**4. Discussion**

The antioxidant potential of *D. indum* fruit was evaluated by measuring the capabilities of various extracts and fractions of the different fruit parts in reducing phosphomolybdic-phosphotungstic acid reagents, neocuproine, 2,2-diphenyl-picrylhydrazyl (DPPH), and inhibiting linoleic acid peroxidation. The gallic acid equivalence (GAE) for reducing phosphomolybdic-phosphotungstic acid reagents was the highest in the DCM fractions for the exocarp (EDF) and mesocarp (MDF), while for the seed, the highest GAE was measured in the methanol fraction (SMF). The GAE of SMF was found to be the highest when compared with other local underutilized fruits of Malaysia, namely *Baccaurea angulata* [3], *Canarium odontophyllum* [37], and *Sandoricum macropodum* [38] in other studies. It can be suggested from this result that most antioxidants in the exocarp and mesocarp of *D. indum* fruit are semipolar in nature while in the seed, most of the antioxidants are polar. Low GAE values in hexane fractions might be due to the higher concentration of non-polar constituents such as fatty acids. This study also showed a strong, positive correlation (*<sup>r</sup>*s = 0.929, *n* = 16, *p* < 0.010) between GAE and TAC values, which is in line with a previous study that proved the good correlation (*r* = 0.966) in herbal teas [39]. Both the GAE and TAC values were determined using different assays of a similar underlying antioxidant mechanism, which measure the reduction of the oxidation number of the transition metal ions by antioxidants achieved via electron transfer [40].

The strong neocuproine reducing activity, which was interpreted as the high total antioxidant capacity (TAC) of SMF, can be attributed to the presence of non-phenolic metabolites, particularly the amino acids that also showed neocuproine reducing activity in another study [41]. The following order was ranked for the DPPH radical scavenging activity of the exocarp and seed: SMF > SCM > ECM > EDF > EMF > EHF. However, this sequence was not exactly replicated in their GAE by which the following order was found: SMF > EDF > ECM > EMF > SCM > EHF. The difference in GAE and DPPH radical scavenging activities can be attributed to the difference in the accessibility of various phenolic antioxidants in the different extracts to the unpaired electron of the divalent nitrogen atom of the DPPH radical. Steric accessibility is a major determinant for redox reactions with DPPH, where small antioxidants that have better access to the radical site than their larger counterparts tend to have higher DPPH-reducing power [42]. This finding corroborates previous studies that reported a negative relationship between the IC50 values of DPPH radical scavenging and GAE [43–45].

Lipid peroxidation involves the formation and propagation of hydroperoxide radicals, which decompose to form byproducts such as malondialdehyde (MDA), ketones, alcohols, and hydrocarbons that can interact with sulfhydryl and amine groups in proteins, causing damage to vital proteins. The distinctive feature of this lipid peroxidation assay lies in the medium used, which is a linoleic acid emulsion, whereas in the GAE, TAC, and DPPH assays, polar media such as water, methanol, and ethanol were used. Moreover, the assay conforms to the antioxidant polar paradox hypothesis, which states that lipid-soluble antioxidants are most effective in emulsions and membranes [46]. It is interesting to note that SMF, which exhibited excellent positive results for all previous assays showed weak linoleic acid peroxidation inhibitory activity with the maximum percentage of inhibition (at 250 μg/mL) below 50%. This finding may be related to the dominance of hydrophilic antioxidants in SMF. The antioxidant activity of compounds in emulsions is attributed to the hydrophilic–lipophilic balance of their polar and hydrocarbon moieties where more lipophilic antioxidants are present in the lipid phase and at the oil-water interface at which interactions between hydroperoxides and prooxidants occur [47].

The strong DPPH radical scavenging activity of SMF can be attributed to the presence of the amino acids detected, namely proline, serine, threonine, pyroglutamic acid, phenylalanine, and glutamic acid. Amino acids were found to exert antioxidant activities by donating protons to electron-deficient radicals [48]. The percentage of amino acids in SMF was four times more than that of the detected phenolics and was higher in the seed than in the exocarp. *D. indum* is a plant in the family Fabaceae, which is a well-known plant family that produces seeds with a high content of proteins and saccharides and are consumed as nutritive food worldwide [49]. The difference in metabolite compositions between EDF and SMF is largely due to the higher percentage of phenolics in EDF (11.48%) than SMF (0.21%). The nine phenolics in EDF can be categorized into two main groups: phenolic acids and phenolic aldehydes. Previous studies have shown that the more the number of hydroxyl groups at the benzene ring, the stronger the DPPH radical scavenging activities of the phenolics and the conjugated carbon skeleton plays an important role in the antioxidant activities of the phenolics. The strength of the in vitro DPPH radical scavenging activities of the phenolics can be ranked in the following decreasing order: hydroxycinnamic acid derivatives > hydroxybenzoic acid derivatives > phenolic aldehydes [50].

Varying percentages of different phenolics have been found in the selected EDF subfractions. Vanillic acid and sinapic acid were consistently detected in EDF and all the selected subfractions, where sub-fraction Di-21 had the highest percentage of vanillic acid (2.07%) while the highest percentage of sinapic acid (3.48%) was found in sub-fraction Di-25. Higher percentages of vanillic acid in EDF, subfractions Di-21 and Di-22 could be related to their weaker DPPH radical scavenging activities than the subfractions with stronger activity, namely subfractions Di-24, Di-25, and Di-26. This finding was parallel to a study that observed an antagonistic effect of vanillic acid when combined with gallic acid, protocatechuic acid, and chlorogenic acid [51]. The stronger DPPH radical scavenging activities of the more polar EDF subfractions viz. Di-24, Di-25, and Di-26 can be related to their higher percentage of hydroxycinnamic acid derivatives (ferulic acid, sinapic acid, and *p*-coumaric acid). Hydroxycinnamic acid derivatives are more potent antioxidants than hydroxybenzoic acid derivatives due to the presence of the additional carbon–carbon double bond next to the benzene ring, thus extending the conjugated π orbital system. This configuration, in turn, stabilizes the resulting phenoxy radicals by resonance and enhances their antioxidant activities [50,52].
