Simultaneous Study of Anti-Ferroptosis and Antioxidant Mechanisms of Butein and (S)-Butin

To elucidate the mechanism of anti-ferroptosis and examine structural optimization in natural phenolics, cellular and chemical assays were performed with 2′-hydroxy chalcone butein and dihydroflavone (S)-butin. C11-BODIPY staining and flow cytometric assays suggest that butein more effectively inhibits ferroptosis in erastin-treated bone marrow-derived mesenchymal stem cells than (S)-butin. Butein also exhibited higher antioxidant percentages than (S)-butin in five antioxidant assays: linoleic acid emulsion assay, Fe3+-reducing antioxidant power assay, Cu2+-reducing antioxidant power assay, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-trapping assay, and α,α-diphenyl-β-picrylhydrazyl radical (DPPH•)-trapping assay. Their reaction products with DPPH• were further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Butein and (S)-butin produced a butein 5,5-dimer (m/z 542, 271, 253, 225, 135, and 91) and a (S)-butin 5′,5′-dimer (m/z 542, 389, 269, 253, and 151), respectively. Interestingly, butein forms a cross dimer with (S)-butin (m/z 542, 523, 433, 419, 415, 406, and 375). Therefore, we conclude that butein and (S)-butin exert anti-ferroptotic action via an antioxidant pathway (especially the hydrogen atom transfer pathway). Following this pathway, butein and (S)-butin yield both self-dimers and cross dimers. Butein displays superior antioxidant or anti-ferroptosis action to (S)-butin. This can be attributed the decrease in π-π conjugation in butein due to saturation of its α,β-double bond and loss of its 2′-hydroxy group upon biocatalytical isomerization.


Introduction
Ferroptosis is a form of cell death activated by iron oxidation [1,2]. Ferroptosis regulation offers a new strategy for the treatment of various diseases, including cancer [3,4] and Alzheimer's disease [5,6]. Recently, ferroptosis inhibitors have been reported, including dietary flavones, such as baicalein [5], that are bio-synthesized from dietary chalcones (especially 2 -OH chalcone) [7]. Our research suggests that the biosynthesis of flavone from 2 -OH chalcone is an antioxidant reduction process [8]. As a result, we postulate that 2 -OH chalcone will be a more effective ferroptosis inhibitor compared to its corresponding flavone. However, no direct evidence has been reported previously. In this study, we used erastin to induce ferroptosis in bone marrow-derived mesenchymal stem cells (BMSCs), which are considered important seed cells to treat degenerative diseases, such as geriatric diseases [16], by transplantation engineering. These have enhanced the clinical characteristics of the study.
In addition to comparing anti-ferroptosis bioactivities for butein and (S)-butin, anti-ferroptosis mechanisms for both will also be discussed. Recent research has indicated that ferroptosis regulation can be affected by the cellular redox environment [17]; anti-ferroptosis was closely associated with radical-trapping antioxidants [18][19][20] or even antioxidant chemical elements (e.g., selenium [21]). This suggests that the anti-ferroptosis mechanism may be mediated by antioxidants. Therefore, in this study, butein and (S)-butin were mixed with α,α-diphenyl-β-picrylhydrazyl radical (DPPH • ), an antioxidant reaction probe, and the product mixture was further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). This cutting-edge approach provides precise m/z values and offers reliable information regarding the anti-ferroptosis mechanism of natural phenolics.
Since LPO affects ferroptosis, butein and its isomer (S)-butin were investigated for LPO inhibition using a linoleic acid system. As shown in Supplementary Materials S1, the two isomers increased the LPO inhibition percentages with increasing concentration. Both the inhibition and formation of LPO are reported to stem from electron-transfer (ET) reactions [37][38][39][40][41]. To explore ET, the two were further investigated using the Fe 3+ -reducing antioxidant power (FRAP) assay and Cu 2+ -reducing antioxidant power (CUPRAC) assay. As shown in Supplementary Materials S1, a direct correlation was found between the dose of butein or (S)-butin and the relative FRAP and CUPRAC percentages, indicating that both butein and (S)-butin had ET antioxidant potentials [42][43][44].
Several reports have suggested that ET is always accompanied by H + -transfer (proton transfer) in biological systems [45][46][47][48]. To evaluate proton transfer with butein and (S)-butin, we used a PTIO • scavenging assay established by our team [49]. In the assay, varying IC 50 values (the concentration with 50% radical inhibition or relative reducing power) were observed for the two isomers at different pH values (4.5, 6.0, and 7.4, shown in Table 1, and Supplementary Materials S1). Such a pH effect indicates that, in aqueous solution, PTIO • radical scavenging could involve H + (i.e., proton). Thus, there was a proton transfer (PT) pathway. The involvement of the PT pathway can be attributed to a fact that phenolics always exhibit weak acidity. In fact, (S)-butin had a pKa value of 7.11 at 3 -OH [50,51]. On the other hand, the PTIO • scavenging reaction in pH 4.5 aqueous solution is indicated as an ET process [49]. The effectiveness of the two isomers at pH 4.5 implies that they also had the potential for ET. In short, the antioxidant process of butein and (S)-butin is considered to be a single process comprising ET accompanied by PT (designated as ET plus PT). The IC 50 value is defined as the concentration with 50% radical inhibition or relative reducing power, calculated by linear regression analysis, and expressed as the mean ± SD (n = 3). The linear regression was analyzed by using Origin 6.0 professional software. The IC 50 values with different superscripts (a or b) among the two isomers are significantly different (p < 0.05). Trolox and l-Ascorbic Acid were used as the positive control. All dose-dependent curves are given in Supplementary Materials S1. N.D., no detected.
A similar difference was also observed regarding the antioxidant bioactivities between butein and (S)-butin. As seen in Supplementary Material 1, both butein and (S)-butin dose-dependently increased the percentages in all five antioxidant assays, including linoleic acid emulsion assay, CUPRAC assay, Fe 3+ -reducing assay, PTIO • radical-trapping assay, and DPPH • radical-trapping assay. Linoleic acid however was one of polyunsaturated fatty acids and has been recently suggested as one target of ferroptosis [84,85]. The effectiveness of butein and (S)-butin in linoleic acid emulsion assay means that the anti-ferroptosis effect of butein and (S)-butin may be related to the LPO-inhibition on linoleic acid. However, as seen in Table 1, butein had lower IC 50 values than (S)-butin in the five antioxidant assays. This indicates that, butein is a stronger LPO-inhibitor or antioxidant than (S)-butin. That their relative anti-ferroptotic activities parallel their relative antioxidant (or LPO-inhibitory) levels further supports the above hypothesis, namely, that the anti-ferroptotic action of butein and (S)-butin occurs via antioxidant mechanisms.
The difference in their activities is likely associated with CHI-mediated isomerization. As shown in Figure 1, isomerization from butein to (S)-butin is a cyclization reaction. Cyclization results in loss of the 2 -OH and thus lowers the antioxidant level [69,86]. More importantly, cyclization also causes saturation of the α,β-double bond. After the α,β-double bond is saturated, the β-carbon has an sp 3 -hybridized tetrahedral configuration. As such, the molecule is no longer planar ( Figure 8). Loss of the exocyclic double bond and loss of molecular planarity reduces the region of π-π conjugation. From resonance theory, saturation of the exocyclic α,β-double bond prevents stabilization of the free radical intermediate after the HAT reaction [62]. We previously demonstrated that these changes greatly decrease the antioxidant level [87,88].

Extraction and Culture of Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs)
The bone marrow-derived mesenchymal stem cells (BMSCs) were extracted and cultured using our routine experimental protocols [89]. Briefly, male Sprague-Dawley rats were collected, and the adherent soft tissues were removed. Both ends of the bones were cut away from the diaphysis with bone scissors. The bone marrow plugs were hydrostatically expelled from the bones by insertion of needles fastened to 10-mL syringes filled with complete medium; the needles were inserted into the distal ends of femora and proximal ends of the tibiae, and the marrow plugs expelled from the opposite ends. The cells were centrifuged and resuspended twice in complete medium; 5 × 10 7 cells in 7-10 mL of complete medium were then introduced into 100-mm culture dishes. Two days later, the medium was changed and the nonadherent cells were discarded. The adherent cells were cultured in SD rat bone marrow mesenchymal stem cell complete medium with glucose, supplemented with 10% (v/v) fetal bovine serum. The cultured cells were seeded and grouped to study the prevention of erastin-induced ferroptosis of butin and (S)-butein.

Prevention of Erastin-Induced Ferroptosis in BMSCs
The erastin-induced ferroptosis model of BMSCs was created based on the recent literature [19,20] with modifications. To measure the anti-ferroptosis bioactivities of butin and (S)-butein, three assays were applied in the study. The three assays referred to the C11-BODIPY assay and flow cytometric assay.
The C11-BODIPY assay was used to characterize the degree of lipid peroxidation and performed using the method [90,91]. In brief, the cultured BMSCs mentioned were seeded at 1 × 10 6 cells per well into 12-well plates. After adherence for 24 h, BMSCs were divided into control, model, and sample groups. In the control group, BMSCs were incubated for 12 h in Stel Basal medium. In the model and sample groups, BMSCs were incubated in the presence of erastin (20 µM). After incubation for 12 h, the mixture of erastin and medium was removed. The BMSCs in the model group were incubated for 12 h in Stel Basal medium while BMSCs in the sample group were incubated for 12 h in Stel Basal medium with the indicated 30 µM sample concentrations. The incubated cells were determined using the fluorescent probe C11-BODIPY (Invitrogen, Molecular Probes). Cells were incubated for 30 min prior to analysis with C11-BODIPY (2.5 µM). Photos were taken under a fluorescence microscope.
The flow cytometric assay was conducted according to previous methods [92][93][94][95]. In brief, the cultured BMSCs (Section 3.2) were seeded at 1 × 10 6 cells per well into 96-well plates. They were washed twice with cold PBS, and then cells were resuspended in 1 × Binding buffer at a concentration of 1 × 10 6 cells/mL. Then, 100 µL of the solution (1 × 10 5 cells) was transferred to a 5-mL culture tube, and 5 µL of FITC Annexin V and 5 µL PI was added. The cells were gently vortexed and incubated for 15 min at room temperature in the dark, and 400 mL of 1×Binding Buffer were added to each tube after adherence for 12 h. BMSCs were divided into control, model, and sample groups. The three groups were analyzed by flow cytometry within 1 h. Each sample test was repeated in three independent wells.

Linoleic Acid Emulsion Assay
The anti-lipid peroxidation effects of butin and (S)-butein were investigated using the linoleic acid emulsion assay [96]. Briefly, linoleic acid emulsion was prepared using linoleic acid and Tween 20. Then, 1.5 mL of linoleic acid emulsion were mixed with 0.15 mL of sample methanolic solution (0.4-2.0 mg/mL) and 0.35 mL of 30% ethanol (v/v). The reaction mixture (total 2 mL) was incubated at room temperature for 72 h. Then, 0.15 mL of the mixture were added to 3.65 mL 75% ethanol (v/v), 0.1 mL NH 4 SCN (30%, w/w), and 0.1 mL FeCl 2 (0.02 M in 3.6% HCl). The resulting mixture was measured with a UV-Vis spectrophotometer (Unico 2600A, Shanghai, China) at 500 nm. The inhibition percentage was calculated by the equation: where A 0 is the absorbance of the control without sample, and A is the absorbance of the reaction mixture with sample.

CUPRAC Assay
The CUPRAC assay was adapted from the method proposed by Apak [97], with small modifications, as described by Tian [98]. Twelve microliters of CuSO 4 solution (0.01 M) and 12 µL of ethanolic neocuproine solution (7.5 mM) were added to a 96-well plate and mixed with different concentrations of samples (0-2.0 µg/mL). The total volume was then adjusted to 100 µL with a CH 3 COONH 4 buffer solution (0.1 M), and mixed again to homogenize the solution. The mixture was maintained at room temperature for 30 min, and the absorbance was measured at 450 nm on a microplate reader (Multiskan FC, Thermo Scientific, Shanghai, China). The relative reducing power of the sample was calculated as follows: Relative reducing e f f ect% = where A max is the maximum absorbance, A min is the minimum absorbance, and A is the absorbance of the sample.

FRAP Assay
The FRAP assay was adapted from Benzie and Strain FRAP [99]. Briefly, the FRAP reagent was freshly prepared by mixing 10 mM TPTZ, 20 mM FeCl 3 , and 0.25 M pH 3.6 acetate buffer at 1:1:10 (volume ratio). The test sample (x = 0-10 µL, 0.5 mg/mL) was added to (20 − x) µL of 95% ethanol followed by 80 µL of FRAP reagent. The absorbance was measured at 595 nm after a 30 min incubation at 37 • C using distilled water as the blank. The relative reducing power of the sample compared to the maximum absorbance was calculated using the formula of Section 3.5.

PTIO • Radical-Trapping Assay
The PTIO • -scavenging spectrophotometer assay was conducted in accordance with our method [49,84]. In brief, the test sample solution (x = 0-10 µL, 0.5 mg/mL) was added to (20 − x) µL of methanol, followed by 80 µL of an aqueous PTIO • solution. The aqueous PTIO • solution was prepared using 0.1 mM of phosphate buffer/methanol (1/4, v/v) solution (pH 4.5, 6.0 and 7.4). The mixture was maintained at 37 • C for 2 h, and the absorbance was then measured at 560 nm using a microplate reader (Multiskan FC, Thermo Scientific, Shanghai, China). The PTIO • inhibition percentage was calculated based on the formula of Section 3.5

DPPH • Radical-Trapping Assay
The DPPH • radical trapping was determined as previously described [100]. Briefly, 80 µL of DPPH • solution (0.1 M) were mixed with methanolic sample solutions at the indicated concentration (x = 0-10 µL, 0.5 mg/mL). The total volume of mixture was adjusted to 100 µL, and maintained at room temperature for 1 min, and the absorbance was measured at 519 nm on a microplate reader. The percentage of DPPH • scavenging activity was calculated using the equation of Section 3.4.

UPLC-ESI-Q-TOF-MS Analysis of RAF Products of Two Isomers Interacting with DPPH •
Butein (2 mg/mL) was subjected to a reaction with DPPH • (5 mg/mL) under the previous conditions [101]. A methanol solution of butein was mixed with a methanolic DPPH • solution (5 mg/mL) with a molar ratio of 2:1, and then incubated for 24 h at room temperature. The product mixture was passed through a 0.22-µm filter.
The filtrate was analyzed using the UPLC-ESI-Q-TOF-MS method [63]. In the UPLC-ESI-Q-TOF-MS analysis, a Phenomenex Luna C 18 column (2.1 mm i.d. × 100 mm, 1.6 µm, Phenomenex Inc., Torrance, CA, USA) was used as the chromatographic column. The sample injection volume was 3 µL. The sample (reaction products) in the chromatographic column was eluted by a mobile phase at a flow rate of 0.2 mL/min. The mobile phase, however, consisted of a mixture of methanol (phase A) and 0.1% formic acid water (phase B). The proportion of phase A and phase B was adjusted using a gradient program: 0-2 min, maintain 30% B; 2-10 min, 30%-0% B; 10-12 min, 0%-30% B. The Q-TOF-MS analysis was performed on a Triple TOF 5600 plus mass spectrometer (AB SCIEX, Framingham, MA, USA) equipped with an ESI source, which was run in the negative ionization mode. The scan range was set at 50-1500 Da. The system was run with the following parameters: Ion spray voltage, −4500 V; ion source heater temperature, 550 • C; curtain gas pressure (CUR, N 2 ), 30 psi; nebulizing gas pressure (GS1, Air), 50 psi; Tis gas pressure (GS2, Air), 50 psi. The declustering potential (DP) was set at −100 V, whereas the collision energy (CE) was set at −45 V with a collision energy spread (CES) of 15 V. The above experiment was repeated using (S)-butin (2 mg/mL), instead of butein (2 mg/mL).

Preferential Conformation Analysis by Computational Chemistry and Molecular Weight Calculation
The preferential conformation was analyzed based on force fields by computational chemistry. In brief, the energy minimization of butein and (S)-butin were respectively calculated through molecular mechanics II (MM2) using the Chem3D Pro14.0 program (PerkinElmer, Waltham, MA, USA) [102,103].
The Q-TOF-MS analysis is characterized by highly accurate m/z values, particularly molecular weights. The molecular weight calculation based on the formula is vital for comparison with the m/z values from the Q-TOF-MS analysis. In the present study, the molecular weight calculations were conducted based on the accurate relative atomic masses. The relative atomic masses of C, H, O, and N were 12.0000, 1.007825, 15.994915, and 14.003074, respectively [70].

Statistical Analysis
The results were reported as the mean ± SD of three independent measurements; the IC 50 values were calculated by linear regression analysis, and independent-sample T-tests were performed to compare the different groups [104]. A p value of less than 0.05 was considered statistically significant. The statistical analyses were performed using the SPSS software 17.0 (SPSS Inc., Chicago, IL, USA) for windows. All of the linear regression analyses described in this paper were processed using version 6.0 of the Origin professional software (OriginLab Corporation, Northampton, MA, USA).

Conclusions
2 -Hydroxy chalcone butein and its isomer dihydroflavone (S)-butin inhibit ferroptosis through antioxidant action. Antioxidant action occurs by the HAT mechanism, as evidenced by the presence of the butein dimer and (S)-butin dimer. Interestingly, butein and (S)-butin can interact with each other to produce a cross dimer. The isomers display different antioxidant or anti-ferroptotic levels; this discrepancy can be attributed to biocatalyzed isomerization, which results in the loss of a phenolic -OH and produces a saturated α,β-double bond, impairing π-π conjugation in (S)-butin.

Conflicts of Interest:
The authors declare that they have no competing interests.

Abbreviations
The following abbreviations are used in this manuscript: