Ursolic Acid Inhibited Cholesterol Esterase and Pancreatic Lipase Activities and Decreased Micellar Cholesterol Solubility In Vitro

Ursolic acid (UA) is a natural triterpene carboxylic acid with an underlying anti-hyperlipidemia efect. Tis study examined the mechanism of interactions of UA with cholesterol esterase (CEase), pancreatic lipase (PL), and micellar cholesterol solubility in vitro . Te half-maximal inhibitory concentration (IC 50 ) of CEase, PL, and micellar cholesterol solubility was determined to be 0.07 ± 0.01 mg/mL, 1.81 ± 0.13mg/mL, and 1.73 ± 0.08mg/mL, respectively. Multispectral combination revealed that UA changed the secondary structure and quenched the intrinsic fuorescence by static quenching of the two enzymes. Te interactions were exothermic reaction as determined by enthalpy. Furthermore, molecular docking confrmed that UA was bound to amino acids of two enzymes at active site through hydrophobic interaction and van der Waals forces. Molecular dynamics (MD) simulation found that CEase and PL rearranged with UA to form stable complexes. Te strong inhibition efect of CEase and PL mediated by UA might provide additional insight into understanding the role of UA in the hypolipidemic efect.


Introduction
Hyperlipidemia is a disease characterized by abnormal lipid metabolism, and excessive lipid accumulation predisposes to infection with atherosclerosis [1].In the process of adipogenesis, cholesteryl esterase (CEase) and pancreatic lipase (PL) in the small intestine are the essential enzymes associated with hyperlipidemia, converting dietary fat into more readily absorbed free cholesterol, monoacylglycerol, and fatty acids [2,3].Tese molecules form mixed cholesterol micelles with bile salts, crossing the brush border membrane of the enterocyte and further entering the lymphatic system to participate in circulation [4,5].Inhibiting CEase and PL activity is expected to decrease lipids levels and the risks of related diseases.Orlistat is widely used in clinical practice as a special kind of CEase and PL inhibitor.It efectively reduces the incidence of obesityrelated diseases, whereas long-term use will induce a number of side efects including diarrhea, nausea, vomiting, and fatty stool [6][7][8].Tus, exploring alternative plant-derived natural inhibitors may be an approach to prevent hyperlipidemia with bright prospects.
Plants have always been a treasure trove of bioactive compounds with diverse therapeutic relevance.Triterpenes, in the form of free glycosides or esters in plants, play a role in regulating blood lipid composition and reducing lipid accumulation.Studies have shown that some naturally derived triterpene saponins had promising inhibitory efects on intestinal esterase activity and lipid accumulation [9][10][11].Ursolic acid (UA) is a natural triterpene carboxylic acid, widely found in the leaves and fruits of the Rhododendron family [12,13].It has a lot of pharmacological efects, including anti-hyperlipidemic, anti-atherosclerotic, and alleviating alcoholic liver injury [14,15].Te antiobesity properties have also been documented by inhibiting adipogenesis in 3T3-L1 adipocytes and adipose tissue hypertrophy in high-fat diet-induced rats [16,17].Te maslinic acid (MA), corosolic acid (CA), and oleanolic acid (OA), structurally similar isomers to UA, improved lipid-lowering biological activities through regulation of Sirt1/AMPK signaling pathway and the expression of PPARc, AdipoR1, and AdipoR2, as well as inhibition of pancreatic lipase (PL) [18][19][20][21].However, no reports have been available on the inhibition efect and mechanism of UA against CEase and PL.
Terefore, this study aimed to evaluate the inhibitory potential and binding afnity of UA on the two enzymes of CEase and PL.Inhibition kinetics and mixed micelle suppression were determined to discover the inhibition types and micelle conformation.Diferent techniques including isothermal titration calorimetry (ITC), forescent spectroscopy, Fourier-transform infrared spectroscopy (FTIR), and computer simulations were used to characterize the combined mechanism of UA with CEase and PL.Te fndings in this article will provide a relevant reference and theoretical basis to explore the functional food and application of UA to enhance the hypolipidemic efect.

CEase and PL Inhibitory Rates.
Te inhibitory rate of UA on CEase was calculated as described previously with slight modifcations [22].Te confguration of p-NPB stock solution (20 mM) was carried out in acetonitrile.CEase was dissolved in 0.1 M PBS bufer (0.1 M NaCl, 5.16 mM sodium taurocholate, pH 7.0).Diferent concentrations of UA (0.01-0.15 mg/mL, 50 μL) were mixed with p-NPB (10 μL) and incubated at 310 K for 10 min.Ten, CEase solution (0.54 mg/mL, 15 μL) was added to the system to initiate the enzyme reaction.After 5 min, the absorbance was measured with an ultraviolet spectrophotometer (UVmini-1240, Shimadzu Instrument Co., Japan) at 405 nm.
Te inhibitory rate of UA against PL was analyzed as described earlier by Zhu et al. and Su et al. [23,24].A fxed concentration of the substrate p-NPL (0.8 mg/mL, 0.6 mL) and various amounts of UA (0.01-4 mg/mL) were mixed with Tris-HCl (0.1 M, pH 8.2).Te mixed solution was incubated continuously at 310 K for 10 min.Te reaction was initiated by adding PL initial solution (5 mg/mL, 200 μL).Te absorbance value was detected (405 nm) after 20 min.Inhibitory rates were calculated according to the following equation: where A and B represent the absorbance values of control group and blank control group and C and D represent the absorbance values of experimental group and blank experimental group.
Competitive inhibition: Mixed inhibition: where V is the reaction velocity; V m is the maximum reaction rate; [S] is the p-NPB/p-NPL concentration; [I] is the UA concentration; K i is the inhibition constant of UA with CEase/PL; K is is the inhibition constant of UA with enzymesubstrate complex; and K m is the Michaelis-Menten constant.
where C 0 is the cholesterol concentration in the absence of UA and C 1 is the cholesterol concentration in presence of UA.
2.4.2.Morphology.Dynamic light scattering (DLS) was used to analyze particle sizes of the mixed micelles by using a Zetasizer Nano ZS instrument (Brookhaven Instruments, Holtsville, UK).For each measurement, the micelles were not further treated by ultracentrifugation but diluted with distilled water at a rate of 1 : 2 (v/v, pH 7.4) [27].Results were performed in triplicate as mean diameter (nm).
Transmission electron microscopy (TEM) was used to observe various atomic structures of solid materials by the fuctuation of electrons [27].Te micelles were diluted 50 times, and then 5 µL suspension was treated with a 200-mesh hole carbon support grid.Excess liquid was absorbed by the flter paper to form thin flm, which was transferred into TEM (Talos G2 200X, Tokyo, Japan).Te low electron beam was used as the light source and the electromagnetic feld as the lens.Diferent light and dark images formed were displayed on vision systems after magnifying and focusing.
2.5.Fourier-Transform Infrared (FTIR) Spectra.Te changes in secondary structure were studied by Termo Nicolet IS50 FTIR Spectrometer (Termo Nicolet Corp., USA).In brief, UA was incubated with CEase and PL separately at a rate 1 : 1 (v/v) for 15 min at 310 K and then lyophilized.After mixing with potassium bromide, samples were prepared by the pellet method.Te wavelength range was 400 to 4000 cm −1 and resolution was 4 cm −1 [28].Termo Scientifc OMNIC software was used for subtracting the blank background and smooth processing in the range of 1400-1800 cm −1 [7].Te second derivative ftting was performed using Origin.9.0.Te main peaks of the protein structure were further resolved by baseline automatic calibration and curve ftting [8].
2.6.Fluorescence Spectroscopy.Te characteristic fuorescence of CEase and PL was detected using an F-7100 fuorescence spectrometer (HITACHI, Japan) based on a previous method [29].Te structures and quenching mechanisms between CEase (0.5 mg/mL) or PL (5 mg/mL) and UA were detected by analyzing the maximum fuorescence intensity change.Te emission wavelength (λ em ) was 310 to 420 nm with fxed excitation wavelength (λ ex ) at 280 nm.Te excitation and emission slits widths were all 5 nm.Fluorescence absorption peaks were recorded at different temperatures (298 K, 304 K, and 310 K), which were frequent and representative for analyzing the fuorescence quenching mechanism [7,30].
Te Stern-Volmer equation was used to analyze the fuorescence quenching ability [31]: where [Q] represent the diferent concentrations of UA; τ 0 is the average lifetime of fuorescence without UA (τ 0 ≈10 −8 s); and K sv and K q represent the Stern-Volmer dynamic quenching constant and the quenching rate constant of UA on CEase and PL, respectively.Te Scatchard equation was used to analyze the binding strength [32]: where F 0 and F represent the fuorescence intensity of the free CEase/PL and complex, respectively; n is the number of binding sites; and K a is the binding constant.

Conformational Studies.
To further investigate the quenching mechanism of characteristic amino acids more intuitively, synchronous and excitation-emission matrix (EEM) fuorescence method was followed [33].Tyrosine (Tyr) and tryptophan (Trp) fuorescence intensities were recorded by varying diference excitation or emission wavelength range.Te emission wavelengths (λ em ) of tryptophan and tyrosine were recorded in the continuous range of 265-420 nm and 220-420 nm, respectively, with fxed excitation wavelength (λ ex ) at 280 nm.Te EEM spectra were collected with excitation and emission wavelength in the range of 200-500 nm.

Isothermal Titration Calorimetry (ITC)
. ITC was conducted using an ITC-200 calorimeter (Malvern, USA) as described by Wu et al. [34].UA (135 µM) was set as the titrant, and CEase or PL solution (13.5 µM) was set as the sample solution.In all, 50 μL titrant was injected sequentially into the titration cell with CEase or PL solution (150 μL).Te time interval of each injection was 150 s and lasted for 4 s between successive injections.Origin 9.0 was used for standardization, including estimating thermodynamic parameters, ftting binding isotherms, and iterative curve ftting.Te Gibbs free energy was analyzed by the following equation: where ΔH and ΔS are enthalpy and entropy change, respectively, and T is the Kelvin temperature.2.10.Dynamic Simulation.Molecular dynamics simulation was performed using GROMACS 2021 to analyze the dynamic trajectories and stability of complex molecules [35].It was considered as an important tool to explore the patterns of binding and interaction.Te structures of the receptor and ligand were derived from the molecular docking results.Complex structures were immersed in the center of a cubic box (TIP3P water molecules).Te charge was neutralized by sodium chloride under the AMBER14SB force feld (310 K).Simulation fles were output in 30,000 ps.

Statistical
Analysis.Data analysis and plotting were performed with the software of OMNIC and Origin.9.0.SPSS 26 was applied at a signifcant level of p < 0.05.Each measurement was repeated three times.

Results and Discussion
3.1.Inhibitory Efect of UA on CEase and PL.Enzyme activity inhibition experiments were carried out in vitro using orlistat as the positive control.As shown in Figures 1(a) (D) and 1(b) (E), the IC 50 value of UA and orlistat on CEase was 0.07 ± 0.01 mg/mL and 0.003 ± 0.010 mg/mL, respectively.Similarly, the IC 50 value of UA and orlistat to PL was 1.81 ± 0.13 mg/mL and 0.05 ± 0.02 mg/mL, respectively.Although less compared to orlistat, the inhibition efect of UA on CEase was higher than that of ginkgolide B (IC 50 : 0.34 ± 0.02 mg/mL) [36], as well as that of UA on PL was higher than that of betulin (IC 50 : 3.19 ± 0.11 mg/mL) and ginsenoside Rd (IC 50 : 4.06 ± 1.41 mg/mL) [21].

Inhibition Types.
Te inhibition types of enzymes generally fall into two categories (reversible and irreversible inhibition) [37].Te relationships between enzyme concentration and reaction rate with diferent UA concentrations are shown in Figures 1(a) (B) and 1(b) (B).All curves of V versus CEase and PL concentration passed through the origin as the concentration of UA increased, indicating that the inhibitions of UA against CEase and PL were reversible and the binding was noncovalent.
Te Lineweaver-Burk plots were used to explore the inhibition mechanisms of UA on the two enzymes.With an increase in UA concentration, the four Lineweaver-Burk lines intersected in the second quadrant, the K m value increased, and V m gradually decreased (Figure 1(a) (C)).Te inhibition type of UA on CEase was mixed inhibition, and UA was bound to the CEase-p-NPB to form the UA-CEasep-NPB ternary compound.Te inhibition constants K i and K is were 2.07 μM and 3.96 μM, respectively, in Figures 1(a) (D) and (E).Te binding afnity of UA with free CEase was greater than that of the CEase-p-NPB complex (K i < K is ).In case of PL, all the straight lines exhibited in Figure 1(b) (C) intersected on the Y-axis.Te K m values gradually increased with increased UA concentration (Table S1), suggesting that the inhibition type of UA on PL was competitive.UA might bind to the PL active site region by inducing the structural change and decreasing catalytic activity of PL [37].

MCI Analysis.
Micelle formation is a pivotal step in the absorption of fatty acids and cholesterol in the small intestine [26].Te MCI was evaluated by preparing artifcial micelles (Figure 2(a)).In the supernatant obtained by ultracentrifugation, signifcant dose-dependent inhibition of micellar solubility was determined with the addition of UA (IC 50 : 1.73 ± 0.08 mg/mL).Te size of micellar particles increased with the addition of UA as shown in Figure 2(b).Te average particle size of micelles increased from 47 nm to 700 nm with the PDI value increasing.Te larger the PDI, the wider the molecular weight distribution [38].Results indicated that UA destroyed the micelle homogeneous state in the solution.
As shown in Figures 2(c) and 2(d), the former as control micelles showed orbicular droplets and the specimens in the presence of UA generated anomalous particles.Te spheroidal structure of micelles was destroyed and cholesterol was released from mixed micelle after treating with UA, suggesting that UA had a considerably destructive potential toward micelle conformation and strong inhibition of cholesterol incorporation in soluble micelles.Te most likely reason for the elimination of cholesterol from micelles was due to the hydrophobic interactions with bile salt [39].
3.4.FTIR Spectra.FTIR spectra were used to obtain information about the secondary structure of CEase and PL.As shown in Figure 3(a), the amide I band peak position of CEase was changed from 1654 to 1648 cm −1 with the addition of UA while the amide II band shifted from 1541 to 1552 cm −1 .Te changes of peak positions illustrated the relocation of the hydrogen bonding pattern and the secondary conformation changes of CEase.In Figure 3(b), the amide I band peak of PL was changed from 1652 to 1648 cm −1 while the amide II band peak changed from 1546 to 1543 cm −1 .Both the amide I and II band peaks showed a blue shift, which revealed that UA decreased the polarity of PL in solution and enhanced the hydrophobicity.
Trough curve ftting amide I band, the percentage of each secondary structure was estimated for further quantitative comparison.Free CEase constituted 34.49% α-helix, 17.48% β-sheet, 27.66% β-turn, 12.54% β-antiparallel, and 7.83% random helix in Figure 3(a), showing that the α-helix was the most abundant structure of amino acids and acted as a scafold to equilibrate the steric confguration of CEase [40].In case of CEase-UA, the contents of β-sheet, α-helix, and β-antiparallel were decreased to 13.72%, 26.98%, and 10.44%, respectively.Meanwhile, the α-helix contents of PL-UA were reduced to 42.85% (Figure 3(b)).According to previous studies, the formation of the intermolecular hydrogen bond was related to the β-sheet structure [41].Te decreased β-sheet indicated a more fexible molecular structure.Alteration in FTIR spectra refected that UA interfered with the hydrogen bonding networks of CEase and PL, and thus the secondary structure of the two enzymes was rearranged.fuorophores (or both) might be afected by UA, so the values of all fuorescence intensity were corrected to avoid the inner flter efect [42].Maximum fuorescence peaks were reduced with increased UA concentration (Figures 3(c) and 3(d)).Tis suggests that UA had a signifcant quenching efect on key lipid-digestive enzymes.Te intrinsic fuorescence of CEase and PL was decreased by 58.18% and 14.29%, respectively, at 0.5 mg/mL UA, implying that the fuorescence quenching ability of UA on CEase was stronger than PL.Stern-Volmer curves and Scatchard equation curves were curve-ftted to analyze the mechanism of fuorescence quenching.A good linear relationship of F 0 /F versus [Q] indicated that the intrinsic fuorescence of CEase and PL quenched by UA existed in a single quenched manner (dynamic or static).In Table 1, the quenching constant K q of CEase and PL was 1.892 ± 0.052 and 1.622 ± 0.294 (×10 11 L•mol −1 •s −1 ), respectively.K q was much larger than the maximum scatter collision quenching constant (2 × 10 10 L•mol −1 •s −1 ), forming slower difusion and more ground-state complexes at high temperatures.Te K sv values of UA to CEase or PL decreased gradually with rising temperature, suggesting that a static quenching happened by forming an enzyme-UA complex.

Fluorescence
Te binding interaction parameters of UA and enzymes were investigated using the Scatchard equation.Te results are shown in Table 1.Te values of binding constant (K a ) of UA on CEase and PL were 1.892 ± 0.052 and 1.622 ± 0.294 (×10 5 L/mol) at 310 K, respectively.UA could bind well to CEase and PL to reduce the activity of both enzymes in vivo at the optimal temperature (310 K) [43,44].Te values of K a decreased with the elevated temperature to inhibit the production of complexes [45], which further explained the static quenching efect of UA on CEase and PL.In addition, the value of n approximately equal to one meant that UA had at least one high-afnity binding site in CEase or PL [46].

Conformational Analysis
3.6.1.Synchronous Fluorescence.Synchronous fuorescence quenching is commonly related to hydrophobic alteration regarding the major amino acid including tyrosine (Tyr) and   Synchronous fuorescence spectra of the interaction between UA and PL are shown in Figures 4(c) and 4(d).UA strongly quenched the fuorescence intensity of both Trp and Tyr in PL.Trp fuorescence peak shows a red shift from 290 to 296 nm with increasing amounts of UA.Te result suggests that UA reinforced the polarity of the microenvironment around Trp residues.Taken together, it was possible that UA interacted with the fuorophores of CEase and PL through hydrophobic interactions and hydrogen bonds, disturbed the microenvironment of amino acids at active sites, and further altered the tertiary structure of CEase and PL.

Excitation-Emission Matrix (EEM) Fluorescence
Spectra.Fluorescence excitation-emission matrix spectra were used to reveal the comprehensive fuorescence structure of free CEase or PL and complexes.As shown in Figures 4(e)-4(h), peak 1 was the fuorescence intensity of the Trp and Tyr residues while peak 2 was the fuorescence intensity of the polypeptide-skeleton structure, respectively.Peaks a and b represented the frst-order (λ ex � λ em ) and second-order (λ ex � 2λ em ) Rayleigh scattering, respectively [48].

Isothermal Titration Calorimetry (ITC).
ITC was used to monitor and record the calorimetric curves and thermodynamic parameters of protein-ligand interactions [50].Te curves of binding isotherms and corresponding heat fow versus time were obtained by injecting aliquots of UA solution into CEase (Figure 5(a)) and PL (Figure 5(b)).Te decreased binding isotherms of UA on Cease/PL showed that the interaction was typically exothermic.Te number of available binding sites on CEase/PL was relatively reduced.Negative ΔG of CEase (−7.67 kJ/mol) and PL (−6.33 kJ/mol) at applied temperatures indicated a spontaneous complexation process (Table S2).Also, the negative ΔH and ΔS demonstrated that hydrogen bond and van der Waals forces were dominant in the interaction of UA on CEase and PL.Te value of ΔH was much lower than that of covalent bond energy (200-400 kJ/mol), further verifying that the interactions of UA with CEase or PL were noncovalent.Te high afnity constants (K itc ) of CEase-UA and PL-UA, 4.09 and 3.15 (×10 5 L/mol), suggested a strong binding, and further studies were needed to analyze the type of actions.K itc of PL-UA was slightly lower than that of CEase-UA and UA had a stronger afnity with CEase at 298 K in comparison with PL.K itc was higher than binding constant (K a ) values shown in Table S2, which might be due to the diversity in sensitivity of experimental instruments [32].

Molecular Simulation
3.8.1.Molecular Docking and Binding Energy.To further confrm the above experimental fndings at the molecular level, the conformation with the highest LibDock score was used to analyze the interaction of macromolecules with inhibitors [51].Te CEase-UA corresponding to the most stable conformations is supplemented in Figure 6(a).Ligands were located within the active pocket of CEase and surrounded by the hydrophobic residues (Leu, Trp, Phe, and Ala) in this region.Te active site of PL contained the catalytic triad Ser152, Asp176, and His 263 [52].Te favorable binding conformation and binding site of the complex were directly observed in Figure 6(b).On locating the potential binding sites, UA formed pi-sigma and van der Waals force with His263 and Ser152, respectively.Te results confrmed that CEase and PL were catalytically active only in specifc steric conformations and UA binds to amino acids at the active sites of both enzymes to reduce the catalytic activity.Te negative average value shown in Table 2 suggested that the binding was stable (CEase-UA, −32.26 ± 14.51 kJ/mol, and PL-UA, −24.69 ± 11.12 kJ/mol) [32].Te higher van der Waals energy and lower electrostatic energy suggested that the stability of CEase-UA and PL-UA mainly depended on van der Waals, which was consistent with the negative ΔH and ΔS in ITC analysis (Figure 5 and Table S2).

Molecular Dynamics Simulation.
Molecular dynamics simulations were analyzed to investigate the molecular arrangement of the optimal docking conformation system.Previously, it was found that when the fuctuation of the root mean square deviation (RMSD) was relatively constant within 1 Å, a stable state of dynamic equilibrium could be achieved, and the lower RMSD value made the protein structure more stable [35].With the extension of simulation time, the RMSD values of free CEase and CEase-UA were 2.21 and 2.15, respectively (Figure 7(a)).On the other hand, the value of PL-UA (1.86) was slightly higher than that of PL (1.84) in Figure 7(b).Results demonstrated that the diference between the two was negligible, which might be due to the disorderly movement of atoms caused by the surface temperature and the presence of water molecules in the molecular dynamics [53].[45].Te values of catalytic site amino acid for Phe324 (CEase) and His263 (PL) were 1.121 Å, 0.884 Å, 0.877 Å, and 0.504 Å for CEase, CEase-UA, PL, and PL-UA complex, respectively, which meant that the addition of UA enhanced the structure stability for both CEase and PL [54].It suggested that the interaction had little efect on the stability of CEase while it stabilized the structure of PL.
Te radius of gyration (Rg) was used to determine the tightness of the complexes of ligand and protein.As shown in Figure 7(e), the average values of Rg for the free enzyme (23.6) are higher than those of CEase-UA (23.4), indicating that the insertion of UA tightened the structure of CEase.Te average Rg of the PL-UA system was decreased compared with that of free PL (Figure 7(f )).Mutual repulsion eventually leads to the extrusion of the double helix between the hydrophobic groups of PL and the hydrophilic residues of UA [32].In general, the Rg values of the complex remained at a low level during the simulation.CEase and PL had good spatial compactness after combining with UA.
Solvent accessible surface area (SASA) is a parameter for estimating the exposure of amino acids to solvents, depending on the primary and secondary structure of the   Values were expressed as mean ± standard deviation.Journal of Food Biochemistry     protein [55].Total SASA values of CEase-UA and PL-UA fuctuated less and the curve leveled of compared to those of the free enzymes in the 5-30 ns (Figures 7(g) and 7(h)), indicating that UA lowered the solvent accessible surface area of both enzymes to induce aggregation of the enzymes and molecular rearrangement.Te MD simulation results showed that UA changed the structure of CEase and PL by reducing the fexibility of conformation, improving tightness and stability.
Quenching.Fluorescence spectroscopy is a sensitive instrument to evaluate the tertiary structure characteristics of CEase and PL.Te fuorescence intensity was closely associated with phenylalanine, tryptophan, and tyrosine.Te excitation and emission light of the

Figure 1 :
Figure 1: Inhibition types and kinetics of UA on CEase (a) and PL (b).Te inhibition rate (A) of CEase and PL by UA and orlistat; reversible test plots (B) of CEase and PL; Lineweaver-Burk plots (C) of UA on CEase and PL.Inset: the linearly ftted secondary plots of slope (D) or intercept versus [I] (E) of CEase.

Figure 2 :
Figure 2: (a) Te micelle solubility inhibition rate; (b) variation of micelle size and polydispersity index; (c) control micelles without UA; (d) micelles plus 0.5 mg/mL UA.Each measurement was repeated three times and data are presented as mean ± SD.

Figure 5 :
Figure 5: ITC for UA binding to CEase (a) and PL (b) at 298 K: (upper) raw data plot of titration heat fow with time (sec); (below) total heat released by titration with UA concentration.Te black line indicates the least squares regression best-ft line.Te histogram shows the changes in the thermodynamic parameters.

Figure 6 :
Figure 6: Molecular docking of CEase-UA (a) and PL-UA (b).Te most appropriate confrmation (A), hydrophobic surface (B), and 2D schematic diagram (C).Te interaction types are explained by the color of the legend.

Figure 7 :
Figure 7: RMSD (a, b), RMSF (c, d), Rg (e, f ), and SASA (g, h) of CEase and PL in free and combined states with UA.
Docking.Molecular docking of UA with CEase and PL was simulated by Discover Studio 3.5 (DS3.5 Accelrys, USA).Te 3D structures of CEase and PL were procured from the Protein Data Bank (CEase ID: 1AQL, PL .com).Water molecules were removed from CEase and PL, and missing hydrogen atoms were added before the simulation.Te interaction model with the highest LibDock score was deemed as the optimal confguration.

Table 2 :
Te averaged binding free energies of the simulated CEase-UA and PL-UA.