Inhibitory characteristics of flavonol-3-O-glycosides from Polygonum aviculare L. (common knotgrass) against porcine pancreatic lipase

Pancreatic lipase (PL) is an enzyme that plays an essential role in the digestion of dietary lipids and is a suitable target for an anti-obesity dietary supplement. The objective of this study was to find a novel source of PL inhibitors from Korean medicinal plants and investigate the PL-inhibitory properties of the active constituents. From among 34 kinds of methanolic crude extracts, Polygonum aviculare L. showed the highest PL-inhibitory activity (63.97 ± 0.05% of inhibition). Solvent fractionation and liquid chromatography/mass spectrometry (LC/MS) analysis identified flavonol-3-O-glycosides, flavonol-3-O-(2″-galloyl)-glycosides, and flavonol aglycones as active constituents. Furthermore, the inhibitory characteristics of the major compounds were investigated in terms of enzyme kinetics and fluorescence quenching. The results suggested that the inhibitory activity of the major compounds is closely related to the tertiary structural change in PL, and that differences in inhibitory activity occurred due to slight discrepancies in their chemical structure.

inal plants were prepared using 80%(v/v) aqueous methanol, which can dissolve both unknown hydrophilic and hydrophobic compounds effectively. The PL-inhibitory activities of these crude extracts were evaluated at the same concentration (1.25 mg/mL), and the results are given in Table 1. Most of the crude extracts (1−30) had PL-inhibitory activity to various extents, and this phenomenon was explained by previous reports on natural PL-inhibitory compounds in plants, such as polyphenols, saponins, and terpenes 9 . The PL reactions reached equilibrium very rapidly even though crude extracts inhibited the reactions (see Supplementary Fig. S1), which means that the putative inhibitor compounds would show a conventional mechanism of inhibition (i.e., fast reversible inhibition). Consequently, P. aviculare showed the significantly highest (p < 0.05) PL inhibition (63.97 ± 0.05%) among any other plant species. P. aviculare has not been mentioned previously as a source of PL inhibitors; hence, it was chosen as the final target for study and analyzed to identify its major compounds.
The methanolic crude extract of P. aviculare was successively fractionated with n-hexane and EtOAc solvents in polarity order. After every solvent fractionation, we were able to obtain about 0.5−1.0 mg of lyophilized n-hexane and EtOAc fractions of P. aviculare uniformly. The relative residual activity (RRA) of PL in the presence of 0.25 mg/mL crude extract and n-hexane fraction was 76.30 ± 2.47% and 72.69 ± 0.66%, respectively, as shown in Fig. 1. Because there was no significant difference between these (p > 0.01), it was considered that inherent active constituents in the crude extract were not specifically fractionated by n-hexane with a high level of relative polarity (0.009) 16 . On the other hand, the EtOAc fraction exhibited significantly higher PL-inhibitory activity (42.62 ± 1.49% of RRA) than those of the crude extract and n-hexane fraction (p < 0.01), which means that EtOAc with a moderate level of polarity (0.228) was able to fractionate a group of compounds including the active constituents required for PL inhibition 16 . According to previous studies, P. aviculare commonly contains many phenolic compounds, such as flavonoids and flavonoid glycosides 17,18 . As these compounds can be fractionated by EtOAc and may have PL-inhibitory activities, we expected that flavonoid compounds in P. aviculare could be major compounds in PL inhibition 12 .

Chemical identification of EtOAc fraction by UPLC-ESI-MS analysis. We performed UPLC-ESI-MS
analysis of the crude extract and EtOAc fraction of P. aviculare to identify the EtOAc-fractionated compounds. The UPLC chromatograms of each sample are shown in Fig. 2. The methanolic crude extract was a mixture of hydrophilic and hydrophobic compounds (see Fig. 2a), whereas the EtOAc fraction only contained compounds with the moderate polarity expected in a family of flavonoids (see Fig. 2b). All distinguishable peaks in the chromatogram were successively analyzed by ESI-QTOF MS, which is suitable for natural compound analysis, and the results were compared with previous reports on the P. aviculare [17][18][19] . Consequently, most of the compounds in the EtOAc fraction were annotated to flavonoids, as expected. Structural information of 10 compounds with a high relative abundance in the EtOAc fraction is provided in Fig. 2c and Table 2. Flavonoids in the EtOAc fraction consisted of flavonol-3-O-glycosides (1−5), flavonol-3-O-(2″-galloyl)-glycosides (6−8), and flavonol aglycones (9−10). The largest proportion was flavonol-3-O-glycosides of kaempferol, quercetin, and myricetin with rhamnose or arabinose at the position of the C-3 hydroxyl group; hence, avicularin (quercetin-3-O-α-L-arabinofuranoside), myricitrin (myricetin-3-O-α-L-rhamnopyranoside), and quercitrin (quercetin-3-O-α-L-rhamnopyranoside) were the major compounds in PL inhibition by P. aviculare. Several weak peaks in the chromatogram require further fractionation to identify all constituents clearly; however, the MS results of major peaks, which probably exert the largest influence on the PL-inhibitory activity, were highly reliable because there is no large discrepancy (below 5 ppm) between the calculated and observed molecular weight for all constituents.
Flavonols are a class of flavonoids that have the 3-hydroxyflavone backbone, and their diversity stems from the different positions of the phenolic hydroxyl groups 20 . As several flavonols and flavonol glycosides have been reported to have PL-inhibitory activity, the same activity of P. aviculare in this study could be explained by its inherent flavonol glycosides 12 . The majority of compounds in the EtOAc fraction of P. aviculare were largely www.nature.com/scientificreports www.nature.com/scientificreports/ unknown about their PL-inhibitory activities; therefore, P. aviculare could be proposed as a novel source of PL inhibitors for anti-obesity agents, based on the inhibitory characteristics of its major compounds. In addition, we checked the total flavonoid contents of the crude extract and EtOAc fraction of P. aviculare. The contents of flavonoids in the crude extract and EtOAc fraction were 27.66 ± 0.72 µg of quercetin equivalent/mg of crude extract and 226.46 ± 4.15 µg of quercetin equivalent/mg of EtOAc fraction, respectively. The total flavonoid contents increased about 8-fold after EtOAc fractionation. This implies that a high content of flavonoids could contribute to PL-inhibitory activity, thus paralleling the results of the UPLC-ESI-MS analysis.
Inhibition kinetics of promising candidates for PL inhibitors. The PL-inhibitory activity of major compounds (avicularin, myricitrin, and quercitrin) and flavonol aglycones (kaempferol, quercetin) in the EtOAc fraction of P. aviculare were converted into the half-maximal inhibitory concentration (IC 50 ) by enzyme kinetics analysis. The IC 50 is a practical measure of the potency of a compound for inhibiting a specific enzyme, and facilitates quantitative comparison of the inhibitory activity of different compounds. As shown in Table 3, the IC 50 values of quercetin, kaempferol, myricitrin, quercitrin, and avicularin against PL were 53.05, 79.38, 92.85, 100.56, and 141.84 µM, respectively. These values were calculated from the RRA of PL in the presence of the compounds at various concentrations ( Supplementary Fig. S2). The PL-inhibitory activities of quercetin, kaempferol, and quercitrin had been reported previously 12,21 ; however, those of myricitrin and avicularin were reported for the first time in this study. Furthermore, the inhibition modes of each compound were elucidated by evaluating the changes of the kinetic parameters from double-reciprocal Lineweaver-Burk plots under the presence of 50 μM and 100 μM of each compound (Fig. 3). Myricitrin, quercitrin, and avicularin which belong to flavonol-3-O-glycosides showed non-competitive or mixed-type inhibition, whereas flavonol aglycones (quercetin and kaempferol) showed competitive inhibition.  www.nature.com/scientificreports www.nature.com/scientificreports/ Collectively, it was found that these compounds showed quite different PL-inhibitory characteristics despite their similar structures (see Fig. 2c). These results indicate that differences in inhibitory activity occurred according to slight discrepancies in structure, such as hydroxylation in B-ring of flavone backbone, the presence of glycosylation at the position of C-3 hydroxyl group, and the type of glycosidic sugar. Especially, quercetin and kaempferol exhibited higher inhibitory activity than myricitrin, quercitrin, and avicularin. Glycosylation can strongly influence the hydrophilicity and steric hindrance of a structure 22 , affecting the interaction between enzyme and inhibitor (i.e., inhibitory activity). Therefore, the presence of rhamnopyranose or arabinofuranose in major compounds rendered their structures inaccessible to PL and lowered the inhibitory activity. Similar to our studies, the glycosylation of flavonoids has been shown to cause a decrease in PL-inhibitory activity in previous studies 21 . In addition, an influence of hydroxylation and glycosidic sugar type on inhibitory activity can be inferred from our data. For the future, experiments focusing on addressing the comparative analysis and in-depth structural analysis to reveal these effects would make explicit the findings from this study.

Molecular interaction between PL and promising candidates for PL inhibitors.
In most cases, the inhibitory activity cannot demonstrate all changes in an enzyme caused by an inhibitor. To understand the interaction between PL and the major compounds in the EtOAc fraction of P. aviculare, fluorescence analysis of PL was performed in the absence and presence of the major compounds and flavonol aglycones. First, the fluorescence emission spectra (excitation at 295 nm) of PL in the presence of each compound were obtained, as shown in Fig. 4. The fluorescence emission intensity of PL decreased with an increase in the concentration of the inhibitory compound, and a slight blue shift (i.e., a decrease in wavelength) was observed in the emission maximum wavelength (2−6 nm). This phenomenon is explained by fluorescence quenching of tryptophan in protein; in other words, the inhibitory compound interacted with PL and the consequent changes in PL structure quenched the intrinsic fluorescence of tryptophan in PL. The anisotropy displayed by tryptophan in protein is sensitive to both the overall rotational diffusion of proteins and the extent of segmental motion during the excited state; for that reason, the intrinsic fluorescence of tryptophan (selectively excited at 295−305 nm) in protein can provide considerable information about protein structure and dynamics 23 . Most importantly, we found that greater fluorescence quenching (especially blue shift) occurred when the IC 50 value of the inhibitory compound was smaller, which implies that the PL-inhibitory activity of the compounds is closely related to the structural change in PL, in particular the tertiary structure.
Second, we derived several parameters (K SV , k q , K A , and n) of fluorescence quenching (see Table 3) from the Stern-Volmer equation (see Supplementary Fig. S3) and its double-logarithmic equation (see Supplementary  Fig. S4). The K SV values at 37 °C were not significantly different from the K SV values at 25 °C, probably due to the strong formation of the protein-quencher complex. On the other hand, the calculated k q values at both temperatures were higher than the maximum diffusion collision quenching rate constant (10 10 M −1 s −1 ). This status can be defined as static quenching, which means that fluorescence quenching was caused by the formation of a complex between protein and molecule 23 . Taken together, the results of the fluorescence analysis suggest that flavonol glycosides and flavonol aglycones in the EtOAc fraction of P. aviculare manifest their PL-inhibitory activities by interacting with PL, thereby forming the complex and ultimately modifying the tertiary structure. Our suggestion corresponds with previous studies on enzyme inhibition by flavonoid compounds, which proposed the formation of a complex between the enzyme and inhibitor as the mode of action 24,25 .     Table 3 also presents the K A and n values of each compound obtained with the double-logarithmic equation. The number of binding sites (n) of each compound was close to one, indicating that only one binding site existed in the PL. The binding constants (K A ) for quercetin, kaempferol, myricitrin, quercitrin, and avicularin were 3.57 × 10 4 , 2.07 × 10 4 , 1.45 × 10 4 , 1.00 × 10 4 , and 0.94 × 10 4 M −1 , respectively. The K A value indicates the ability to combine with protein, and thus may be closely related to inhibitory activity. As expected, the relation between these K A values and the IC 50 values corroborated our suggestion regarding the PL-inhibitory characteristics of flavonol glycosides and flavonol aglycones, because the order of the K A value was contrary to the order of the IC 50 value (i.e., quercetin > kaempferol > myricitrin > quercitrin > avicularin for PL-inhibitory activity). In addition, CD analysis was performed to understand the effect of the major compounds on the secondary structure of PL. As shown in Table 4, the composition of secondary structures of PL was as follows: α-helix (18.7%), antiparallel β-sheet (33.0%), parallel β-sheet (12.8%), β-turn (20.0%), and random coil (43.8%). However, no significant discrepancy was found in the secondary structure of PL, compared with the results of other studies [24][25][26] . Hence, the effect of the major compounds on the secondary structure of PL was negligible.

Molecular weight
In conclusion, we have proposed P. aviculare as a novel source of PL inhibitors. The EtOAc fraction of P. aviculare exerted high PL-inhibitory activity and it was composed of flavonol-3-O-glycosides, flavonol-3-O-( 2″-galloyl)-glycosides, and flavonol aglycones. Furthermore, inhibitory characteristics of major compounds in P. aviculare were investigated by enzyme kinetics and fluorescence quenching analysis. The scope of this study was limited in terms of in vitro PL inhibition; however, the findings have a number of important implications for future practice, such as in vivo PL inhibition and anti-obesity functionality. Therefore, as well as demonstrating a series of scientific processes for identifying a novel source of PL-inhibitors from natural sources, our results could provide information aiding effective utilization of P. aviculare as an anti-obesity agent based on PL inhibition. Additionally, since the plant P. aviculare are quite affordable in Korea as well as across many countries in temperate regions of Eurasia and North America, it would be an excellent material for practical utilization. Solvent extraction and fractionation. The Korean plant sample (10 g) was homogenized with 200 mL of 80%(v/v) aqueous methanol at room temperature for 24 h and then filtered through a 0.5 µm polytetrafluoroethylene (PTFE) membrane filter (Advantec MFS Co., Tokyo, Japan). The filtrate was concentrated by an EYELA N-1100 rotary evaporator (Tokyo Rikakikai Co., Tokyo, Japan), and the concentrate was lyophilized by an FD 8512 freeze dryer (IlShin BioBase Co., Gyeonggi-do, Republic of Korea) at −76 °C to obtain a crude methanol extract of the plant sample. The obtained crude extract (7 g) was fractionated by suspending it in water (200 mL) and n-hexane (200 mL) for 2 h, and the n-hexane layer was separated using a separatory funnel. The EtOAc layer was successively separated from the aqueous layer using the same method. The n-hexane and EtOAc layers were lyophilized to obtain each solid fraction.   Table 3. Half-maximal inhibitory concentrations (IC 50 ) and fluorescence quenching parameters of major compounds and flavonol aglycones. R 1 , R 2 , and R 3 are substituent positions on the basic structure of flavonol in www.nature.com/scientificreports www.nature.com/scientificreports/ Determination of total flavonoid content. The total flavonoid contents of the crude extract and the EtOAc fraction were determined by the aluminum chloride (AlCl 3 ) colorimetric method with a slight modification 27 . A volume of 0.5 mL of the sample was mixed with 0.1 mL of 10%(w/v) AlCl 3 solution, 0.1 mL of 0.1 mM potassium acetate, 1.5 mL of methanol, and 2.8 mL of distilled water. After incubation of 30 min, the absorbance of the mixture was measured at 415 nm with a UV-2450 ultraviolet-visible spectrophotometer (Shimadzu Co., Kyoto, Japan). The total flavonoid content was expressed as µg quercetin equivalents per mg dry weight of the sample, and the standard curve is shown in Supplementary Fig. S5. All prepared solutions were filtered through a membrane filter (0.45 µm).  Pancreatic lipase inhibition assay. The PL assay using 4-MUO as a substrate was conducted as described previously with slight modification 28,29 . Lipase from porcine pancreas type II (i.e., pancreatic lipase) was dissolved in 50 mM Tris-HCl buffer (pH 8.0) to give 10 mg/mL, and centrifuged at 5,000 g for 10 min to remove insoluble material before the enzyme assay. Then, 50 µL of 0.5 mM 4-MUO solution dissolved in the above buffer and 100 µL of the sample solution dissolved in methanol (i.e., the inhibitor solution) were mixed in 96-well microplate to form the reaction system. The enzymatic hydrolysis was initiated by adding 50 µL of the PL solution to the reaction system (final 250 units/mL) at 37 °C. Fluorescence of 4-methylumbelliferone (4-MU), which is a product of hydrolysis of 4-MUO, was detected at an excitation wavelength of 320 nm and an emission wavelength of 455 nm using a SpectraMax ® i3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). The inhibitory activities against PL were calculated for comparison with each other, as follows:

Methods
where A 0 is the relative fluorescence unit after the reaction for 30 min in the absence of sample (i.e., only methanol) and A S is the fluorescence unit in the presence of sample.
To determine the IC 50 , the RRA of PL in the presence of the sample at various concentrations was evaluated. The reaction of PL was measured by the same procedure described above, and the RRA was calculated from the following equation: where ν 0 is the initial velocity of the reaction in the absence of sample (i.e., only methanol) and ν S is the initial velocity in the presence of sample. Nonlinear regression curve fitting of RRA against sample concentration was performed using the SigmaPlot software (ver. 12.5; Systat Software Co., CA, USA) and the IC 50 was determined. The type of inhibition was elucidated by evaluating the changes of kinetic parameters from Lineweaver-Burk plot as follows: where [S] is the concentration of substrate, v is the initial velocity of the reaction, V max is the maximum initial velocity when substrate approaches infinite concentration, and K m is the dissociation constant of the enzyme-substrate complex (i.e., the Michaelis constant).
Fluorescence emission spectroscopy. The Table 4. Conformational changes in the secondary structure of pancreatic lipase in the presence of each major compounds and flavonol aglycones.
www.nature.com/scientificreports www.nature.com/scientificreports/ widths were set at 9 and 15 nm, respectively. The fluorescence data were analyzed by fitting to the Stern-Volmer equation for analysis of fluorescence quenching 23 : where F 0 and F is the fluorescence intensity in the absence and presence of quencher, respectively, k q is the bimolecular quenching constant, τ 0 is the lifetime of fluorescence in the absence of quencher, K SV is the Stern-Volmer quenching constant, and [Q] is the concentration of quencher. K SV was obtained from the slope of a plot of F 0 /F versus [Q] by linear regression curve fitting, and k q was calculated by K SV , where τ 0 is equal to 1.59 ns 31 . The binding constant (K A ) and number of binding sites (n) were obtained according to a double-logarithmic equation 32 : Statistical analysis. Statistical analysis was performed using SPSS statistics software (ver. 23.0; IBM Co., Armonk, NY, USA). All experiments were conducted in triplicate. The data were subjected to one-way analysis of variance (ANOVA), and significant differences among mean values were compared using Duncan's multiple range test.