ic acid, gallocatechin, epigallocatechin, caffeine, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate, which were not detected in the HE of C. japonica seed coats. The largest peak of ME corresponded to catechin. Additionally, Figure 3 displays the chromatograms of the hexaneextracted compounds and control standards. The chromatogram indicates that tocotrienols and tocopherols were extracted by hexane from the seed coat of C. japonica, which was composed of αtocopherol, γ-tocopherol, β-tocopherol, δ-tocopherol, α-tocotrienol, γ-tocotrienol, and δ-tocotrienol. The results were correlated to a previous report by Hu and Yang (2018) [26], who reported that the major lipophilic antioxidant phytochemicals in C. oleifera oil were α-tocopherol and γ-tocopherol. The amounts of phytochemical compounds in the ME and HE extracts is shown in Table 3. The current results indicated that the catechin and related compounds (hydrophilic antioxidants) in the ME of C. japonica seed coats possessed both ROS-and RNS-scavenging activities. These compounds also suppressed the production of ROS and RNS in the cell-based study of C. japonica seed coats, as represented by both the hydrophilic and lipophilic antioxidant phytochemicals. Catechin and the related compounds all have phenolic hydroxyl groups that were able to stabilize the free radicals [23]. The inhibition on RNS production in RAW 264.7 cells and the scavenging effect on intracellular ROS production by C. japonica seed coat extracts corresponded to antioxidant phytochemical compounds. Catechin and related compounds all have phenolic hydroxyl groups that are able to stabilize the free radicals [27]. The phenolic hydroxyl groups of catechin and its derivatives can react with ROS and RNS in a termination reaction, resulting in a breakdown of the cycle of new radical production [28]. Furthermore, the tocotrienols and tocopherols present in the HE of C. japonica seed coats included a lipophilic antioxidant. The HE exhibited a higher antioxidant activity in the cellbased study than in the in vitro study. Vitamin E isoforms (tocopherols and tocotrienols) were incorporated into cellular membranes and exhibited intracellular antioxidant effects. Additionally, tocopherols and tocotrienols exhibited similar mobilities within the membranes; however, tocotrienols were more easily transferred through the membranes and incorporated into the membranes [29,30]. Therefore, we hypothesized the antioxidant activity from the combination of hydrophilic and lipophilic antioxidant phytochemicals of C. japonica seeds.  Figure 2 displays the HPLC chromatogram identifying catechin and its derivatives. The ME of C. japonica seed coats contained hydrophilic antioxidant compounds, namely gallic acid, gallocatechin, epigallocatechin, caffeine, catechin, epicatechin, epigallocatechin gallate, and epicatechin gallate, which were not detected in the HE of C. japonica seed coats. The largest peak of ME corresponded to catechin. Additionally, Figure 3 displays the chromatograms of the hexane-extracted compounds and control standards. The chromatogram indicates that tocotrienols and tocopherols were extracted by hexane from the seed coat of C. japonica, which was composed of α-tocopherol, γ-tocopherol, β-tocopherol, δ-tocopherol, α-tocotrienol, γ-tocotrienol, and δ-tocotrienol. The results were correlated to a previous report by Hu and Yang (2018) [26], who reported that the major lipophilic antioxidant phytochemicals in C. oleifera oil were α-tocopherol and γ-tocopherol. The amounts of phytochemical compounds in the ME and HE extracts is shown in Table 3. The current results indicated that the catechin and related compounds (hydrophilic antioxidants) in the ME of C. japonica seed coats possessed both ROS-and RNS-scavenging activities. These compounds also suppressed the production of ROS and RNS in the cell-based study of C. japonica seed coats, as represented by both the hydrophilic and lipophilic antioxidant phytochemicals. Catechin and the related compounds all have phenolic hydroxyl groups that were able to stabilize the free radicals [23]. The inhibition on RNS production in RAW 264.7 cells and the scavenging effect on intracellular ROS production by C. japonica seed coat extracts corresponded to antioxidant phytochemical compounds. Catechin and related compounds all have phenolic hydroxyl groups that are able to stabilize the free radicals [27]. The phenolic hydroxyl groups of catechin and its derivatives can react with ROS and RNS in a termination reaction, resulting in a breakdown of the cycle of new radical production [28]. Furthermore, the tocotrienols and tocopherols present in the HE of C. japonica seed coats included a lipophilic antioxidant. The HE exhibited a higher antioxidant activity in the cell-based study than in the in vitro study. Vitamin E isoforms (tocopherols and tocotrienols) were incorporated into cellular membranes and exhibited intracellular antioxidant effects. Additionally, tocopherols and tocotrienols exhibited similar mobilities within the membranes; however, tocotrienols were more easily transferred through the membranes and incorporated into the membranes [29,30]. Therefore, we hypothesized the antioxidant activity from the combination of hydrophilic and lipophilic antioxidant phytochemicals of C. japonica seeds.  Figure 4 displays the wide-range EPR spectrum in order to determine any paramagnetic species of whole C. japonica seeds at ambient temperature. The magnetic field center used was 240.0 mT with a 300 mT sweep width. The EPR spectrum had one distinguishable signal with a broad line. The signal was stable, and the EPR results were similar for at least a month. No Mn 2+ signal was observed in the seed coats, as previously described in other studies [8,10,11]. The signal was strong and reproducible with a g value of about 2.002, which is indicative of stable organic radicals [6,8]. The g value gives a clue as to the state of the unpaired electron in the compound.

EPR of Stable Radicals in C. japonica Seed Coat
In addition, antioxidants such as phenolic compounds that lose the hydrogen of the OH-group become intermediate radicals.   Figure 4 displays the wide-range EPR spectrum in order to determine any paramagnetic species of whole C. japonica seeds at ambient temperature. The magnetic field center used was 240.0 mT with a 300 mT sweep width. The EPR spectrum had one distinguishable signal with a broad line. The signal was stable, and the EPR results were similar for at least a month. No Mn 2+ signal was observed in the seed coats, as previously described in other studies [8,10,11]. The signal was strong and reproducible with a g value of about 2.002, which is indicative of stable organic radicals [6,8]. The g value gives a clue as to the state of the unpaired electron in the compound.

EPR of Stable Radicals in C. japonica Seed Coat
Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 14 intermediate) is stable and otherwise not reactive. This suggests that these radicals can be generated under oxidative conditions and that antioxidant-related organic compounds were present in the seed at ambient temperature. Stable radicals detected by EPR can be the products of antioxidant reactions with ROS and/or the oxidation of compounds in the seed coats. There is a possible explanation for stable radical production. We propose that a stable (or reactive) intermediate may be responsible for antioxidant reactions and the scavenging activity in the seed coats, as suggested previously [31]. In most cases, unpaired electrons or radicals can be delocalized within a molecule for stability. In addition, plant physiological processes produce ROS [31,32], which, together with nitric oxide, regulate various processes in plants [29]. Figure 5 displays the EPR spectra obtained at g values of ~2.002 of the C. japonica seed coats at ambient temperature. Additionally, the spin (radical) concentration was estimated using a TEMPOL (known concentration) solution in a capillary tube (outer diameter 1.0 mm, inner diameter 0.9 mm). The number of spins per gram was approximately 3 × 10 18. The calculation procedure was performed as described previously [11,12]. The radical species between the seed coats and cotyledon are not the same species, as each composition is not the same. The radical intensity of the seed coats is approximately 22 times stronger than that of the cotyledon, considering the sample weight measured. In addition, antioxidants such as phenolic compounds that lose the hydrogen of the OH-group become intermediate radicals. The unpaired electron (radical) is delocalized throughout the 2p z orbitals of the conjugated double bond. The delocalization of the unpaired electron (radical intermediate) is stable and otherwise not reactive. This suggests that these radicals can be generated under oxidative conditions and that antioxidant-related organic compounds were present in the seed at ambient temperature.
Stable radicals detected by EPR can be the products of antioxidant reactions with ROS and/or the oxidation of compounds in the seed coats. There is a possible explanation for stable radical production. We propose that a stable (or reactive) intermediate may be responsible for antioxidant reactions and the scavenging activity in the seed coats, as suggested previously [31]. In most cases, unpaired electrons or radicals can be delocalized within a molecule for stability. In addition, plant physiological processes produce ROS [31,32],