High Resolution Mass Spectroscopy-Based Secondary Metabolite Profiling of Nymphaea nouchali (Burm. f) Stem Attenuates Oxidative Stress via Regulation of MAPK/Nrf2/HO-1/ROS Pathway

The secondary metabolites profiling of Nymphaea nouchali stem (NNSE) extract was carried out using a high-resolution mass spectroscopic technique. The antioxidant effects of NNSE, as well as the underlying mechanisms, were also investigated in tert-butyl hydroperoxide (t-BHP)-stimulated oxidative stress in RAW264.7 cells. Tandem mass spectroscopy with (−) negative mode tentatively revealed the presence of 54 secondary metabolites in NNSE. Among them, phenolic acids and flavonoids were predominant. Phenolic acids (brevifolincarboxylic acid, p-coumaroyltartaric acid, niazinin B, lalioside, 3-feruloylquinic acid, and gallic acid-O-rutinoside), flavonoids (elephantorrhizol, apigenin-6-C-galactoside 8-C-arabinoside, and vicenin-2), sialic acid (2-deoxy-2,3-dehydro-N-acetylneuraminic acid), and terpenoid (α-γ-onoceradienedione) were identified in NNSE for the first time. Unbridled reactive oxygen species/nitrogen species (ROS/RNS) and redox imbalances participate in the induction and development of many oxidative stress-linked diseases. The NNSE exhibited significant free radical scavenging capabilities and was also able to reduce t-BHP-induced cellular generation in RAW264.7 cells. The NNSE prevented oxidative stress by inducing the endogenous antioxidant system and the levels of heme oxygenase-1 (HO-1) by upregulating Nrf2 through the modulation of mitogen-activated protein kinases (MAPK), such as phosphorylated p38 and c-Jun N terminal kinase. Collectively, these results indicate that the NNSE exhibits potent effects in preventing oxidative stress-stimulated diseases and disorders through the modulation of the MAPK/Nrf2/HO-1 signaling pathway. Our findings provide new insights into the cytoprotective effects and mechanisms of Nymphaea nouchali stem extract against oxidative stress, which may be a useful remedy for oxidative stress-induced disorders.


Introduction
Reactive oxygen and nitrogen species (ROS/RNS) are important for maintaining cellular homeostasis, but unbridled ROS/RNS and redox imbalances participate in the induction

Plant Materials and Extraction
N. nouchali stem (NNS) plants were collected from Khulna in Bangladesh, identified by the National Herbarium of Bangladesh, and stored in our laboratory for future reference. To prepare an ethanolic extract, 50 g of coarse NNS powder was extracted with 500 mL of ethanol under reflux for 1 h (three times). The mixture was filtered, dried in a rotary vacuum evaporator, lyophilized, and stored at −20 • C. The ethanolic extract residue (NNSE) was dissolved in deionized H 2 O to obtain a 30 mg/mL stock solution. For ESI-MS/MS analysis, a stock solution (10 mg/mL) was prepared in 100% HPLC-grade ethanol and then diluted using a 70% ethanolic solution. Homogenization was carried out by vortexing for 1 min, followed by sonication for 5 min in a sonication bath (Powersonic 410, Hwashin Technology Co., Seoul, Korea) [11].

Electro Spray Ionization (ESI)-Mass Spectroscopy Analysis
A Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA) was used for negative-mode ESI-MS experiments. A 500 µL graduated syringe (Hamilton Company Inc., Reno, NV, USA) and a syringe pump (Model 11, Harvard, Holliston, MA, USA) were used to immerse the sample through the ESI source at 15 µL/min. The characteristic negative-mode ESI-MS conditions were a mass resolution of 140,000 (full width at half maximum, FWHM), sheath gas flow rate of 5, sweep gas flow rate of 0, auxiliary gas flow rate of 0, spray voltage of 4.20 kV, capillary temperature of 320 • C, S-lens Rf level, and automatic gain control of 5 E 6. Nitrogen gas with high purity (99.99%) was used for the sheath, auxiliary, and sweep gas flow. For negative modes, external calibrations were performed using a Pierce Velos solution (Thermo Fisher Scientific) in the ESI source.
Three different stepped normalized collision energies (NCE = 10, 30, and 40) were used to perform MS/MS experiments with the same instrument. The instrument was operated in (−) mode, and the other operative parameters for MS/MS experiments were as follows: sheath gas flow rate of 10, auxiliary gas flow rate of 0 (arbitrary units), spray voltage of 3.50 kV, capillary temperature of 320 • C, and an S-lens Rf level of 50 [2,12].

Data Processing
Mass spectrum data acquired from the orbitrap mass spectrometer were organized using Xcalibur 3.1 along with foundation 3.1 (Thermo Fisher Scientific Inc. Rockford, IL, USA). The m/z peaks were tentatively identified by matching their exact (theoretical) masses of deprotonated (M-H) adducts with measured m/z values and ESI-MS/MS fragmentation patterns from an in-house MS/MS database, and online databases such as FooDB (https: //foodb.ca/, accessed date 2 April 2021) and METLIN database (https://metlin.scripps. edu/landing_page.php?pgcontent=mainPage, accessed date 2 April 2021). Compound structures were drawn using ChemDraw Professional 15.0 (PerkinElmer, Waltham, MA, USA).

Radical Scavenging Activity Assays
To assess the free radical scavenging capability of NNSE, DPPH, ABTS, superoxide, and hydroxyl-radical, scavenging assays were carried out using a previously described protocol [4]. Ascorbic acid, quercetin, and gallic acid were treated as standard antioxidants for DPPH, ABTS, and superoxide-and hydroxyl-radical scavenging assays, respectively. The percent inhibition was computed using the following equation: Radical − scavenging activity (% inhibition) =   Abs control − Abs sample Abs control   × 100 (1) 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.
According to the method described by Alam et al. [2], the cupric-reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays were used to define the reducing power capacity and were expressed as an ascorbic acid-equivalent antioxidant value (µM), using an ascorbic acid standard curve.

Cell Culture and Intracellular ROS Generation Assay
RAW 264.7 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10 % FBS and streptomycin-penicillin (100 µg/mL each; Hyclone) at 37 • C and 5% CO 2 . The cells (5 × 10 5 cells/mL) were seeded into 96-well plates for 12 h, followed by treatment with NNSE (1-30 µg/mL) for 24 h with or without t-butyl hydroperoxide (t-BHP). An MTT assay and 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) method was used to evaluate the cellular toxicity and generation of t-BHP-induced ROS as a cellular oxidative stress biomarker, respectively, as described previously [2].

Western Blot Analysis
A radioimmunoprecipitation assay buffer was used to lyse and harvest the cells. A nuclear and cytoplasmic extraction kit (Sigma-Aldrich Co. St. Louis, MO, USA) was used to extract the nuclear and cytosolic protein. Protein content was quantified using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Equivalent amounts (30 µg) of protein were subjected to western blot analysis as described in our previous report using various antibodies (Supplementary Data, Table S1) [2].

Statistical Analysis
Data were expressed as the mean ± standard deviation (SD; n = 3). One-way analysis of variance, followed by Tukey's multiple-comparisons test, was performed using SigmaPlot software (SigmaPlot, Ver 12.5, Systat Software, Inc., Chicago, IL, USA) to determine the significance for the differentiation and fusion indices. The hierarchy of activity (a < b < c) indicates statistical differences between the means. p < 0.05 was considered statistically significant.

Secondary Metabolite Profiling of NNSE
Secondary metabolites of NNSE were identified and characterized by ESI-MS/MS in negative mode, which is a standard procedure for revealing the structure of compounds from extracts. As shown in Table 1, there were 54 compounds identified on the basis of their MS 2 information provided by the precursor ion's mass, their fragments, known fragmentation patterns for the given classes of compounds, neutral mass loss, comparison with the available literature, and searching in online databases. The identified compounds were classified into phenolic acids, flavonoids, amino acids, dicarboxylic acids, fatty acids, sugar, flavoring agents, sialic acid, terpenoid, and others. Phenolic acid glycoside began its fragmentation by segmentation of the glycosidic bond and provided the m/z of the phenolic acid and the corresponding loss of sugar molecule mass (−162 Da). Moreover, phenolic acid produced its characteristic product ion by losing the neutral mass of hydroxyl (−18 Da), methyl (−15 Da), or carboxylic (−44 Da) moiety [13]. Compound 1, 2, 3, 4, and 5 yielded a molecular ion peak [M-H] − at m/z 135.0444, 137.0227, 153.0186, 167.0344, and 169.0134 and fragmentation ions at m/z 91.05, 93.03, 106.02, 123.01, and 125.02, respectively, because of neutral loss of CO 2 confirmed the presence of methyl benzoic acid, salicylic acid, protocatechuic acid, vanillic acid, and gallic acid, respectively [12][13][14]. Compound 6 produced a precursor ion peak [M-H] − at m/z 183.0290 and diagnostic ions at m/z 139.0401 and 123.0088 by losing a CO 2 and CH 3 group and was identified as methoxygallate [15]. Compound 7 was confirmed as brevifolincarboxylic acid having a molecular ion [M-H] − at m/z 291.0141 and yielding a daughter ion at m/z 247.02 by loss of CO 2 and m/z 219, and 191.04 from successive losses of CO ( Figure 1A). Compound 10 yielded an ion [M-H] − at m/z 305.0300, generating a characteristic peak at m/z 273.01 from a loss of CH 3 OH, whereas other quasi-molecular ions were formed from a consecutive loss of CO and was identified as methyl brevifolincarboxylic acid [13,16].      [2,12]. Based on the literature [2,11,12] and compared with data from the FooDB  46,47,48,49,50,51,52,53, and 54 were identified as glyceric acid, sorbic acid, salicylaldehyde, methyl benzoic acid, hydroxynicotinic acid, ribonic acid, shikimic acid, quinic acid, and N-undecanoylglycine.

Radical Scavenging Activities of NNSE Extracts
The antioxidant activities of phytochemicals involve various molecular mechanisms. Thus, various methods should be used to assess the antioxidant potential of plant extracts. In this study, the antioxidant potential of NNSE was analyzed using DPPH-, ABTS-, superoxide-and hydroxyl-radical scavenging assays, along with FRAP, CUPRAC, and ORAC assays. As shown in Figure 4A,B, NNSE exhibited a dose-dependent and significant DPPH-and superoxide-radical scavenging potential with IC 50 values of 44.59 ± 1.29 µg/mL and 18.50 ± 0.40 µg/mL, whereas the positive control, ascorbic acid, had an IC 50 value of 16.58± 0.24 µg/mL in the DPPH-radical scavenging assay and gallic acid had an IC 50 value of 14.34 ± 0.70 µg/mL in the superoxide-radical scavenging assay, respectively. These results suggest that NNSE exhibits antioxidant potential through a hydrogen atom transfer mechanism. Moreover, Figure 4C,D show that NNSE has a significant and concentration-dependent ability to scavenge ABTS-and hydroxyl-radical with IC 50 values of 73.51 ± 1.07 µg/mL and 9.48 ± 0.36 µg/mL, respectively, whereas ascorbic acid and quercetin (positive control) had an IC 50 value of 14.49 ± 0.55 µg/mL for the ABTS-radical scavenging assay and 4.13 ± 0.06 µg/mL for the hydroxyl-radical scavenging assay. Based on these results, we speculated that NNSE also uses a single electron transfer mechanism to perform its antioxidant activity. Furthermore, CUPRAC, FRAP, and ORAC assays were performed to assess the reducing capability of NNSE. NNSE exhibited 7.42 ± 0.10 and 11.69 ± 0.26 µM ascorbic acid equivalents reducing power in the CUPRAC and FRAP assays at 100 µg/mL, respectively ( Figure 4E). NNSE exhibited 7.02 ± 0.56 mg Trolox equivalents/g antioxidant potential at 100 µg/mL in the ORAC assay ( Figure 4F).

Attenuation of t-BHP Induced Cellular Oxidative Stress by NNSE
Cellular oxidative stress was induced by t-BHP, a short-chain lipid peroxide analog, and is widely accepted as a model to evaluate the alteration of cellular mechanisms caused by oxidative stress in cells and tissues [40]. As shown in Figure 5A, treatment with t-BHP caused significant cell death, whereas pretreatment with NNSE and gallic acid attenuated the cellular toxicity at nontoxic doses (supplementary data S2). Furthermore, Figure 5B shows that NNSE exhibits the capability of mitigating the production of cellular ROS in a dose-dependent manner similar to that of gallic acid (50 µg/mL). and is widely accepted as a model to evaluate the alteration of cellular mechanisms caused by oxidative stress in cells and tissues [40]. As shown in Figure 5A, treatment with t-BHP caused significant cell death, whereas pretreatment with NNSE and gallic acid attenuated the cellular toxicity at nontoxic doses (supplementary data S2). Furthermore, Figure 5B shows that NNSE exhibits the capability of mitigating the production of cellular ROS in a dose-dependent manner similar to that of gallic acid (50 μg/mL). Superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione (GSH) are considered as first-line antioxidant defense systems and play an important role in maintaining the cellular redox environment [41]. As demonstrated in Figure 5C and 3D, t-BHP treatment significantly ameliorated the levels of SOD1, catalase, and GPx-1 protein, whereas pretreatment with NNSE significantly reversed this trend in a concentration-dependent manner. The endogenous antioxidant protein levels were also induced Superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione (GSH) are considered as first-line antioxidant defense systems and play an important role in maintaining the cellular redox environment [41]. As demonstrated in Figure 5C,D, t-BHP treatment significantly ameliorated the levels of SOD1, catalase, and GPx-1 protein, whereas pretreatment with NNSE significantly reversed this trend in a concentration-dependent manner. The endogenous antioxidant protein levels were also induced by gallic acid in the t-BHP model. These data support the enhancement of antioxidant enzyme proteins by NNSE, resulting in the maintenance of the cellular redox balance and attenuating oxidative stress-induced cell death. Further evidence has revealed that polyphenolic compound-rich medicinal plants/food can ameliorate SOD1, CAT, and GPx activity to minimize oxidative stress [42,43]. Our previous report revealed that a methanolic extract of the N. nouchali flower and leaves increased the transcription and translation of the SOD, CAT, and GPx enzymes [4,29]. Furthermore, mounting evidence suggests that the administration of N. alba, N. pubescens, and N. stellata flowers attenuates hepatotoxicity by augmenting the activity of endogenous enzymes such as SOD, CAT, and GPx [33,44,45].
Multiple lines of evidence recommend that polyphenolics, such as protocatechuic acid, vanillic acid, gallic acid, naringenin, 5-O-caffeoylquinic acid, catechin, taxifolin, and vicenin-2 have the capability to augment the endogenous antioxidant system, leading to cellular protection from oxidative stress [46][47][48][49][50][51][52]. Therefore, it is hypothesized that the augmentation of first-line antioxidant enzymes by NNSE, resulting from an abundance of phenolics and flavonoids, may contribute to the beneficial effects of NNSE against oxidative stress.

NNSE Induces Phase II Enzymes through Nrf2 Regulation
Several reports have indicated that phase II detoxifying/antioxidant enzymes, such as heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) quinone oxidoreductase-1 (NQO1), play an important role in detoxifying ROS and is modulated by Nrf2, a central regulator of ARE-driven antioxidant gene expression [53]. Accordingly, to judge whether NNSE has the potential to boost phase II antioxidant enzymes through the regulation of Nrf2, immunoblotting analysis was performed. As shown in Figure 6A, NNSE treatment significantly increased the levels of HO-1 and NQO1 protein in a dose-dependent manner, similar to that of gallic acid. In a resting state, Nrf2 activity is firmly controlled in the cytosol by Kelch-like ECH associating protein 1 (Keap1) as an adaptor protein for Cullin-3 (Cul3)-dependent E3 ubiquitin ligase enzyme, which is responsible for Nrf2 ubiquitination and degradation [4,53]. Thus, western blot analysis was performed to evaluate the function of NNSE in preventing cytosolic Nrf2 degradation and enhancing the nuclear translocation of Nrf2. Figure 6B indicates the time-dependent attenuation of Keap1 protein in the cytoplasm in association with Nrf2 enrichment by NNSE treatment, which peaked at 4 h. In addition, NNSE treatment significantly increased nuclear Nrf2 content in association with minimized cyto-Nrf2 levels ( Figure 6C). Furthermore, to validate the potential of NNSE to stimulate phase II antioxidant enzymes by modulating Nrf2, knocked-down expression of Nrf2 using a small interfering RNA (siRNA) technique was performed (Supplementary Figure S3). Nrf2 protein levels were considerably reduced by si-Nrf2 treatment, which was not restored even after treatment with NNSE ( Figure 6D). Furthermore, as shown in Figure 6E,F, si-Nrf2 treatment significantly reduced the levels of HO-1 and NQO1 proteins, and NNSE treatment was unable to normalize basal HO-1 and NQO1 protein levels. This observation indicates that NNSE may disrupt the proteasomal degradation of Nrf2 in the cytoplasm by Keap1 and may facilitate the nuclear translocation of Nrf2, resulting in upregulation of HO-1 expression.

NNSE Activates MAPKs and Regulates Nuclear Translocation of Nrf2, Leading to Reduced Oxidative Stress
Many studies have demonstrated that phosphorylation of MAPKs, such as ERK, JNK, and p38, can positively regulate phase II antioxidant enzyme expression by activating the ARE/Nrf2 mechanism in various cell types [54,55]. Here, immunoblot analysis was performed to identify the signaling pathways participating in the regulation of phase II antioxidant enzyme activity in NNSE-treated cells. As presented in Figure 7A, NNSE treatment significantly enhanced the phosphorylation of p38 and JNK from 0.5 to 2 h, with a peak at 1 h, whereas NNSE treatment did not result in ERK phosphorylation. To determine whether p38 and JNK can regulate HO-1 expression by modulating Nrf2, cells were treated with each specific inhibitor before stimulation with NNSE. As shown in Figure 7B, both the p38 and JNK inhibitor (SB239063 and SP600125, respectively) markedly suppressed HO-1 and Nrf2 expression, which was increased by NNSE treatment. This indicates that p38 and JNK phosphorylation may regulate the induction of HO-1 through modulation of Nrf2 signaling in RAW 264.7 cells. Furthermore, to validate the role of MAPK/Nrf2/HO-1 signaling in the reduction in oxidative stress, cells were treated with each specific inhibitor before stimulation with NNSE, and intracellular ROS generation induced by t-BHP treatment was measured. Interestingly, t-BHP stimulation significantly increased cellular ROS generation, which was strongly ameliorated by NNSE ( Figure 7C,D, third column), whereas treatment with p38 and JNK inhibitors (SB239063 and SP600125, respectively) reversed this trend ( Figure 7C). Moreover, treatment with the HO-1 inducer, CoPP, strongly and significantly abolished t-BHP-induced generation of cellular ROS, and this trend was reversed by HO-1 and Nrf2 inhibitors (SnPP and Brusatol, respectively) ( Figure 7D). These data strongly suggest that phosphorylation of p38 and JNK by NNSE can regulate Nrf2/HO-1 signaling, which accounts for cell subsistence against oxidative stress in RAW 264.7 cells. Several studies have revealed that extracts from various medicinal plants/food, such as N. nouchali flower and leaves, N. alba, N. lotus, N. pubescens, and N. stellata flower extract, result in the activation of Nrf2-mediated phase II enzyme expression in Raw 264.7 cells [4,29,33]. Furthermore, protocatechuic acid, vanillic acid, gallic acid, naringenin, 5-Ocaffeoylquinic acid, catechin, taxifolin, and vicenin-2 can modulate the Nrf2/ARE/HO-1 signaling cascade and attenuate oxidative stress-mediated kidney and hepatic cell death [46][47][48]52].

NNSE Activates MAPKs and Regulates Nuclear Translocation of Nrf2, Leading to Reduced Oxidative Stress
Many studies have demonstrated that phosphorylation of MAPKs, such as ERK, JNK, and p38, can positively regulate phase II antioxidant enzyme expression by activating the ARE/Nrf2 mechanism in various cell types [54,55]. Here, immunoblot analysis was performed to identify the signaling pathways participating in the regulation of phase II antioxidant enzyme activity in NNSE-treated cells. As presented in Figure 7A, NNSE treatment significantly enhanced the phosphorylation of p38 and JNK from 0.5 to 2 h, with a peak at 1 h, whereas NNSE treatment did not result in ERK phosphorylation. To determine whether p38 and JNK can regulate HO-1 expression by modulating Nrf2, cells were cells were treated with NNSE (50 µg/mL) for various times, and the levels of cytoplasmic Keap1 and Nrf2 protein were confirmed by western blot analysis. (C) Cells were treated with NNSE and gallic acid for 4 h, and nuclear translocation of Nrf2 was measured by immunoblotting assay. Cells were treated with NNSE in the presence or absence of si-Nrf2 RNA, and the level of (D) Nrf2, and (E) HO-1, and NQO1 protein was measured by western blot analysis. (F) The relative expression of HO-1 and NQO1 protein was quantified by Image J software. Values are expressed as the mean ± SD (n = 3), and different letters are considered statistically significant (p < 0.05) to one another. GA, gallic acid. 7C,D, third column), whereas treatment with p38 and JNK inhibitors (SB239063 and SP600125, respectively) reversed this trend ( Figure 7C). Moreover, treatment with the HO-1 inducer, CoPP, strongly and significantly abolished t-BHP-induced generation of cellular ROS, and this trend was reversed by HO-1 and Nrf2 inhibitors (SnPP and Brusatol, respectively) ( Figure 7D). These data strongly suggest that phosphorylation of p38 and JNK by NNSE can regulate Nrf2/HO-1 signaling, which accounts for cell subsistence against oxidative stress in RAW 264.7 cells. Figure 7. Activation of p38 and JNK by NNSE results in Nrf2 translocation. (A) RAW 264.7 cells were treated with NNSE (50 µg/mL) for various times, and kinase activity was determined by immunoblot assay. (B) Cells were treated with NNSE and specific inhibitors, SB239063 (p38 inhibitor) and SP600125 (JNK inhibitor), for 1 h, and Nrf2 and HO-1 protein levels were analyzed by western blot analysis. (C) Cells were treated with NNSE and the specific inhibitors, SB239063 and SP600125, and cellular ROS generation was quantified by the DCFH-DA methods. (D) Cells were treated with NNSE and CoPP (HO-1 activator), SnPP (HO-1 inhibitor), and brusatol (Nrf2 inhibitor), and cellular ROS generation was quantified by the DCFH-DA methods. Values are expressed as the mean ± SD (n = 3), and different letters are considered statistically significant (p < 0.05) to one another.

Conclusions
Oxidative stress is considered one of the major contributing factors to the development and progression of several acute and chronic disorders. Thus, it is anticipated that antioxidants may have valuable health effects as prophylactic agents. Here, ESI-MS/MS analysis revealed the abundance of secondary metabolites in NNSE and demonstrated excellent antioxidant activity in cell-free assays and at the cellular level. Furthermore, Figure 7. Activation of p38 and JNK by NNSE results in Nrf2 translocation. (A) RAW 264.7 cells were treated with NNSE (50 µg/mL) for various times, and kinase activity was determined by immunoblot assay. (B) Cells were treated with NNSE and specific inhibitors, SB239063 (p38 inhibitor) and SP600125 (JNK inhibitor), for 1 h, and Nrf2 and HO-1 protein levels were analyzed by western blot analysis. (C) Cells were treated with NNSE and the specific inhibitors, SB239063 and SP600125, and cellular ROS generation was quantified by the DCFH-DA methods. (D) Cells were treated with NNSE and CoPP (HO-1 activator), SnPP (HO-1 inhibitor), and brusatol (Nrf2 inhibitor), and cellular ROS generation was quantified by the DCFH-DA methods. Values are expressed as the mean ± SD (n = 3), and different letters are considered statistically significant (p < 0.05) to one another.

Conclusions
Oxidative stress is considered one of the major contributing factors to the development and progression of several acute and chronic disorders. Thus, it is anticipated that antioxidants may have valuable health effects as prophylactic agents. Here, ESI-MS/MS analysis revealed the abundance of secondary metabolites in NNSE and demonstrated excellent antioxidant activity in cell-free assays and at the cellular level. Furthermore, NNSE has the potential to reduce the oxidative burden by attenuating the first-line antioxidant system and activating the MAPK-Nrf2-HO-1 signaling cascade, resulting in the suppression of cellular ROS generation. Our findings provide new insights into the cytoprotective effects and mechanisms of Nymphaea nouchali stem extract against oxidative stress, which may be a useful remedy for oxidative stress-induced disorders.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox10050719/s1, Figure S1: Total phenolic and flavonoid content of NNSE, Figure S2: Effect of NNSE on cell viability in RAW264.7 cells, Figure S3: Nrf2 expression using si-RNA in RAW264.7 cells, Table S1. List of the primary antibodies used in the study.