Effectiveness of Prenyl Group on Flavonoids from Epimedium koreanum Nakai on Bacterial Neuraminidase Inhibition

In this study, the inhibitory potential of bacterial neuraminidase (NA) was observed on the leaves of Epimedium koreanum Nakai, which is a popular ingredient in traditional herbal medicine. This study attempted to isolate the relevant, responsible metabolites and elucidate their inhibition mechanism. The methanol extraction process yielded eight flavonoids (1–8), of which compounds 7 and 8 were new compounds named koreanoside F and koreanoside G, respectively. All the compounds (1–8) showed a significant inhibition to bacterial NA with IC50 values of 0.17–106.3 µM. In particular, the prenyl group on the flavonoids played a critical role in bacterial NA inhibition. Epimedokoreanin B (compound 1, IC50 = 0.17 µM) with two prenyl groups on C8 and C5′ of luteolin was 500 times more effective than luteolin (IC50 = 85.6 µM). A similar trend was observed on compound 2 (IC50 = 0.68 µM) versus dihydrokaempferol (IC50 = 500.4 µM) and compound 3 (IC50 = 12.6 µM) versus apigenin (IC50 = 107.5 µM). Kinetic parameters (Km, Vmax, and Kik/Kiv) evaluated that all the compounds apart from compound 5 showed noncompetitive inhibition. Compound 5 was proven to be a mixed type inhibitor. In an enzyme binding affinity experiment using fluorescence, affinity constants (KSV) were tightly related to inhibitory activities.


Introduction
The neuraminidases (EC 3.2.1.18) are enzymes that catalyze the hydrolysis of terminal neuraminic acid from a variety of glycoproteins and gangliosides. Bacterial neuraminidase (NA) preferentially cleaves 5-N-acetylneuraminic acid (Neu5Ac) from cell membrane glycoproteins via the linkage between neuraminic acid and α→3 or α2→6 galactose [1][2][3]. Bacterial neuraminidase is expressed among particular pathogens, such as Streptococcus pneumonia, Pseudomonas aeruginosa, and Clostridium perfringens, and it cleaves neuraminic acid from glycoconjugates. The key function of bacterial NA is to help promote mucosal infection via biofilm formation [4]. Chen et al. reported that bacterial NA inhibition blocks the desialylation of CD24 that occurring in sepsis, which preserves the Siglec inhibitory circuit and suppresses the inflammatory response. At the same time, bacterial NA acts with bacterial virulence, implicating biofilms formation and bacterial defense against antibiotics [5]. Consequently, bacterial NA implicated in numerous pathways involved in bacterial infection and its inhibitors will provide one of the ways to treat bacterial pathogenic diseases depending on the neuraminic acid hydrolysis [6]. Previously, our group has succeeded in isolating and characterizing the naturally occurring neuraminidase inhibitors, which are xanthones, pterocarpans, and geranylated flavonoids from Garcinia mangostana [7], Sophora flavescens [8], and Paulownia tomentosa [9].
Epimedium koreanum Nakai belongs to the Berberidaceae family and has a unique feature, having three branches and three leaves on each branch. It grows in Southeast Asian countries [10]. The aboveground parts of E. koreanum Nakai (leaves and stem) have been used as a medicinal herb for a general tonic against infertility, as well as against inflammatory diseases including cardiovascular diseases and arthritis [11,12]. Nowadays, the leaves are consumed as a popular medicinal herb. Its species continues to be a rich source of phenolic metabolites, of which prenylated flavonoids are the major constituents. Based on the composition of the phenolic metabolites, they display a broad spectrum of biological activities, such as antioxidative, anticancer, immunomodulatory, and neuroprotective functions [13,14].
In this study, we isolated eight prenylated flavonoids from using a methanol extraction process on the leaves of E. koreanum Nakai, and their structures were fully characterized by spectroscopic methods. All the isolated compounds were examined for bacterial NA inhibition and kinetic behavior. In particular, we observed a critical role of the prenyl group on the flavonoids in enzyme inhibition.
Molecules 2019, 24, x FOR PEER REVIEW 2 of 14 the naturally occurring neuraminidase inhibitors, which are xanthones, pterocarpans, and geranylated flavonoids from Garcinia mangostana [7], Sophora flavescens [8], and Paulownia tomentosa [9]. Epimedium koreanum Nakai belongs to the Berberidaceae family and has a unique feature, having three branches and three leaves on each branch. It grows in Southeast Asian countries [10]. The aboveground parts of E. koreanum Nakai (leaves and stem) have been used as a medicinal herb for a general tonic against infertility, as well as against inflammatory diseases including cardiovascular diseases and arthritis [11,12]. Nowadays, the leaves are consumed as a popular medicinal herb. Its species continues to be a rich source of phenolic metabolites, of which prenylated flavonoids are the major constituents. Based on the composition of the phenolic metabolites, they display a broad spectrum of biological activities, such as antioxidative, anticancer, immunomodulatory, and neuroprotective functions [13,14].
In this study, we isolated eight prenylated flavonoids from using a methanol extraction process on the leaves of E. koreanum Nakai, and their structures were fully characterized by spectroscopic methods. All the isolated compounds were examined for bacterial NA inhibition and kinetic behavior. In particular, we observed a critical role of the prenyl group on the flavonoids in enzyme inhibition.

Bacterial Neuraminidase Inhibitory Activities
The isolated prenylated flavonoids (1-8) were tested for enzymatic inhibitory activity against bacterial NA. The enzyme activity was assayed according to a standard literature procedure by following the hydrolysis of 4-methylumbeliferyl-α-D-N-acetylneuraminic acid sodium salt hydrate [15]. The inhibitory profiles of the compounds (1-8) and three mother compounds (luteolin, dihydrokaempferol, and apigenin) are shown in Table 2. All the compounds (1-8) exhibited a potent bacterial NA inhibition with IC 50 values of 0.17~106.3 µM. Table 2. Inhibitory effects of the compounds (1-8) on bacterial neuraminidase activity.

Dihydrokaempferol
Apigenin Quercetin 4 500.4 ± 3.8 107.5 ± 1.8 20.2 ± 0.8 NT NT NT 1 All the compounds were examined in a set of experiments repeated three times; IC50 values of compounds represent the concentration that caused 50% enzyme activity loss. 2 Values of inhibition constant. 3 NT means not tested. 4 Quercetin was used as a positive control.
Three prenylated flavonoids (1-3) inhibited bacterial NA significantly with IC50s of 0.17, 0.68, and 12.6 µM, respectively. They showed much better inhibition than the glycoside compounds (4-6). In particular, compound 1 (IC50 = 0.17 µM), having two prenyl groups on C8 and C5′ of luteolin, was 500 times more effective than the mother skeleton, luteolin (IC50 = 85.6 µM), as shown in Figure 3a. Compound 2 (IC50 = 0.68 µM) was 700 times more potent in comparison with dihydrokaempferol (IC50 = 500.4 µM) (Figure 3b). The prenyl group effectiveness on bacterial NA inhibition was also observed between compound 3 (IC50 = 12.6 µM) and apigenin (IC50 = 107.5 µM) (Figure 3c). Taken together, the prenyl group on the flavonoids played a critical role in bacterial NA inhibition. The two new compounds (7 and 8) also exhibited potent inhibition with IC50s of 2.9 µM and 16.7 µM, respectively. Particularly, the structural differences between compounds 7 and 8 was in the presence of the methoxy group or hydroxyl group on C-2′′, but the methoxy group (7) was 5 times more effective compared with the hydroxyl group (8).  The enzyme inhibition properties of compounds 1-8 were modeled using double-reciprocal plots of Lineweaver-Burk and Dixon plots. As shown in Figure 5a, the analysis of compound 1 exhibited that Vmax decreased without changing Km in the presence of the increasing concentration of The enzyme inhibition properties of compounds 1-8 were modeled using double-reciprocal plots of Lineweaver-Burk and Dixon plots. As shown in Figure 5a, the analysis of compound 1 exhibited that V max decreased without changing K m in the presence of the increasing concentration of the inhibitor. As can be seen in the graph ( Figure 5), −1/K m (the x-intercept) was unaffected by the concentration, whereas 1/V max became more positive. This behavior indicates that compound 1 exhibits noncompetitive inhibition characteristics for NA. The K i value of compound 1 was calculated as 0.15 µM by Dixon plots (Figure 5b). Similar trends can also be observed in all the compounds bearing sugar moieties on both A and C rings, apart from compound 5. A similar analysis of compound 5 showed a series of lines, which intercept to the left of the vertical axis and above the horizontal axis, indicating that compound 5 was a mixed-type inhibitor (Figure 5c). The K i value was estimated as 36.8 µM by Dixon plots (Figure 5d). To further confirm noncompetitive and mixed-type behavior, the results were applied to Yang′s method (Table 3) [16]. In this procedure, Km and Vmax are plotted against the inhibitor concentration. The new kinetic constant Kik can be fitted to Equation (1), while Kiv can be fitted to Equation (2). From the results of the fit, the Kik/Kiv ratio were between 6.68 and 16.04 for compound 1, which is further consistent with noncompetitive behavior. Compound 5 showed typical mixed-type behavior with 2.02~3.66 of Kik/Kiv [16]. To further confirm noncompetitive and mixed-type behavior, the results were applied to Yang's method (Table 3) [16]. In this procedure, K m and V max are plotted against the inhibitor concentration. The new kinetic constant K ik can be fitted to Equation (1), while K iv can be fitted to Equation (2). From the results of the fit, the K ik /K iv ratio were between 6.68 and 16.04 for compound 1, which is further consistent with noncompetitive behavior. Compound 5 showed typical mixed-type behavior with 2.02~3.66 of K ik /K iv [16]. Table 3. Effect of different concentrations of compounds 1 and 5 on V max , K m , and the K ik /K iv ratio using bacterial neuraminidase.

Binding Affinities between Bacterial Neuraminidase and Compounds
Proteins have intrinsic fluorescence mainly due to tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues [17]. The intrinsic fluorescence of the protein often changes as a function of the ligand concentrations. We investigated the enzyme binding affinities of the inhibitors on bacterial NA by a fluorescence quenching (FQ) effect. Bacterial neuraminidase has three fluorescent residues, eight Trp ( [18]. There was no significant emission from any of the other components of the assay mixture under the measurement condition (i.e., emission from 300 to 400 nm). The binding affinity (K SV ) was analyzed using the Stern-Volmer Equation (Equation (3)). Figure 6 shows typical Stern-Volmer plots of compounds 1, 2, and dihydrokaempferol on bacterial NA. Evidently, the fluorescence intensities were dramatically decreased for the best performing inhibitors 1 (IC 50 = 0.17 µM), 2 (IC 50 = 0.68 µM), proportional to the increasing of the concentration. The FQ effect of the lowest active compound (dihydrokaempferol, IC 50 = 500.4 µM) was insignificant. The FQ effects of the remaining compounds are displayed in Table 4 and the Supplementary Materials. The binding affinities of the Stern-Volmer constants (K SV ) of inhibitors could be ranked in the following order 1 > 2 > 3 > 4, which is in agreement with the order of their inhibition potencies (IC 50 s) as shown in Figure 6d. The binding constant (K A ) and the number of binding sites (n) were calculated by Equation (4). The binding constants (K A ) were also correlated with inhibitory potencies (IC 50 s) as shown in Table 4.

Extraction and Isolation
The dried leaves of E. koreanum Nakai (0.5 kg) were extracted using methanol (10 l) at room temperature for 1 week to obtain a crude extract (48 g). The crude extract was suspended in water and successively partitioned into ethyl acetate to afford a dark residue (18 g). The ethyl acetate fraction (15 g) was subjected to column chromatography on silica gel (8 × 40 cm, 500 g) and eluted with a gradient flow of n-hexane/ethyl acetate (20:1 to 1:2, v/v) to give 20 fractions (A1-A20, each 200 mL). The fractions A8-15 (6.

Bacterial Neuraminidase Inhibitory Activity Assay
Neuraminidase from Clostridium perfringens (C. welchii) (EC 3.2.1.18) was evaluated as described previously with slight modification [21]. 4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid sodium salt hydrate was used as the substrate. The fluorescence was measured using a UV-Vis spectrophotometer (Spectra Max M3, Molecular Devise, Sunnyvale, CA, USA) with a 96-well black immuno-microplate (SPL life science, Pocheon, Korea) at 37 • C with an emission wavelength of 450 nm and an excitation wavelength of 365 nm. All the samples were dissolved in DMSO at 10 mM and diluted to the required concentration. First, 20 µl of 1 mM of an aqueous solution of the substrate (K m = 100 µM) was mixed with 160 µl of 50 mM sodium acetate buffer (pH 5.0). Then, 10 µl of the inhibitors and 10 µl of neuraminidase (0.2 units/mL) were added respectively to the mixture. Each assay was conducted as 3 separate replicates. The inhibitor concentration, leading to a 50% activity loss (IC 50 ), was obtained by the following equation: Activity (%) = 100 [1 + ([I]/IC 50 )].

Enzyme Kinetics and Progress Linear Determinations
To determine the enzyme inhibition kinetics, an experiment was performed having different substrate and inhibitor concentration ranges. To find out each curve parameter, a nonlinear regression program was used for data analysis using Sigma Plot. Similarly, K m and V max were derived from the Lineweaver-Burk plot. Additionally, the K i value was calculated from Dixon plots. The K ik and K iv rate constants were calculated according to Equations (1) and (2) proposed by Yang et al. [16].

Binding Affinity between Bacterial Neuraminidase and Compounds
180 µl of 50 mM sodium acetate buffer (pH 5.0) with 10 µl of 0.5 unit/mL neuraminidase from Clostridium perfringens were accurately added into the 96-well black immuno-plates; then, different 10 µl concentrations (15.6~250 µM) of inhibitor were added. The spectra for the fluorescent emissions were recorded from 300 to 400 nm with emission slits adjusted to 2.0 nm, and the excitation was 260 nm using a spectrophotometer (Spectra Max M3). All the experiments were performed in triplicate. Fluorescence quenching is described by the Stern-Volmer equation. The value of n approximates to one, indicating that only a single binding site exists in bacterial neuraminidase for inhibitors [22].

Statistical Analysis
All the experiments were conducted in triplicate. The results were subjected to variance analysis using Sigma Plot (version 10.0, Systat Software, Inc., San Jose, CA, USA). Differences were considered significant at p < 0.05.

Conclusions
In conclusion, we have undertaken a thorough investigation of bacterial neuraminidase inhibition by E. koreanum Nakai, an important medicinal plant. The principal components were identified as prenylated flavonoids, including two new ones, named koreanoside F and koreanoside G. They showed mainly noncompetitive behavior that was demonstrated with the kinetic parameters V max , K m , K ik , and K iv . The binding affinities (K SV ) of the inhibitors were measured by a fluorescence quenching effect. In particular, the prenyl group on the flavonoids played a critical role in bacterial NA inhibition. The most active compound 1 (IC 50 = 0.17 µM) was 500 times more effective than its mother skeleton (IC 50 = 85.6 µM). We are hopeful that prenylated flavonoids can be of use as a lead structure for neuraminidase inhibitors.