Antioxidant Activities of Plant Extracts ( Ammannia multiﬂora , Ammannia coccinea , and Salix gracilistyla ) Activate the Nrf2/HO-1 Signaling Pathway

: To identify potent plant extracts with strong antioxidant activity, we evaluated the free radical scavenging activity of 184 plant extracts obtained from the Freshwater Bioresources Culture Collection (FBCC) of Nakdonggang National Institute of Biological Resources (Republic of Korea), as various plant extracts have been used therapeutically to prevent chronic diseases associated with oxidative stress. From them, three plant extracts (FBCC-EP858 from Ammannia multiﬂora , FBCC-EP920 from Ammannia coccinea , and FBCC-EP1014 from Salix gracilistyla ) were selected based on their abilities to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with more than 80% efﬁciency. We found that these extracts had in vitro half maximal inhibitory concentration (IC 50 ) values ranging from 11.89 to 14.26 µ g/mL and strong total antioxidant activity (corresponding to approximately 0.18, 0.22, and 0.23 mM Trolox, respectively). We also studied the effect of these extracts on RAW 264.7 macrophages and found that FBCC-EP920 signiﬁcantly downregulated relative cell viability at a concentration of 100 µ g/mL. However, the other two extracts, FBCC-EP858 and FBCC-EP1014, did not affect cell viability at the same concentration. Additionally, all three extracts inhibited hydrogen peroxide (H 2 O 2 )-induced reactive oxygen species (ROS) production and depolarization of mitochondrial membrane potential in RAW 264.7 macrophages. An additional experiment in zebraﬁsh larvae showed that the three extracts reduced 2 (cid:48) ,7 (cid:48) -dichlorodihydroﬂuorescein diacetate (DCFDA) ﬂuorescent intensity induced by H 2 O 2 . The extracts also upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and an HO-1 inhibitor, zinc protoporphyrin (ZnPP), attenuated the extract-induced antioxidant activity both in vivo and in vitro. Taken together, these ﬁndings suggest that the extracts from A. multiﬂora , A. coccinea , and S. gracilistyla have potential free radical scavenging and antioxidant capacities both in vivo and in vitro by activating the Nrf2/HO-1 signaling pathway. These results could be useful for the prevention and treatment of various oxidative stress-mediated human diseases.


Introduction
Oxidative stress induced by excessive reactive oxygen species (ROS) contributes to inflammatory diseases and metabolic syndromes, including vascular and neurodegenerative diseases and various cancers [1,2]. Specifically, aberrant ROS production causes cellular lipid oxidation and DNA damage, which affect cellular malfunction in cell division and differentiation, ion-homeostasis, regulation of apoptosis, and inflammation [3,4]. To maintain homeostasis of ROS levels, several antioxidant systems and agents are present extensive study on their antioxidative activities with detailed molecular mechanisms has not been conducted yet.

DPPH Radical Scavenging Assay
DPPH radical scavenging activity was measured by testing three extracts (FBCC-EP858, FBCC-EP920, and FBCC-EP1014) and comparing them to the positive control, which was 20 µM ascorbic acid. Briefly, 120 µM DPPH in 95% ethyl alcohol was freshly prepared, and then 190 µL DPPH was mixed with 10 µL each extract (ranging from 0 to 100 µg/mL) at room temperature for 10 min. The absorbance value was then measured at the wavelength of 517 nm (BioTek Instruments, Inc., Winooski, VT, USA). The half maximal inhibitory concentration (IC 50 ) was calculated using GraphPad Prism 9 software (GraphPad Software, Boston, MA, USA).

Total Antioxidant Capacity
The reduction rate of Cu 2+ was measured using an OxiTec Total Antioxidant Capacity Assay Kit, with Trolox as the standard for comparison. Briefly, 100 µL reaction buffer and 100 µL copper reagent were mixed and then treated with 100 µL each extract (ranging from 0 to 100 µg/mL) for 30 min at room temperature. A standard curve of Trolox was prepared, and the concentration of the extract was calculated corresponding to Trolox concentration. Ascorbic acid (400 µM) was used as a representative antioxidant positive control. For the blank control, the reaction buffer was replaced with ethanol. Each solution (120 µL) was transferred to a 96-microplate, and the absorbance was read at a wavelength of 450 nm.

Cell Culture and Relative Cell Viability
RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 5% FBS at 37 • C in 5% CO 2 . The cells were seeded at a density of 5 × 10 4 cells/mL, and cell viability was determined using a WST-8 (highly sensitive water-soluble tetrazolium salt) Viability Assay Kit. Briefly, RAW 264.7 macrophages were treated with various concentrations (0-100 µg/mL) of each extract. After a 24 h incubation, 10 µL of WST-8 solution was added to the cell culture media and incubated for 1 h. Absorbance was measured at a wavelength of 450 nm (BioTek Instruments, Inc.).

Flow Cytometry Analysis
RAW 264.7 macrophages (5 × 10 4 cells/mL) were treated with various concentrations (0-100 µg/mL) of FBCC-EP858 and FBCC-EP1014 for 20 h and 200 µM H 2 O 2 treated for 4 h. The maximum concentration of FBCC-EP920 was set at 50 µg/mL since a concentration of 100 µg/mL resulted in a slight decrease in relative cell viability.

ROS Production
Cellular ROS production was analyzed using a Muse Oxidative Stress Kit (Luminex, Austin, TX, USA). Briefly, cells were suspended in 1× assay buffer and incubated at 37 • C for 30 min. Cells exhibiting ROS (ROS + ) were then analyzed using a Muse Cell Analyzer (Luminex) [31].

Depolarization of Mitochondrial Membrane Potential
Depolarization of mitochondrial membrane potential was measured using a Muse MitoPotential Kit (Luminex). Briefly, cells were suspended in 1× assay buffer and mixed thoroughly with MitoPotential working solution. The cells were incubated at 37 • C for 20 min, and MitoPotential 7-ADD reagent was added for 5 min at room temperature. Cells exhibiting depolarized mitochondrial membrane potential were finally measured using a Muse Cell Analyzer [32].

Western Blotting
Total cellular proteins were prepared using RIPA Lysis Buffer (ROCKLAND, Pottstown, PA, USA) with protease inhibitors (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentration was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and an equal amount of protein (20 µg) was loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific). The membrane was incubated with primary and secondary antibodies and developed using a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) [34].

Measurement of ROS Production in Zebrafish Larvae
Animal Care and Use Committee of Jeju National University (Jeju Special Self-governing Province, Republic of Korea) approved a zebrafish study for DCFDA staining (Approval No. 2022-0084). All zebrafish experiments followed the approval guidelines described previously [35]. In this experiment, zebrafish larvae were used to investigate the effects of each extract on ROS production. Zebrafish at three days post fertilization (dpf) were treated with each extract for 2 h before being treated with 1 mM H 2 O 2 for 22 h. Then, the zebrafish were stained with 20 µM DCFDA for 30 min and visualized using a CELENA S Digital Imaging System (Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea). In a parallel experiment, an HO-1 inhibitor, ZnPP, was used to evaluate HO-1-mediated antioxidant activity. Each extract was incubated for 2 h, followed by treatment with ZnPP for 1 h, and then 1 mM H 2 O 2 for 22 h.

Statistical Analysis
The western blots and RT-PCR results were quantified using ImageJ 1.50i (National Institute of Health, Bethesda, MD, USA, www.imagej.net, accessed on 22 July 2022), and all data were analyzed using SigmaPlot version 12.5 (Systat Software, San Jose, CA, USA, www.systatsoftware.com, accessed on 28 July 2022). The data represent the mean of at least three independent experiments, and significant differences were determined using Student's t-test and an unpaired one-way ANOVA test with Bonferroni correction. The significance levels are denoted by different symbols ( ### , ***, and +++ p < 0.001, ## and ** p < 0.01, and * p < 0.05).

FBCC-EP858, FBCC-EP920, and FBCC-EP1014 Regulate the Viability of RAW 264.7 Macrophages Depending on Their Concentrations
Prior to evaluating the cellular antioxidant activity of FBCC-EP858, FBCC-EP920, and FBCC-EP1014, the relative viability of RAW 264.7 cells was assessed using the WST-8 assay. As depicted in Figure 2, FBCC-EP858 and FBCC-1014 did not exhibit any significant effect on cell morphology or relative cell viability. However, FBCC-EP920 at a concentration of 100 µg/mL decreased the cell number ( Figure 2A) and relative cell viability (85.0 ± 3.2%, Figure 2B). For further investigation, FBCC-EP920 was used at a maximum concentration of 50 µg/mL, while the highest concentration of FBCC-EP858 and FBCC-1014 was set at 100 µg/mL. The quantification of cell viability was conducted using WST-8 assay. Each value indicates the mean ± SEM from three independent experiments. Significant differences among the groups were determined using an unpaired one-way ANOVA with Bonferroni correction. ## p < 0.01 vs. untreated cells.  To evaluate the cellular antioxidant activity of FBCC-EP858, FBCC-EP920, and FBCC-EP1014, we investigated whether the extracts could downregulate ROS + cell population in H 2 O 2 -exposed RAW 264.7 cells using flow cytometry. As shown in Figure 3A, the ROS + cell population (red peaks) significantly increased in H 2 O 2 -treated RAW 264.7 cells, and treatment with FBCC-EP858, FBCC-EP920, and FBCC-EP1014 downregulated the ROS + cell population in a concentration-dependent manner. At a maximum concentration, FBCC-EP858, FBCC-EP920, and FBCC-EP1014 dramatically attenuated the ROS + cell population from approximately 30.5% to 16.5 ± 0.5%, 18.3 ± 0.4%, and 17.5 ± 0.8%, respectively ( Figure 3B). We also measured the in vivo antioxidant activity of these extracts in H 2 O 2treated zebrafish larvae using DCFDA staining. Treatment with H 2 O 2 increased DCFDA fluorescence (green) in zebrafish larvae, and FBCC-EP858, FBCC-EP920, and FBCC-EP1014 gradually diminished the fluorescence intensity in a concentration-dependent manner ( Figure 3C,D). The results indicate that FBCC-EP858, FBCC-EP920, and FBCC-EP1014 can attenuate H 2 O 2 -induced ROS production both in vitro and in vivo.

FBCC-EP858, FBCC-EP920, and FBCC-EP1014 Maintain Mitochondrial Membrane Potential in H 2 O 2 -Treated RAW 264.7 Macrophages
As oxidative stress is responsible for ROS-mediated cellular damage and depolarization of mitochondrial membrane potential [36], we investigated whether the reduction of ROS by FBCC-EP858, FBCC-EP920, and FBCC-EP1014 could preserve mitochondrial membrane potential in H 2 O 2 -treated RAW 264.7 cells. As illustrated in Figure 4A,B, H 2 O 2 caused a significant increase in the total cell population with depolarized mitochondria membrane potential. However, treatment with FBCC-EP858, FBCC-EP920, and FBCC-EP1014 decreased the population and maintained mitochondrial membrane potential. These results suggest that FBCC-EP858, FBCC-EP920, and FBCC-EP1014 are able to prevent oxidative stress-induced depolarization of mitochondrial membrane potential.
concentrations of FBCC-EP858, FBCC-EP920, and FBCC-EP1014 were applied to 3 dpf zebrafish larvae 2 h before the addition of 1 mM H2O2 for 22 h. (C) The larvae were stained using 20 µM DCFDA for 30 min and visualized using a CELENA S Digital Imaging System. (D) The DCFDA fluorescence intensity was calculated using ImagJ 1.50i. Each value represents the mean ± SEM from three independent experiments. Significant difference: ### p < 0.001 vs. untreated group (Student′s t-test) and *** p < 0.001 vs. H2O2-treated group (one-way ANOVA).

HO-1 Inhibition Reduces the Antioxidant Activity of FBCC-EP858, FBCC-EP920, and FBCC-EP1014
To confirm whether the reduction of ROS production by FBCC-EP858, FBCC-EP920, and FBCC-EP1014 is related to the activation of the Nrf2/HO-1 pathway, we investigated the effect of an HO-1 inhibitor, ZnPP, on ROS production. As shown in Figure 7A,B, H 2 O 2 treatment increased the population of ROS + cells, and treatment with FBCC-EP858, FBCC-EP920, and FBCC-EP1014 reduced the population ( Figure 7A,B). However, treatment with ZnPP reversed the extract-induced reduction in the ROS + cell population, causing an increase in the population. Additionally, DCFDA fluorescence intensity induced by H 2 O 2 was significantly elevated in zebrafish larvae, and treatment with FBCC-EP858, FBCC-EP920, and FBCC-EP1014 attenuated the intensity ( Figure 7C,D). Consistent with the data in Figure 7A,B, treatment with ZnPP reversed the three extract-induced reductions in DCFDA fluorescence intensity, causing a reversal increase. These results suggest that the antioxidant activity induced by FBCC-EP858, FBCC-EP920, and FBCC-EP1014 is related to the activation of the Nrf2/HO-1 pathway.

Discussion
Oxidative stress is a condition that arises due to the imbalance between the produc-

Discussion
Oxidative stress is a condition that arises due to the imbalance between the production of ROS and the antioxidant defense system in the body. ROS can be generated endogenously during cellular metabolism or from exogenous sources such as exposure to cigarette smoke, ozone exposure, hypoxia, ionizing radiation, and heavy metal ions [37]. Oxidative stress has been implicated in various age-related and metabolic diseases, and targeting oxidative stress is a promising strategy for the prevention and treatment of this disease [1,38,39]. Antioxidant therapy, including plant-derived antioxidant agents, has been used in clinical trials due to its low toxicity and relevance to overall health and diseases [40]. In this study, we tested the DPPH radical scavenging activity of 184 plant extracts and identified three extracts (FBCC-EP858 from A. multiflora, FBCC-EP920 from A. coccinea, and FBCC-EP1014 from S. gracilistyla) that possessed powerful antioxidant activity. A. multiflora, A. coccinea, and S. gracilistyla are plants known for their traditional medicinal uses, and their antioxidant properties were previously unknown or not well-characterized [21,27]. We found that these extracts enhanced antioxidant activity both in vitro and in vivo by activating the Nrf2/HO-1 pathway. Overall, the study provides insight into the potential of plant-derived antioxidants as a therapeutic approach for oxidative stress-mediated human diseases.
Plant-derived natural antioxidants have been recognized as a potential therapeutic approach to reducing oxidative stress and associated diseases [17]. Cui et al. reported that plant-derived antioxidants protected the nervous system from aging by alleviating ROS production [41]. Akbarti et al. demonstrated that antioxidants supplied by foods or herbal supplements attenuated oxidative stress-associated chronic and degenerative diseases, such as cardiovascular, autoimmune, and neuronal diseases [42]. Additionally, the antioxidant properties of plant-derived compounds have received a great deal of attention in cosmetics because these antioxidants protect skin fibroblasts from ultraviolet-mediated ROS production and inhibit melanin biosynthesis [43,44]. In this regard, natural products targeting Nrf2 have received significant attention due to their potential to activate the antioxidant and detoxifying enzymes against oxidative stress and prevent ROS-mediated diseases such as inflammation, diabetes, and cancers [20,45]. In preclinical studies, some agents stimulating the Nrf2-HO-1 axis have shown promise in limiting inflammatory and oxidative stress biomarkers, and clinical trials in humans are underway [11]. The three powerful plant-derived antioxidant extracts (FBCC-EP858, FBCC-EP920, and FBCC-EP1014) identified in this study could potentially be used as antioxidant supplements due to their reported anti-inflammatory and antioxidant properties. Particularly, it was reported that A. multiflora contains anti-inflammatory and antioxidant compounds, such as rhamnetin and 3-rhamnosyl glucoside [26], and A. coccinea contains affluent flavonoid glycosides [46]. S. gracilistyla was proven to possess free radical scavenging and skin-whitening properties from the extracts of the stem [29]. Further analysis of the metabolites contained in each extract is necessary to identify their potential effects.

Conclusions
In conclusion, we selected three extracts with the strongest in vitro antioxidant activity from 184 plant extracts and verified their antioxidant properties on cells and zebrafish larvae. Furthermore, we confirmed that these three extracts exhibited powerful antioxidant effects through the activation of Nrf2 and HO-1. These findings suggest that three plant extracts have the potential as natural antioxidant supplements to prevent and treat various diseases caused by oxidative stress. Additionally, we conducted several bioactive assays on the extracts, revealing equivalent levels of antioxidant activity but differing levels of inhibition or activity in lipoxygenase, collagenase, α-glucosidase, acetaldehyde dehydrogenase, and acetylcholine esterase (Supplementary Table S2). Further studies are needed to investigate the specific phytochemicals responsible for the antioxidant activity and to elucidate the molecular mechanisms underlying the protective effects of these extracts. Additionally, clinical trials are required to confirm their safety and efficacy in humans. Nevertheless, these results provide promising evidence for the potential of natural products as alternative sources of antioxidant therapy.