Inhibitory Potential of Quercetin Derivatives Isolated from the Aerial Parts of Siegesbeckia pubescens Makino against Bacterial Neuraminidase

This study aimed to isolate bacterial neuraminidase (BNA) inhibitory O-methylated quercetin derivatives from the aerial parts of S. pubescens. All the isolated compounds were identified as O-methylated quercetin (1–4), which were exhibited to be noncompetitive inhibitors against BNA, with IC50 ranging from 14.0 to 84.1 μM. The responsible compounds (1–4) showed a significant correlation between BNA inhibitory effects and the number of O-methyl groups on quercetin; mono (1, IC50 = 14.0 μM) > di (2 and 3, IC50 = 24.3 and 25.8 μM) > tri (4, IC50 = 84.1 μM). In addition, the binding affinities between BNA and inhibitors (1–4) were also examined by fluorescence quenching effect with the related constants (KSV, KA, and n). The most active inhibitor 1 possessed a KSV with 0.0252 × 105 L mol−1. Furthermore, the relative distribution of BNA inhibitory O-methylated quercetins (1–4) in S. pubescens extract was evaluated using LC-Q-TOF/MS analysis.


Introduction
Neuraminidase (sialidase, EC 3.2.1.18), belonging to the hydrolases class, is mainly present in bacteria and viruses [1,2]. They are responsible for the cleavage of glycolconjugated proteins on the host cell membrane to release sialic acid [3][4][5]. In particular, bacterial neuraminidase (BNA) contributes to bacterial cell-to-cell signaling [6], which leads to the generation of self-extracellular polymeric substances (EPSs) that cause bacterium to adhere to one another, which is known as biofilms [7,8]. In addition, neuraminidase is also secreted by bacterial pathogens for the colonization of biofilms [9], which causes differences in the immune defense system by being present in bacterially infected sites of the human body [10]. Depending on the infecting pathogen, BNA can cause various inflammatory diseases, including pneumonia, enteritis, and sepsis [11]. Although antibiotics provide an effective treatment for pathogenic infections, biofilms within pathogens provide protection from the antibacterial effects, enabling their survival [12][13][14]. Therefore, preventing the formation of biofilm through the inhibition of BNA activity is essential for the successful treatment of infectious, chronic inflammatory diseases [15]. Many recent studies have reported on the development of bacterial neuraminidase inhibitors from various natural sources, such as chromenone derivatives from F. philippinensis [16], ugonins from H. zeylanica [17], anthraquinone from P. cuspidatum [18], rotenoids from A. fruticose [19], etc.
Siegesbeckia pubescens is an annual plant native to East Asia, mainly Korea, Japan, and China [20]. The plant typically grows naturally in mountain and field areas [21], where it is collected during mature flowering for use in traditional medicine [22]. The dried plants of the aerial parts have been used as a remedy for various inflammations related to bones, joints, muscles, and rheumatoid pain [23]. From many previous reports, the therapeutic effects of S. pubescens are known to be associated with the well-known abundance of secondary metabolites, such as diterpenes, sesquiterpenes, and quercetin derivatives [24]. There is evidence of anti-inflammation effects from previous reports, which show that terpenes have the potential to inhibit Pam 3 CSK 4 -induced inflammation [25], reduce oxidation stress in vivo [26], inhibit elastase release [27], and attenuate postoperative inflammation. Quercetin derivatives also play a role in anti-inflammatory effects by inhibiting the production of pro-inflammatory cytokines and mediators, such as nitric oxide and prostaglandin E2 [20,28]. In particular, O-methylated quercetins in S. pubescens have not been reported to have BNA inhibitory capacities.
This study aimed to explore the inhibitory effects of O-methylated quercetins from the aerial parts of S. pubescens against BNA to evaluate its anti-inflammatory ability. Four O-methylated quercetins (1-4) responsible for BNA inhibition were isolated and identified using spectroscopic data. The kinetic studies of inhibitors were characterized by doublereciprocal plots against BNA. Binding affinities between BNA and isolated compounds were investigated by fluorescence quenching. Moreover, the natural abundance of BNA inhibitors (1-4) from the aerial parts of S. pubescens were analyzed using LC-Q-TOF/MS.

Inhibitory Effects of Quercetin Derivatives against Bacterial Neuraminidase
Bacterial neuraminidase plays an important role through the production of sialic acid from host cells. Thus, inhibition of BNA was related to bacterial infectious inflammation. All of the isolated quercetin derivatives (1-4) exhibited dose-dependent inhibitory effects against BNA, with IC 50 values of 14.0~84.1 µM (Figure 2A (4). The most active BNA inhibitor was compound 1, which contained an O-methyl group. However, no differences in inhibitory effects were observed between compounds 2 and 3 with O-dimethyl moieties. Among them, the lowest level of activity was detected in O-trimethyl quercetin 4. A higher level of inhibitory activity against BNA was detected for 3-O-methyl quercetin, compared with quercetin (IC 50 = 26.1 µM) as a positive control, which had the mother skeleton of compound 1 (Table 1).  In addition, the inhibition modes and related constants of inhibitors were verified by double-reciprocal plots, including Lineweaver-Burk and Dixon plots. All of the isolated (O-methylated) quercetins (1-4) were confirmed as noncompetitive inhibitors, which exert inhibitory activities by binding to the enzyme-substrate complex. In the Lineweaver-Burk plot, the x-axis is the reciprocal of the substrate concentration derived from the K m value, and the y-axis is the reciprocal of the value for maximum velocity. As shown in Figure 2C, the constant K m value and the decreasing V max , indicating a noncompetitive mode, were observed for inhibitor 1, which was the most active. In addition, the enzyme inhibition constant (K i ) was obtained by the determination of substrate concentrations between inhibitor concentrations and V max values in the Dixon plot. Thus, it was confirmed that the K i value of inhibitor 1 was 13.8 µM ( Figure 2D). Other inhibitors (2-4) had K i values of 24.7, 22.4, and 79.5 µM, respectively. In comparison with O-methyl substituents on the quercetin, the BNA inhibitory activities increased in the order of mono > quercetin, and di-> tri-. Four O-methylated quercetins (1-4) exhibited noncompetitive inhibition modes, where they were bound to an allosteric site on an enzyme complex, with the substrate confirmed by Lineweaver-Burk plots. Moreover, K i values derived from the Dixon plot showed they were similar to IC 50 , reverifying them as noncompetitive inhibitors. Overall, 3-O-methyl quercetin (1) was the lead BNA inhibitor and was a more active metabolite than none, di-, or tri-type methylated quercetins.

Plant Materials and Chemicals
Collection of the aerial part of S. pubescens was conducted at a local mountain in Chungcheongbuk-do, Republic of Korea in March 2022. At that time, S. pubescens was in the yellow flowers blooming growth stage. The collected plant was dried under dark conditions at room temperature for the isolation of the metabolites. Quercetin, methanol-d 4 , chloroform-d, neuraminidase from Clostridium perfringens, 2 -(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate, and dimethyl sulfoxide were purchased from Sigma Aldrich (St. Louis, MO, USA). Organic solvents, including hexane, chloroform, ethyl acetate, acetone, and methanol used for the separation, isolation, and purification of metabolites were purchased from Daejung Chemical and Metals (Siheung-si, Gyeonggido, Republic of Korea). Analytical-grade acetonitrile and water were purchased from Honeywell (Charlotte, NC, USA).

Bacterial Neuraminidase Inhibition Assay and Kinetics
Measurement of the inhibition of bacterial neuraminidase (BNA) activity was performed according to previous reports [29]. The BNA assay was performed using fluorescence (FS) of excitation and emission at 365 nm and 450 nm, respectively. At the fixed wavelength, the production of umbelliferon from sodium 2-(4-methylumbelliferyl)-N-acetylneuraminate as a florescent substrate was monitored for the determination of  (1) Double-reciprocal plots were used to elucidate the results of kinetic studies of BNA inhibitory activities. Inhibitory modes were determined using the Lineweaver-Burk plot. The inhibition constant was derived from the origin of the Dixon plot. The reciprocal plots were displayed using the relationship between the Michaelis-Menten constant (K m ) and the maximal velocity (V max ), according to different concentrations of inhibitors, based on the half-maximal inhibitory concentration (IC 50 ) value. The progressive curves were visualized using Sigma Plot ver. 10.0.

Fluorescence Quenching Experiments
BNA contains specific amino acid residues, including tryptophan, phenylalanine, and tyrosine, possibly as fluorescence quenching. In the interaction between the enzyme and the inhibitor, three amino acid residues showed reduced fluorescence intensities near 360 nm of emission wavelength, as indicated by the 290 nm of excitation wavelength. For the FQ analysis, 10 µL of 0.2 unit/mL neuraminidase from Clostridium perfringens and 10 µL of various concentrations of inhibitors containing individual IC 50 ranges were mixed in 180 µL of sodium acetate buffer without substrate. Based on the results, the related parameters were calculated as follows, using Equations (2) and (3).
where F 0 and F are fluorescence emission intensities in the absence and presence of quercetin derivatives, respectively; [Q] is the concentration of quenchers with the same means as the inhibitors; K SV is the Stern-Volmer quenching constant; K A is the binding constant between the enzyme and the quenchers; and n is the binding number between the enzyme binding site and the inhibitors.

LC-Q-TOF/MS Analysis
Shimadzu NEXERA (Shimadzu, Kyoto, Japan) was used as the equipment for LC analysis, and a Poroshell 120 EC-C18 column (2.1 mm × 100 mm, 2.7 µm, Agilent, Santa Clara, CA, USA) was used as the column for analysis. Purified water (A) containing 0.1% acetic acid and acetonitrile (B) containing 0.1% acetic acid were used as the mobile phase, with a flow rate of 1 mL/min. The solvent condition utilized a gradient solvent system that increased the ratio of the mobile phase composition B from 0 to 100% for 40 min. SCIEX X500R Q-TOF equipment was used for Q-TOF/MS, and MS conditions were set to 5.5 kV for capillary voltage and 450 • C for temperature in positive ionization mode. MS conditions were set to a collision energy of 10 V and a desolvation gas flow of 800 L/h at a temperature of 400 • C.

Statistical Analysis
Measurements of inhibitory activities against BNA, enzyme kinetics, and binding affinities were performed in triplicate. The mean, deviations, and p-values (<0.05) obtained in the results were expressed using SigmaPlot ver. 10.0 (Systate Software Inc., Chicago, IL, USA).

Conclusions
In this study, BNA inhibitory O-methylated quercetins were isolated from the aerial parts of S. pubescens. The chemical structures of isolated compounds were identified as 3-O-methyl quercetin (1), 3,4 -O-dimethyl quercetin (2), 3,7-O-dimethyl quercetin (3), and 3,7,4 -O-trimethyl quercetin (4) by fully spectroscopic data. BNA inhibitory effects showed a close relationship with the number of methyl groups on quercetin: 1 (IC 50 = 14.0 µM, mono) > 2 (IC 50 = 25.8 µM, di), and 3 (IC 50 = 24.3 µM, di) > 4 (IC 50 = 84.1 µM, tri). All of the compounds (1-4) were identified as noncompetitive inhibitors, which bind with the enzyme and substrate complex. In addition, inhibitors (1-4) that showed that binding affinities (K SV ) with BNA were closely associated with inhibitory effects against BNA (IC 50 ). Finally, the natural abundance of quercetin derivatives from S. pubescens extract was confirmed by the result showing that the most active BNA inhibitor (3-O-methyl quercetin, 1) contained the highest contents of BPC using LC-Q-TOF/MS. This was the first report where O-methylated quercetins were responsible for BNA inhibition in the appropriate skeleton. The findings of this study suggest that the active metabolites from the plant have the potential to be used as anti-inflammatory lead compounds to associate with pathogenesis from bacterial infection. Further studies are necessary to elucidate the mechanism of action on the cell experiments and in vivo.