Extract of Acanthopanax senticosus and Its Components Interacting with Sulfide, Cysteine and Glutathione Increase Their Antioxidant Potencies and Inhibit Polysulfide-Induced Cleavage of Plasmid DNA

Aqueous root extract from Acanthopanax senticosus (ASRE) has a wide range of medicinal effects. The present work was aimed at studying the influence of sulfide, cysteine and glutathione on the antioxidant properties of ASRE and some of its selected phytochemical components. Reduction of the 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (●cPTIO) stable radical and plasmid DNA (pDNA) cleavage in vitro assays were used to evaluate antioxidant and DNA-damaging properties of ASRE and its individual components. We found that the interaction of ASRE and its two components, caffeic acid and chlorogenic acid (but not protocatechuic acid and eleutheroside B or E), with H2S/HS−, cysteine or glutathione significantly increased the reduction of the ●cPTIO radical. In contrast, the potency of ASRE and its selected components was not affected by Na2S4, oxidized glutathione, cystine or methionine, indicating that the thiol group is a prerequisite for the promotion of the antioxidant effects. ASRE interacting with H2S/HS− or cysteine displayed a bell-shaped effect in the pDNA cleavage assay. However, ASRE and its components inhibited pDNA cleavage induced by polysulfides. In conclusion, we suggest that cysteine, glutathione and H2S/HS− increase antioxidant properties of ASRE and that changes of their concentrations and the thiol/disulfide ratio can influence the resulting biological effects of ASRE.


Introduction
Acanthopanax senticosus (Rupr. et Maxim.) Maxim. (AS), synonymous with Eleutherococcus senticosus, and its extract are widely used in countries such as China, Korea, Japan and Russia for specific pharmacologic effects. It contains various phytochemical compounds that ensure its broad-spectrum effects. The numerous and diverse pharmacological activity of AS and its individual components involve for example, anti-tumour, anti-inflammatory, anti-radiation and cardiovascular protection potencies. In addition, AS positively influences the central nervous system, prevents and treats respiratory infections and improves physical fatigue effects [1][2][3]. AS extracts, as adaptogens, increase adaptability and survival of organisms under stress, where observed antioxidant properties can positively contribute to the effects [2,[4][5][6][7][8]. Polysaccharides, flavones and several dozen individual compounds were isolated from AS having numerous biological effects [2,8]. The AS component chlorogenic acid (CGA) is a naturally occurring nonflavonoid polyphenol that exerts numerous biological effects, e.g., antiviral, antitumor, antibacterial, antioxidant, antiinflammatory, 2. Results 2.1. Experiments Using ASRE 2.1.1. ASRE Interacting with H 2 S Reduces • cPTIO Na 2 S used in our study dissociates in aqueous solution and reacts with H + to yield H 2 S, HS − , and a trace of S 2− . We use the term H 2 S to describe the total mixture of H 2 S, HS − , and S 2− forms. Since H 2 S is endogenously produced in organisms and exogenous H 2 S donors are being considered to be used in medicine, we have studied the interaction of H 2 S with ASRE and the ability of the generated products to reduce the • cPTIO stable radical. The UV-VIS spectrum of ASRE has a relevant absorbance (ABS) peak at <400 nm ( Figure 1A). In our study, the concentration of 75 µg mL −1 ASRE had an absorbance (ABS) at 322 nm equal to 0.6. The ASRE/ • cPTIO mixture had an additional ABS peak of • cPTIO at 560 nm ( Figure 1A). During reduction of • cPTIO, peaks at 358 and 560 nm decreased ( Figure 1B). Since the peak at 560 nm did not significantly interfere with the spectrum of ASRE or its individual components, it was used to study the antioxidant properties of ASRE and its selected individual components interacting with H 2 S, Na 2 S 4 , Cys, GSH, glutathione oxidized (GSSG), cystine and methionine (MET).

ASRE Interacting with Cys or GSH (But Not with GSSG, MET or Cystine) Reduces • cPTIO
Since H 2 S increased the reduction of • cPTIO in the presence of ASRE, Cys and GSH (having a thiol group) were subsequently studied as well. Cys (400 µmol L −1 ) alone had a minor effect on • cPTIO reduction ( Figure 3A). Similarly to H 2 S, the addition of ASRE to Cys significantly increased the reduction of • cPTIO, with the resulting effect being strongly ASRE-dose-dependent at a constant Cys/ • cPTIO ratio ( Figure 3A). However, at a constant ASRE/ • cPTIO ratio, saturation at a higher Cys concentration was reached ( Figure 3B). Next, the ability of GSH, GSSG, MET and cystine to reduce • cPTIO was evaluated. GSH (400 µmol L −1 ) on its own did not reduce • cPTIO ( Figure 3C). However, the addition of ASRE to the GSH/ • cPTIO mixture caused the reduction of this radical. In contrast, the mixture of GSSG, MET or cystine (400 µmol L −1 ) with ASRE had only a negligible effect on • cPTIO reduction ( Figure 3C). Potency of the compounds to increase the • cPTIOreducing effect of ASRE was in the order of: H 2 S/ASRE > Cys/ASRE > GSH/ASRE > ASRE ( Figure 3D).

Effect of ASRE on Na 2 S 4 Induced Reduction of • cPTIO
Recently, endogenous hydropersulfides and related polysulfides were recognized as important biological mediators [22,23]. Therefore, we studied the ability of the polysulfide Na 2 S 4 /ASRE mixture to reduce • cPTIO. Previously, we have shown that Na 2 S 4 effectively reduces • cPTIO [28]. The potency of 20 µmol L −1 Na 2 S 4 was comparable to that of~400 µmol L −1 Trolox. The presence of ASRE in the Na 2 S 4 / • cPTIO mixture slightly decreased the concentration of • cPTIO when compared to that with the Na 2 S 4 / • cPTIO mixture ( Figure 4A). However, the dynamics and extent of the • cPTIO decrease in the presence of the Na 2 S 4 /ASRE/ • cPTIO mixture was less than the cumulative effects of ASRE/ • cPTIO and Na 2 S 4 / • cPTIO ( Figure 4B), indicating that ASRE slightly inhibited the potency of Na 2 S 4 to reduce • cPTIO. Next, we examined whether ASRE on its own or in the mixture with H 2 S, polysulfides, Cys or GSH could directly cleave pDNA in vitro, where the contribution of other (unknown) biologically important molecules and/or pathways is eliminated. Briefly, the pDNA cleavage assay can detect any activity that attacks and disrupts the sugar-phosphate backbone of DNA. ASRE alone had a minor but significant cleavage effect on pDNA with a bell-shaped profile. However, in the presence of H 2 S and Cys, and to a minor extent with GSH, pDNA cleavage by ASRE increased in a concentration-dependent manner ( Figure 5). H 2 S, Cys or GSH (100 µmol L −1 ) on their own did not cleave pDNA at all. In contrast to these com-pounds, polysulfides can effectively cleave pDNA in the order Na 2 S 4 ≥ Na 2 S 3 > Na 2 S 2 . Importantly, we observed that polysulfide-mediated pDNA cleavage was prevented by ASRE in a concentration-dependent manner ( Figure 5). Concentrationdependent effect of ASRE without (black) and with 100 µmol L −1 GSH (dark yellow), Cys (pink), Na 2 S (green), Na 2 S 2 (red), Na 2 S 3 (cyan) and Na 2 S 4 (blue). The final concentration of pDNA was 0.2 µg in 20 µL. I R of ncDNA form represents the relative intensity of the nicked circular pDNA. Horizontal black marks indicate the means ± SD, n = 3-6. Statistical significance of the effects of ASRE alone was evaluated by one-way ANOVA (p < 0.0001) followed by Dunnett's test with a significant difference for 0.47 (p < 0.01), 0.94 (p < 0.0001) and 1.41 (p < 0.01) mg mL −1 ASRE. Next, the potency of five individual components of ASRE, the CA, CGA, PCA, EB and EE, either on their own or in the mixture with H 2 S, to reduce • cPTIO was assessed. UV-VIS spectra of ASRE, CA, CGA, PCA, EB and EE are shown in Figure 6A. CGA alone had a modest radical-reducing effect. However, addition of H 2 S (≥5 µmol L −1 ) to a constant CGA/ • cPTIO mixture ratio significantly increased the reduction of • cPTIO in an H 2 S concentration-dependent manner ( Figure 6B). Furthermore, adding CGA to the constant H 2 S/ • cPTIO molar ratio increased the reduction of • cPTIO in a concentration-dependent manner. Similar results were observed for CA, which manifested a modest radical-reducing effect ( Figure 7A), but an addition of H 2 S (≥5 µmol L −1 ) into a constant CA/ • cPTIO mixture ratio significantly increased the reduction of • cPTIO in a concentration-dependent manner ( Figure 6C). The effect of PCA on • cPTIO reduction was minor and only slightly increased after the addition of H 2 S. EB and EE alone or in a mixture with H 2 S had no effect on • cPTIO concentration ( Figure 6C). The order of potency to reduce • cPTIO was CA ≥ CGA > PCA ≥ EB = EE = 0 ( Figure 7A). Potency to reduce • cPTIO in a mixture with H 2 S was as follows: CA~CGA > PCA > EB = EE = 0 ( Figure 6C).   (Figures 4A and 7B). The presence of ASRE components, CA, CGA, PCA, EB and EE in the • cPTIO/Na 2 S 4 mixture slightly decreased the • cPTIO concentration. However, the rate and extent of the • cPTIO decrease in the presence of Na 2 S 4 /ASRE component/ • cPTIO mixture was similar to the cumulative effects of the ASRE component/ • cPTIO and Na 2 S 4 / • cPTIO ( Figure 7B,C). Data for EB and EE are not shown. This indicates that ASRE components do not influence the potency of Na 2 S 4 to reduce • cPTIO.

CA, CGA and PCA (But Not EB or EE) in the Mixture with Cys or GSH Reduce • cPTIO
In addition, we compared the potency of five selected individual components of ASRE to reduce • cPTIO after their interaction with Cys ( Figure 8A) and GSH ( Figure 8B). The order of their potency to reduce • cPTIO in the mixture with Cys was: CA > CGA > PCA > EB~EE~Cys = 0. The order of their potency to reduce • cPTIO in the mixture with GSH was: CA~CGA > PCA > EB~EE~GSH = 0. When the data on the potency of the ASRE components mixed with H 2 S ( Figure 6) were included into the comparison, the order of • cPTIO-reducing potency was as follows: ASRE component/H 2 S > ASRE component/Cys > ASRE component/GSH.

Effect of the ASRE Components on pDNA Cleavage
Because of its low solubility in the buffer, EE was not included in the pDNA cleavage experiments. H 2 S (100 µmol L −1 ) alone or mixed with the increased concentrations of CA, CGA, PCA or EB, did not induce cleavage of pDNA. Polysulfides (100 µmol L −1 ) induced pDNA cleavage in the order of potency Na 2 S 4 > Na 2 S 2 , which is in accord with Figure 5. Notably, CA, CGA or PCA inhibited polysulfide-induced pDNA cleavage in a concentration-dependent manner (Figure 9). However, in the presence of polysulfides, EB slightly increased pDNA cleavage at lower concentrations and decreased it at higher concentrations ( Figure 9D).

Discussion
AS and its phytochemical individual components possess numerous diverse pharmacological effects, including antioxidant properties, yet their molecular mechanism is not fully understood [2]. Herein, we provide evidence that the interaction of ASRE and some of its individual components with H 2 S, Cys and GSH, but not with Na 2 S 4 , GSSG, MET or cystine, significantly increased its antioxidant potency to the level comparable or even more effective than that of Trolox. Similarly, an increased antioxidant potency has been observed after the interaction of H 2 S with NO, selenite, phthalic selenoanhydride or doxycycline [28][29][30][31]. However, detailed information on how the biologically active compounds stimulate the antioxidant potency of ASRE is not available yet. H 2 S is produced endogenously and exerts relevant biological effects and functions. The H 2 S concentration in plasma is less than 1 µmol L −1 . However, in local microenvironments where it is enzymatically produced, it can reach higher levels [32]. Therefore, the physiological significance of the H 2 S/ASRE antioxidant effects will need to be determined.
The concentration of total Cys in the serum/plasma is~250 µmol L −1 [33]. GSH is the most abundant nonprotein thiol in mammalian cells, reaching an intracellular concentration in the mmol L −1 range, whereas its plasma concentration does not exceed micromolar range [34]. The increased antioxidant effect of the Cys/ASRE and GSH/ASRE mixture found in vitro are within the physiological concentrations of Cys and GSH and can thus be physiologically relevant in the thiols/ASRE interactions in a living organism.
Whereas the H 2 S/ASRE interaction increased the reduction of the • cPTIO radical, the Na 2 S 4 /ASRE interaction had an opposite minor effect: it inhibited the reduction of • cPTIO induced by Na 2 S 4 . Since Na 2 S 4 alone possesses high potency to reduce • cPTIO, we assume that the inhibitory effect of ASRE may result from "scavenging" of Na 2 S 4 leading to a decrease of Na 2 S 4 redox capacity in the system.
In the pDNA cleavage assay, high concentrations of the compounds were used in order to detect any cleavage. However, it should be noticed that even tiny DNA damage levels, not detectable by our pDNA cleavage assay, may have significant physiological consequences. Minor pDNA cleavage caused by ASRE on its own may indicate that it contains active unknown species responsible for the effects. The molecular mechanism of the bell-shaped increase of pDNA cleavage by interaction of ASRE with H 2 S or Cys is unknown. Similarly, the physiological significance of pDNA cleavage in vitro in a complex biological system, at in situ concentrations of ASRE, is unknown. Na 2 S n induced pDNA cleavage in the same manner as in our previous studies [28,31]. ASRE and its components, CA, CGA and PCA, but not EB, decreased the concentration of • cPTIO. Similarly, ASRE, CA, CGA and PCA, but not EB at a lower concentration, inhibited polysulfide-induced pDNA cleavage. If we assume that the pDNA cleavage reaction requires the action of certain radicals, we may suggest that the inhibitory effect of ASRE, CA, CGA, PCA, and to less extend of EB, on pDNA cleavage can result from the "scavenging" of the radicals by these compounds.
It is of interest to know which particular ASRE components are responsible for the ASRE antioxidant effects. ASRE and several dozen compounds isolated from ASRE possess numerous biological effects, including antioxidant properties. Dried root ethanolic extract from AS has been found to scavenge superoxide anion and hydroxyl radicals [4]. CA and CGA isolated from AS have antioxidant activities [5], whereas EB and EE have no significant antioxidant (anti-DPPH) properties [6,8]. To extend these observations, CA, CGA, PCA, EB and EE as ASRE components [2] were selected for our study. We confirmed some of the previous observations, but we also found the important fact that the antioxidant properties of CA and CGA were strongly enhanced after interaction with H 2 S, Cys or GSH. This increase is similar to that observed for ASRE, implying that these two agents (and possibly others that were not studied) are responsible for increasing their antioxidant properties and inhibiting polysulfide-induced pDNA cleavage. PCA had only a partial effect compared to that of CA or CGA, while EB and EE had no effect at all. CA, CGA and to a lesser extent PCA interacted with H 2 S, Cys and GSH but not with GSSG, MET or cystine, indicating that the presence of thiol groups was a prerequisite for the observed effects.
The concentrations of H 2 S, Cys and GSH differ in different parts of a living organism and can change in different pathological conditions [32][33][34]. From our results, we hypothesize that the antioxidant properties of ASRE and its components depend on the concentrations of H 2 S, Cys, and GSH in situ. Two central thiol/disulfide redox couples in human plasma, Cys/cystine and GSH/GSSG, vary little among healthy individuals [35]. However, changes of the thiol/disulfide ratio regulate and are implicated in many biological processes and diseases, including enzyme catalysis, gene expression, and pathway signaling [36,37]. If the increase of the antioxidant properties of the ASRE/thiol mixture found in vitro takes place in a living organism, then changes of the thiol/disulfide ratio can influence the biological effects of ASRE and its components.
In conclusion, we found that H 2 S/HS − , Cys and GSH interacting with ASRE, CA and CGA increased their antioxidant properties and modulated cleavage of plasmid DNA. These findings can contribute to understanding many biological effects of ASRE, especially under conditions where radicals play a significant role, and suggest that antioxidant properties of ASRE and its components depend on the concentrations of H 2 S, Cys and GSH, and changes of the thiol/disulfide ratio in situ. We may assume that the interaction of H 2 S/HS − , Cys and GSH with ASRE, CA and CGA can contribute to their numerous biological effects. However, whether and to what extent the in vitro data on • cPTIO reduction in a phosphate buffer and pDNA cleavage can be directly implicated in the complex biological environment requires further study.

Preparation of Aqueous Root Extract from Acanthopanax Senticosus (ASRE)
Dried roots of Acanthopanax senticosus (AS, Siberian ginseng) were obtained from the arboretum in Liptovsky Hradok (Slovakia) in November 2021. The roots were crushed in liquid nitrogen to obtain fine pieces. Five hundred milligrams of these pieces were incubated in 10 mL of the buffer consisting of 0.9% NaCl and 10 mmol L −1 sodium phosphate (pH 7.0) at 80 • C for 120 min. After incubation and cooling of the solution, the upper clear ASRE extract was collected (without a very top layer, where light insoluble particles floated). The extract was divided into 400 µL aliquots and stored at -20 • C for several days. It contained~7.5 mg dry matter per mL. UV-VIS spectra of ASRE were used to adjust its concentration in experiments from three different ASRE preparations.

UV-VIS of • cPTIO Radical
To obtain 1 mL of the working solution, 1-50 µL of stock solution of the studied compounds was added to the appropriate volume (950-999 µL) of 100 mmol L −1 sodium phosphate buffer (pH 7.4, 37 • C) containing the final concentrations of 100 µmol L −1 • cPTIO and 100 µmol L −1 DTPA. UV-VIS absorption spectra (900-200 nm) were recorded every 30 s for 30 min with a Shimadzu 1800 (Kyoto, Japan) spectrometer at 37 • C. In all experiments, the absorbance (ABS) path length of 10 mm was used. Reduction of the • cPTIO radical was determined as the decrease of the absorbance at 560 nm [29,38]. Concentration and stability of the studied compounds were checked by their UV-VIS spectra at the beginning of particular experiment in the range of 200-900 nm.

Plasmid DNA Cleavage
A pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs, Inc., N3033 L, Ipswich, MA, USA) was performed as reported previously [31]. In this assay, all samples contained 0.2 µg pDNA in the final sodium phosphate buffer (25 mmol L −1 sodium phosphate, 50 µmol L −1 DTPA, pH 7.4). Five microliters of the Na 2 S, Na 2 S n , Cys or GSH aqueous stock solution (in final 100 µmol L −1 ) was added to 15 µL of pDNA solution containing ASRE (in mg mL −1 : 0, 0.23, 0.47, 0.94, 1.41 or 1.88) or its chemical component (in mmol L −1 : 0.2, 0.4, 0.8, 1.2 or 1.6). All listed concentrations were final and calculated for a 20 µL sample. All ASRE components were prepared in water, except PA, which was dissolved in 100 mmol L −1 sodium phosphate. The resulting samples were incubated for 30 min at 37 • C. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Integrated densities of all pBR322 forms in each lane were quantified using Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA cleavage efficiency.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.