oxygen is generated in the skin upon UV-radiation and it induces hyperoxidation, oxygen cytotxicity and decreases the antioxidant activity . The higher IC50 values (Table 2) of the studied extracts than the reference compound lipoic acid indicated that the extracts of the fruits of T. chebula, T. belerica and E. officinalis, with T. belerica being the most effective among them, have singlet oxygen scavenging activity but poor compared to standard lipoic acid, as also found in Figure 7.
Hypochlorous acid is another harmful ROS. At the sites of inflammation, the oxidation of Cl- ions by the neutrophil enzyme myeloperoxidase results in the production of this ROS , which breaks down the heme prosthetic group and inactivates the antioxidant enzyme catalase. The obtained results (Figure 8) indicate that the standard ascorbic acid is a comparable scavenger to the fruit extracts (Table 2). So, it is anticipated that T. chebula, T. belerica and E. officinalis are efficient scavengers of HOCl, with T. belerica being the most effective, just like in case of singlet oxygen.
The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the activity of antioxidants has been attributed to various mechanisms such as prevention of chain initiation, decomposition of peroxides, reducing capacity and radical scavenging . As shown in figure 9, the reducing power of the fruit extracts were compared with standard ascorbic acid and it was found that reducing capacity of the fruit extracts were although not better than standard, yet showed considerable activity with E. officinalis as the best among the three studied extracts.
The results indicate that the fruit extracts contain significant amount of flavonoids and phenolic content, in the order T. belerica > E. officinalis > T. chebula and E. officinalis > T. belerica > T. chebula for the flavonoid and phenolic contents, respectively. Both of these compounds have good antioxidant potential and their effects on human nutrition and health are considerable. The mechanism of action of flavonoids is through scavenging or chelating process . Phenolic contents are also very important plant constituents because of their scavenging ability due to their hydroxyl groups . Moreover, ascorbic acid acting as a chain breaking antioxidant impairs with the formation of free radicals in the process of formation of intracellular substances throughout the body, including collagen, bone matrix and tooth dentine . From the results, the trend for the ascorbic acid content was found to be E. officinalis > T. chebula > T. belerica.
We also observed that treating mice with total extracts of medicinal plants increased the activity of all antioxidant enzymes examined, including SOD, CAT, GST and GSH. These enzymes are modulated in various diseases by free radical attack, thus maintaining the balance between the rates of radical generation and scavenging. It is of particular interest to note that SOD catalyzes the breakdown of O2
. to O2 and H2O2, and thus prevents the formation of OH., and thereby, has been implicated as an essential defense against the potential oxygen toxicity. SOD catalyzes the breakdown of endogenous cytotoxic superoxide radicals to H2O2 which is further degraded by CAT. Thus, they play a crucial role in maintaining the physiological levels of O2 and H2O2. GSH, in conjunction with GST, has a basic role in cellular defense against deleterious free radicals and other oxidant species . GST catalyzes the conjugation of thiol group of glutathione to electrophilic substrates, and thereby detoxifies endogenous compounds such as peroxidized lipids . The present study supports the antioxidant potency of the fruit extract as evidenced by the increased level of these antioxidant systems in extract treated mice.