Phytochemical Characterization of Dillenia indica L. Bark by Paper Spray Ionization-Mass Spectrometry and Evaluation of Its Antioxidant Potential Against t-BHP-Induced Oxidative Stress in RAW 264.7 Cells

The antioxidant effects of the ethyl acetate fraction of Dillenia indica bark (DIBEt) and the underlying mechanisms were investigated in tert-butyl hydroperoxide (t-BHP)-stimulated oxidative stress in RAW 264.7 cells. Paper spray ionization-mass spectroscopy with positive-ion mode tentatively revealed 27 secondary metabolites in D. indica bark extract; predominant among them were alkaloids, phenolic acids, and flavonoids. A new triterpenoid (nutriacholic acid) was confirmed in DIBEt for the first time. DIBEt had strong free radical-scavenging capabilities and was also able to reduce t-BHP-induced cellular reactive oxygen species (ROS) generation in RAW 264.7 cells. DIBEt was found to prevent oxidative stress by boosting the levels of heme oxygenase-1 (HO-1) through the up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) via the regulation of extracellular signal-regulated kinase (ERK) phosphorylation in RAW 264.7 cells. These results support the potential of DIBEt for defense against oxidative stress-stimulated diseases.


Introduction
Among the various signaling molecules, reactive oxygen species (ROS) and reactive nitrogen species (RNS) play critical roles in maintaining cellular homeostasis. Redox imbalance precisely participates in the pathogenesis and pathophysiology of numerous chronic diseases [1]. However, macrophage cells serve as the first line of defense in infected cells, and activated macrophages are a major source of ROS and RNS triggers epigenetic modifications, leading to the pathogenesis of chronic diseases [2]. Thus, activated macrophage models can identify the active components for functional diet development through a multiple-target strategy [2]. Natural medicinal products have been exploited

Plant Materials and Extraction
DIB was collected from Jahangirnagar University, Bangladesh, in August 2018 and taxonomically identified by the National Herbarium of Bangladesh (voucher specimen no. 49403) and retained in the laboratory for future reference. Dried and coarsely powdered barks (100 g) were extracted by shaking with ethanol at 60 • C for 12 h (three times) and dried in a rotary vacuum evaporator. The ethanolic extract (DIBE; 18.21 g) was suspended in 200 mL deionized water and partitioned sequentially with n-hexane, chloroform, and ethyl acetate using separating funnels in a stepwise manner. After vacuum filtration, the solutions were concentrated in a rotary vacuum evaporator. The n-hexane fraction (DIBH; 1.70 g), chloroform extract (DIBC; 2.44 g), ethyl acetate fraction (DIBEt; 8.55 g), and aqueous fraction (DIBW; 5.18 g) were dissolved in deionized water at 30 mg/mL concentration.
DIBEt was dissolved in deionized water and then diluted with HPLC-grade ethanol at 10 mg/mL concentration for PSI-tandem MS (MS/MS). The sample solution was vortexed for 1 min and sonicated for 5 min in a Powersonic 410 sonication bath (Hwashin Technology Co., Gyeonggi, Korea) for a homogeneous mixture.

PSI-MS
A 2 µL stock solution (10 mg/mL) was loaded using a disposable glass Pasteur pipette (Volac; Poulten & Graf Ltd., Barking, UK) onto the center of a chromatographic paper tip (Whatman 1 Chr., Kent, UK). The positive-ion mode of the Q-Exactive orbitrap MS (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used to collect the data over the range of m/z 50-600. To make a sharp tip, the chromatographic paper was cut into dimensions of 6 mm base and 14 mm height. A syringe pump (Fusion 100T; Chemyx, Stafford, TX, USA) was used to load the ethanol solvent onto the sample-loaded paper at a flow rate of 15 µL/min. A spray voltage of 4.5 kV was directly applied to the paper tip for the ionization of the sample. The other parameters for the PSI experiment were as follows: capillary temperature 300, S-lens RF level 50, mass resolution 140,000 (full-width at half-maximum), and maximum injection time 150 ms. The automatic gain control was set to 1 × 10 6 .
To perform the MS/MS experiments, three different stepped normalized collision energies (10, 30, and 50) were used with the same instrument. The instrument was operated in the positive-ion mode. The other operative parameters for the MS/MS experiments were as follows: sheath and auxiliary gas flow rate 10 and 0 (arbitrary units), respectively; spray voltage 3.60 kV; capillary temperature 300; and S-lens RF level 50.

Data Processing
Mass spectral data obtained from the orbitrap MS were processed using Xcalibur 3.1 with Foundation 3.1 (Thermo Fisher Scientific). Compounds were tentatively identified by matching their exact (calculated) masses of protonated (M + H) adducts with measured m/z values and PSI-MS/MS fragmentation patterns from the in-house MS/MS database, and online databases such as the Human Metabolome Database [15] and METLIN [16], and the literature. The compound structures were drawn using ChemDraw Professional 15.0 (PerkinElmer, Waltham, MA, USA).

Radical-Scavenging Activity Assays
DPPH, ABTS, superoxide, and hydroxyl radical-scavenging assays were conducted to evaluate the free radical-scavenging potential of DIB extract following the procedures outlined by Alam et al. [3]. Ascorbic acid and quercetin were used as positive controls for DPPH and ABTS and superoxide and hydroxyl radical-scavenging assays, respectively. The following equation was adapted to calculate the percent inhibition: where Abs control is the absorbance of the control sample and Abs sample is the absorbance of the experimental sample. All samples were analyzed in triplicate.
To determine the reducing power potential, ferric reducing antioxidant power (FRAP) and cupric reducing antioxidant capacity (CUPRAC) assays were performed according to the method described by Alam et al. [17]. The reducing power potential was expressed as the ascorbic acid-equivalent antioxidant value (µM) calculated from the standard curve of ascorbic acid. The oxygen radical absorbance capacity (ORAC) assay [18] was performed using Trolox, a water-soluble analog of vitamin E, as a positive control. The antioxidant potentiality was calculated as the Trolox-equivalent antioxidant value (µM).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). RT & Go Mastermix (MP Biomedicals, Seoul, Korea) was used to prepare cDNA by implementing the manufacturer's protocols. As described in Supplementary Table S1, various primers were used to perform RT-PCR using a PCR Thermal Cycler Dice TP600 (Takara Bio, Inc., Otsu, Japan) [17].

Western Blot Analysis
Cells were lysed and harvested using radioimmunoprecipitation assay buffer. Nuclear and cytosolic proteins were extracted by applying the nuclear and cytoplasmic extraction kit (Sigma-Aldrich, St. Louis, MO, USA). The bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) was used to confirm the protein content. An adequate amount of protein (30 µg) was subjected to Western blot analysis, as described in a previous report using various antibodies (Supplementary Table S2) [20].

Statistical Analysis
Statistical analysis was performed using SigmaPlot version 12.5 (Systat Software, Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation (SD; n = 3). One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was performed to determine the significance of the differentiation and fusion indices. p < 0.05 was considered significant.

Radical-Scavenging Activities of DIB Extracts
Various molecular mechanisms and/or the synergism between them may cause the attribution of antioxidant activity of the secondary metabolites present in plants. Thus, the evaluation of the antioxidant activity of plant extracts should be performed via several methods. DPPH, ABTS, superoxide and hydroxyl radical-scavenging assays, and FRAP, CUPRAC, and ORAC assays were performed to assess the antioxidant potential of various organic and aqueous DIB extracts. All organic and aqueous DIB extracts significantly scavenged DPPH and ABTS radicals in a dose-dependent manner ( Figure 3A; Supplementary Figure S5). DIBEt showed 7.34-and 3.81-fold higher DPPH and ABTS radical-scavenging activities, respectively, than ascorbic acid used as a positive control, with IC 50 of 1.87 ± 0.13 and 7.58 ± 0.10 µg/mL for DIBEt and 13.79 ± 0.87 and 28.90 ± 0.16 µg/mL for ascorbic acid, respectively. Other extracts showed DPPH and ABTS radical-scavenging activities in the following order: DIBE (IC 50 , 5.12 ± 0.68 µg/mL) > DIBW (IC 50 [12,23]. Compared to previous studies, this study revealed that DIB is more powerful to scavenge DPPH radicals. The superoxide and hydroxyl radical-scavenging abilities of DIB extracts were evaluated by the PMS-NADH superoxide-generating system and Fenton reaction in a dose-dependent manner, respectively ( Figure 3B; Supplementary Figure S6). DIBEt had 5.70-and 7.10-fold higher superoxide and hydroxyl radical-scavenging potential than quercetin, with IC 50 of 2.47 ± 0.05 and 1.58 ± 0.06 µg/mL for DIBEt and 14.12 ± 0.77 and 11.21 ± 1.06 µg/mL for quercetin, respectively. Other extracts had superoxide and hydroxyl radical-scavenging activities in the following order: DIBE (IC 50 , 14.78 ± 1.15 µg/mL) > DIBW (IC 50 , 14.48 ± 0.17 µg/mL) > DIBC(IC 50 , >100 µg/mL) > DIBH (IC 50 , >100 µg/mL) and DIBE (IC 50 , 7.85 ± 0.02 µg/mL) > DIBW (IC 50 , 9.54 ± 0.09 µg/mL) >DIBC (IC 50 , 22.41 ± 2.17 µg/mL) > DIBH (IC 50 , 23.85 ± 0.47 µg/mL), respectively. Das et al. [23] reported that the methanolic extract of D. indica fruits had powerful superoxide and hydroxyl radical-scavenging activities with IC 50 of 51.49 and 51.82 µg/mL, respectively. In contrast, this study showed that various organic and aqueous DIB extracts are more powerful to scavenge superoxide and hydroxyl radicals.   Studies revealed that phenolic compounds, such as phenol and flavonoids, have strong redox properties capable of quenching singlet and triplet oxygen, adsorbing and neutralizing free radicals, and/or decomposing peroxides, resulting in superior antioxidant potential [17,24,25]. Thus, total phenolic and flavonoid contents were found in the plant chosen for the study (Supplementary data S8). In addition, Pearson coefficient (ρ) and linear regression analyses were performed to correlate the polyphenol, flavonoid, and antioxidant activities of DIB extracts. The results showed substantial correlation for DPPH, ABTS, and hydroxyl radical-scavenging activities (ρ = −0.939, −0.918, and −0.853, respectively) and moderate correlation for superoxide radical-scavenging activity (ρ = −0.622). A negative ρ-value (−1) stands for a perfect positive correlation, as a correlation between polyphenol and free radical-scavenging abilities was found using IC50. The data were also supported by previous studies describing that the antioxidant activity is highly influenced by the presence of total phenol content, and has a linear correlation between phenolic content and antioxidant activity, but the total flavonoid content provided a mixed function [3,26].
Studies revealed that phenolic compounds, such as phenol and flavonoids, have strong redox properties capable of quenching singlet and triplet oxygen, adsorbing and neutralizing free radicals, and/or decomposing peroxides, resulting in superior antioxidant potential [17,24,25]. Thus, total phenolic and flavonoid contents were found in the plant chosen for the study (Supplementary data S8). In addition, Pearson coefficient (ρ) and linear regression analyses were performed to correlate the polyphenol, flavonoid, and antioxidant activities of DIB extracts. The results showed substantial correlation for DPPH, ABTS, and hydroxyl radical-scavenging activities (ρ = −0.939, −0.918, and −0.853, respectively) and moderate correlation for superoxide radical-scavenging activity (ρ = −0.622). A negative ρ-value (−1) stands for a perfect positive correlation, as a correlation between polyphenol and free radical-scavenging abilities was found using IC 50 . The data were also supported by previous studies describing that the antioxidant activity is highly influenced by the presence of total phenol content, and has a linear correlation between phenolic content and antioxidant activity, but the total flavonoid content provided a mixed function [3,26].

DIBEt Attenuates t-BHP-Induced Cellular Oxidative Stress
Among all extracts, DIBEt has the highest antioxidant potential triggered to evaluate the potential of DIBEt to scavenge oxidative stress-induced cellular ROS generation with cellular toxicity. t-BHP, a short-chain analog of lipid peroxide, is widely accepted as a model substance to induce oxidative stress in cells and tissues and evaluate the molecular mechanisms of cellular alterations caused by oxidative stress [35]. More than 95% of cell viability was noted at DIBEt concentrations up to 50 µg/mL ( Figure 4A; Supplementary Figure S9). In addition, Figure 4B demonstrates that DIBEt has an immense potential to attenuate oxidative stress-induced cellular ROS generation in a concentration-dependent manner with that of gallic acid (30 µg/mL) without showing cellular toxicity. Excessive ROS formation triggers oxidative stress, leading to cell death. Detailed research has revealed that antioxidants mitigated the deleterious effects of ROS and retard many effects that cause cellular death [3,36,37].

Effects of DIBEt on Antioxidant Enzyme Expression in RAW 264.7 Cells
The first-line antioxidant defense system, including superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione, critically maintains cellular redox homeostasis [37]. Various stimuli can induce HO-1 expression, conferring cell protection from oxidative stress by maintaining antioxidant/oxidant homeostasis [38]. In Figure 4C,D, the mRNA expression and protein levels of the first-line antioxidant enzymes SOD1, catalase, and GPx-1 and the phase II enzyme HO-1 were extremely extenuated in t-BHP-treated cells, respectively, but DIBEt treatment dose-dependently reversed this trend. Naringenin also enhanced the mRNA expression and protein levels of first-line antioxidant enzymes in the t-BHP model. These data suggest that increased mRNA and protein levels of antioxidant enzymes by DIBEt plays a critical role in cellular homeostasis and protects against oxidative stress-induced cell death.
Studies revealed that medicinal plants or foods possess abundant polyphenolic compounds that boost SOD1, CAT, and GPx activities, decreasing oxidative stress [39,40]. Evidence showed that polyphenolic acids, such as naringenin, kaempferol, apigenin, and formononetin aglycone, and their glycosidic form and caffeoylisocitric acid have a potential to boost first-line antioxidant proteins, leading to cell protection against oxidative stress [41][42][43][44][45]. Therefore, the presence of phenolic acids and flavonoids in DIBEt may play a major role in boosting the expression of first-line antioxidant enzymes/proteins as the principal mechanism accounting for protecting DIBEt against oxidative stress.
Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 17 Figure 4. Protective effects of DIBEt against t-BHP-induced cell toxicity and intracellular ROS generation through the up-regulation of antioxidant enzymes via Nrf2 activation. Cells were treated with DIBEt and gallic acid (GA) at the indicated concentrations for 12 h and then exposed to 100 μM t-BHP for 6 h. Cell viability percentage (A) and intracellular ROS (B) were determined by MTT assay and the DCFH-DA method, respectively. Mean ± SD (n = 3). # p < 0.001, compared to no treatment; ** p < 0.05, compared to t-BHP treatment. SOD1, catalase, GPx, and phase II antioxidant enzyme (such as HO-1) mRNA (C) and protein expression (D) were analyzed by RT-PCR and Western blot, respectively. Nrf2 protein expression (E) was measured by Western blot. Cells were treated with an Nrf2 inhibitor (brusatol) with and without DIBEt and naringenin (NAR). Nrf2 and HO-1 protein levels were analyzed by Western blot (F). Mean ± SD (n = 3). # p < 0.001, compared to no treatment; ** p < 0.05, compared to sample treatment. (+): presence; (-): absence.
Studies revealed that medicinal plants or foods possess abundant polyphenolic compounds that boost SOD1, CAT, and GPx activities, decreasing oxidative stress [39,40]. Evidence showed that polyphenolic acids, such as naringenin, kaempferol, apigenin, and formononetin aglycone, and their glycosidic form and caffeoylisocitric acid have a potential to boost first-line antioxidant proteins, leading to cell protection against oxidative stress [41][42][43][44][45]. Therefore, the presence of phenolic acids and flavonoids in DIBEt may play a major role in boosting the expression of first-line antioxidant enzymes/proteins as the principal mechanism accounting for protecting DIBEt against oxidative stress.

Effects of DIBEt on Phase II Enzymes Mediated by Nrf2 Nuclear Translocation in RAW 264.7 Cells
An important transcription factor, Nrf2, binds to the ARE in the promoter regions of cytoprotective genes and acts as a master regulator of antioxidative responses [46]. Thus, immunoblotting was performed to evaluate the role of DIBEt on Nrf2 regulation. In Figure 4E, the nuclear Nrf2 content was markedly increased in association with decreased cyto-Nrf2 levels after DIBEt treatment. Furthermore, brusatol, a pharmacological inhibitor of Nrf2, was used to confirm the role of DIBEt on the activation of phase II detoxifying enzymes through Nrf2 regulation. Nrf2 protein levels were greatly diminished by brusatol treatment, which was not reestablished despite and then exposed to 100 µM t-BHP for 6 h. Cell viability percentage (A) and intracellular ROS (B) were determined by MTT assay and the DCFH-DA method, respectively. Mean ± SD (n = 3). # p < 0.001, compared to no treatment; ** p < 0.05, compared to t-BHP treatment. SOD1, catalase, GPx, and phase II antioxidant enzyme (such as HO-1) mRNA (C) and protein expression (D) were analyzed by RT-PCR and Western blot, respectively. Nrf2 protein expression (E) was measured by Western blot. Cells were treated with an Nrf2 inhibitor (brusatol) with and without DIBEt and naringenin (NAR). Nrf2 and HO-1 protein levels were analyzed by Western blot (F). Mean ± SD (n = 3). # p < 0.001, compared to no treatment; ** p < 0.05, compared to sample treatment. (+): presence; (-): absence.

Effects of DIBEt on Phase II Enzymes Mediated by Nrf2 Nuclear Translocation in RAW 264.7 Cells
An important transcription factor, Nrf2, binds to the ARE in the promoter regions of cytoprotective genes and acts as a master regulator of antioxidative responses [46]. Thus, immunoblotting was performed to evaluate the role of DIBEt on Nrf2 regulation. In Figure 4E, the nuclear Nrf2 content was markedly increased in association with decreased cyto-Nrf2 levels after DIBEt treatment. Furthermore, brusatol, a pharmacological inhibitor of Nrf2, was used to confirm the role of DIBEt on the activation of phase II detoxifying enzymes through Nrf2 regulation. Nrf2 protein levels were greatly diminished by brusatol treatment, which was not reestablished despite the application of DIBEt and naringenin ( Figure 4F). Furthermore, DIBEt and naringenin treatment was also unable to restore the basal HO-1 protein levels in brusatol-treated cells ( Figure 4F). This observation conferred that DIBEt might disrupt the proteasomal degradation of Nrf2 in the cytoplasm by Keap1 and facilitate Nrf2 nuclear translocation, resulting in the up-regulation of HO-1 expression. Studies revealed that extracts from various medicinal plants or foods, such as Nymphaea nouchali flowers, Lannea coromandelica bark, and Ginkgo biloba bark, can activate Nrf2-mediated phase II enzyme expression in RAW 264.7, Hepa-1c1c7, and Hep G2 cells [3,17,47]. Furthermore, naringenin, kaempferol, apigenin, and formononetin aglycone and their glycosidic form can modulate the Nrf2/ARE/HO-1 signaling cascade, leading to the attenuation of oxidative stress-mediated melanocytes and kidney and neuronal death [43][44][45].

DIBEt Regulates Nrf2 Translocation Via Activation of MAPK to Lessen Oxidative Stress
MAPKs act as a downstream effector in antioxidant responses. The activation of MAPKs can manifest the activation of Nrf2. In-vitro and in-vivo studies have revealed that extracellular signal-regulated kinase (ERK), JNK, and p38 MAPK positively regulate ARE-containing reporter or detoxifying genes via Nrf2-dependent mechanisms [48,49]. In Figure 5A, ERK1/2 was phosphorylated from 15 to 180 min and peaked at 30 min after DIBEt exposure, whereas p38 and JNK phosphorylation was absent in DIBEt-treated cells. Furthermore, to confirm the role of ERK1/2 phosphorylation in Nrf2 translocation to the nucleus and induction of HO-1 expression, U0126, a specific inhibitor of ERK1/2, was used in DIBEt-treated cells. As expected, in Figure 5B, Nrf2 nuclear translocation and subsequently HO-1 expression were successfully enhanced in DIBEt-treated cells, which were strongly mitigated in U0126-treated cells, suggesting that ERK activation plays a critical role in DIBEt-induced Nrf2 translocation into the nucleus and subsequent boost of HO-1 expression in RAW 264.7 cells. Moreover, according to previous reports, dietary antioxidants can cause MAPK activation accountable for cell protection against oxidative stress [50].
the application of DIBEt and naringenin ( Figure 4F). Furthermore, DIBEt and naringenin treatment was also unable to restore the basal HO-1 protein levels in brusatol-treated cells ( Figure 4F). This observation conferred that DIBEt might disrupt the proteasomal degradation of Nrf2 in the cytoplasm by Keap1 and facilitate Nrf2 nuclear translocation, resulting in the up-regulation of HO-1 expression. Studies revealed that extracts from various medicinal plants or foods, such as Nymphaea nouchali flowers, Lannea coromandelica bark, and Ginkgo biloba bark, can activate Nrf2-mediated phase II enzyme expression in RAW 264.7, Hepa-1c1c7, and Hep G2 cells [3,17,47]. Furthermore, naringenin, kaempferol, apigenin, and formononetin aglycone and their glycosidic form can modulate the Nrf2/ARE/HO-1 signaling cascade, leading to the attenuation of oxidative stressmediated melanocytes and kidney and neuronal death [43][44][45].

DIBEt Regulates Nrf2 Translocation Via Activation of MAPK to Lessen Oxidative Stress
MAPKs act as a downstream effector in antioxidant responses. The activation of MAPKs can manifest the activation of Nrf2. In-vitro and in-vivo studies have revealed that extracellular signalregulated kinase (ERK), JNK, and p38 MAPK positively regulate ARE-containing reporter or detoxifying genes via Nrf2-dependent mechanisms [48,49]. In Figure 5A, ERK1/2 was phosphorylated from 15 to 180 min and peaked at 30 min after DIBEt exposure, whereas p38 and JNK phosphorylation was absent in DIBEt-treated cells. Furthermore, to confirm the role of ERK1/2 phosphorylation in Nrf2 translocation to the nucleus and induction of HO-1 expression, U0126, a specific inhibitor of ERK1/2, was used in DIBEt-treated cells. As expected, in Figure 5B, Nrf2 nuclear translocation and subsequently HO-1 expression were successfully enhanced in DIBEttreated cells, which were strongly mitigated in U0126-treated cells, suggesting that ERK activation plays a critical role in DIBEt-induced Nrf2 translocation into the nucleus and subsequent boost of HO-1 expression in RAW 264.7 cells. Moreover, according to previous reports, dietary antioxidants can cause MAPK activation accountable for cell protection against oxidative stress [50]. Immunoblotting was performed to evaluate kinase activity (A). Cells were treated with DIBEt in the presence and absence of the specific inhibitor U0126. The protein levels of Nrf2 and HO-1 were analyzed by Western blot (B). Mean ± SD (n = 3). # p < 0.001; ** p < 0.05, compared to no treatment. Statistical analysis was performed using one-way ANOVA. Figure 5. DIBEt facilitates Nrf2 translocation by activating ERK1/2. RAW 264.7 cells were pretreated with DIBEt (10 µg/mL) at the indicated times. Immunoblotting was performed to evaluate kinase activity (A). Cells were treated with DIBEt in the presence and absence of the specific inhibitor U0126. The protein levels of Nrf2 and HO-1 were analyzed by Western blot (B). Mean ± SD (n = 3). # p < 0.001; ** p < 0.05, compared to no treatment. Statistical analysis was performed using one-way ANOVA.

Conclusions
Oxidative stress is a major causative condition for the development and progression of numerous acute and chronic clinical disorders. Thus, antioxidants may cause health benefits as prophylactic agents. In this study, DIBEt contained various polyphenolic compounds as confirmed by PSI-MS/MS and showed superior antioxidant activity in cell-free and cellular levels. DIBEt treatment successfully lessened oxidative stress and cell death, most likely by (i) reducing ROS generation and (ii) boosting the expression of endogenous antioxidant enzymes and/or Nrf2-mediated HO-1 expression. DIBEt pretreatment may cause the activation of ERK1/2 signaling pathways involved in the cytoprotective effects of DIBEt. The findings provide new insights into the cytoprotective effects and mechanisms of DIB against oxidative stress, which may be used as treatment for oxidative stress-induced disorders.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/11/1099/s1, Figure S1: Mass fragmentation of nitrogen compounds in DIB extracts. Figure S2: Mass fragmentation of flavonoids in DIB extracts. Figure S3: Mass fragmentation of phenolic acids in DIB extracts. Figure S4: Mass fragmentation of ethyl maltol, 11-dodecenoic acid, and the triterpenoid nutriacholic acid in DIB extracts. Figure S5: DPPH and ABTS radical-scavenging activities of various organic and aqueous DIB extracts. Figure S6: Superoxide and hydroxyl radical-scavenging activities of various organic and aqueous DIB extracts. Figure S7: CUPRAC and FRAP activities of various organic and aqueous DIB extracts. Figure S8: Total phenol (A) and flavonoid (B) content of various organic and aqueous DIB extracts. Figure S9: Cell viability of DIBEt. Table S1: List of primer sequences used in this study. Table S2: List of antibodies used in this study.