Modulatory Effect of Pyrus pyrifolia Fruit and its Phenolics on Key Enzymes against Metabolic Syndrome: Bioassay-Guided Approach, HPLC Analysis, and In Silico Study

This study aims to isolate the active constituents of Pyrus pyrifolia Nakai fruits using a bioassay-guided fractionation approach, test their activity in vitro against key enzymes for metabolic disorders, and support it with molecular docking simulations. The antioxidant potential of the methanolic extract (ME), its polar (PF), and non-polar fractions (NPF), along with the inhibitory activity against α-glucosidase, α-amylase, lipase, angiotensin I converting enzyme (ACE), renin, inducible nitric oxide synthase (iNOS), and xanthine oxidase (XO) were assessed. The PF exhibited the highest antioxidant and enzyme inhibitory activity. Purification of PF yielded rutin, isoquercitrin, isorhamnetin-3-O-β-D-glucoside, chlorogenic acid, quercetin, and cinnamic acid. HPLC-UV analysis of the PF allowed for the quantification of 15 phenolic compounds, including the isolated compounds. Cinnamic acid was the most powerful antioxidant in all assays and potent enzyme inhibitor against the tested enzymes (α-glucosidase, α-amylase, lipase, ACE, renin, iNOS, and XO). Additionally, it showed high affinity to target α-glucosidase and ACE active sites with high docking scores (calculated total binding free energy (ΔGbind) -23.11 kcal/mol and − 20.03 kcal/mol, respectively]. A 20-ns molecular dynamics simulation using MM-GBSA analysis revealed a stable conformation and binding patterns in a stimulating environment of cinnamic acid. Interestingly, the isolated compounds’ dynamic investigations including RMSD, RMSF, and Rg demonstrated a stable ligand − protein complex to the active site of iNOS with ΔGbind ranging from − 68.85 kcal/mol to -13.47 kcal/mol. These findings support the notion that P. pyrifolia fruit is a functional food with multifactorial therapeutic agents against metabolic syndrome-associated diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s11130-023-01069-3.

all of analytical grade. An electrothermal 9100 (UK) was used for the determination of melting points (uncorrected). A Jenway model 6800 spectrophotometer was utilized for recording UV spectra. A Tecan, microplate reader, ((Infinite F50, Switzerland)) was used to measure the absorbances. A Bruker NMR system was used for 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz).
The NMR spectra were determined in CD3OH and DMSO-d6. Chemical shifts are given in δ (ppm) relative to the internal standard TMS. Phenolic acids and flavonoids used in HPLC analysis as well as all other reagents for biological investigations were obtained from Sigma Chemical Company (CA, USA).

Determination of total phenolic (TPC) and flavonoid (TFC) contents of P. pyrifolia fruits methanolic extract (ME), non-polar (NPF), and polar (PF) fractions
The TPC was determined by using the Folin-Ciocalteu method according to the procedure described previously [1] and was calculated as gallic acid equivalent (GAE). While the TFC was evaluated by using AlCl3 method [2] and was expressed as quercetin equivalent (QE).

Inhibitory activity against key enzymes related to metabolic syndrome (MS)
All assays were performed in 96-well plates. Stock solutions of the extract (100 mg/mL), fractions (100 mg/mL), and isolated compounds (1000 µM) were prepared. Tested samples that exceeded 50% inhibition at these concentrations were serially diluted to determine their half-maximal inhibitory concentration (IC50). The percentage of enzyme inhibition was calculated according to the equation: A is the activity of the enzyme without tested samples, B is the control of A with neither tested samples nor enzyme, C, and D denote the activity of the test solutions with and without the tested enzyme, respectively.
Starch [50 μL, 1% phosphate-buffered saline (PBS)] was mixed as substrate with 13 U mL −1 pancreatic α-amylase prepared in PBS, and 50 μL of test solution. A blank was performed using PBS. To initiate the reaction, the enzyme was added to the assay solution after being pre-incubated separately at 37 °C in a water bath for 10 min. Dinitrosalicylic acid reagent (1 mL) was added and heated at 85 °C for 15 min. Then, it was cooled to room temperature and distilled water (1 mL) was added. Acarbose was used as a reference drug. The absorbance was recorded at 540 nm.

Pancreatic lipase inhibitory activity
The pancreatic lipase inhibitory activity was assessed using p-nitrophenyl dodecanoate (p-NPD) as substrate and porcine pancreatic lipase (Abnova, Taipei, Taiwan) which was described previously [7]. Orlistat was used as a reference drug. The tested samples were diluted in DMSO were incubated with 10 g L−1 PL (diluted in 0.05 mol L−1 Tris-HCl buffer pH 8.0, containing 0.010 mol L−1 CaCl2 and 0.025 mol L−1 NaCl) for 20 min at 37 °C. p-NPP (0.008 mol/L, diluted in 0.5% TritonX 100) (m v−1) was added to initiate the reaction. The absorbances were measured at 410 nm for 30 min at 37 °C.

Angiotensin conversion enzyme (ACE) inhibitory activity
Assessment of ACE inhibitory activity was performed using an earlier published method [8] using ACE1 solution (EC: 3.4.15.1, Biovision, California, United States) and histidine-l-hippuryl-lleucine-chloride (HHL) as a chromogenic synthetic substrate. Zofenopril was used as a reference drug. Forty μL of the enzyme solution (2 mU ACE produced in 0.1-M Na borate buffer) was mixed with 20 μL of each tested dilution of each sample and incubated at 37°C for 10 minutes, followed by the addition of 40 μL HHL substrate (0.8 mM/L) and incubation for 1 hour at 37 °C. Sixty μL of 0.5 M sodium hydroxide was added to stop the process. The blank solution for each sample was prepared by substituting the buffer solution for the enzyme solution. Methanol was used to make the control solutions in place of the sample. Fluorescence was measured at excitation (360 nm) and emission wavelengths (500 nm).

Renin inhibitory activity
The renin inhibitory activity was examined according to a previously described method [9] using renin-inhibitor screening assay kit (BPS Bioscince, San Diego, CA, USA) and quinapril as a reference drug. Recombinant renin enzyme (50 μL) was dissolved in 50-mM Tris-HCl buffer (pH 8.0) and 100-mM NaCl (assay buffer) and stored at -80°C for further analysis. Twenty μL of renin substrate (A500 μM in DMSO), 150 μL of assay buffer, and 10 μL of each sample were used to prepare test solutions (10mg/ml in methanol). The blank samples were made using 10 μL of the sample, 20 μL of the substrate, and 160 μL of assay buffer. The positive control samples were created using 10 μL of methanol, 20 μL of substrate, and 150 μL of assay buffer. The positive control and tested samples were combined with 10 μL of the renin solution for the assay in order to catalyze the reaction. After that, the reaction mixture was incubated for 45 minutes at 37 °C.
The fluorescence generated at the 340 nm excitation and 490 nm emission wavelengths was measured using a microplate reader.

Xanthine oxidase (XO) inhibitory activity
Xanthine oxidase (XO) inhibitory potential was carried out according to a previous designed procedure [10] using xanthine oxidase (XO, EC 1.17.3.2; Biovision, USA). Allopurinol was applied as a reference drug. Immediately prior to use, a mixture was made up of 50 μL of sample solution, 35 μL of phosphate buffer (70 mM, pH = 7.5), and 30 μL of new enzyme solution (0.01 units/mL in the same buffer). The reaction was started by adding 60 μL of the substrate solution (150 μM xanthine in the same buffer) following a pre-incubation at 25 °C for 15 minutes. After that, the mixture was incubated at 25 °C for 30 min. After adding 25 μL of 1 M HCl to terminate the reaction, the absorbance was measured at 290 nm with a microplate reader as previously mentioned. The same procedure was used to prepare a blank, except after pipetting HCl, the enzyme solution was added.

Inducible nitric oxide synthase (iNOS) inhibitory activity
iNOS inhibitory activity was determined in mouse macrophage cells (RAW264.7, Shanghai BOGO Industrial Co., Ltd., China) according to a previously designed method [11]. Phenol redfree RPMI medium from Thermo-Fisher was used to develop macrophages. It was supplemented with 10% bovine calf serum, 100 U/mL of penicillin G sodium from Sigma-Aldrich, and 100 g/mL of streptomycin (Sigma-Aldrich). Cells (50,000/well) were seeded onto 96-well plates, which were then incubated for 24 hours. The cells were treated with the tested samples for 30 min. The cells were induced by lipopolysaccharide (LPS) (Sigma-Aldrich) (5 g/mL), and the incubation process lasted for 24 hours. By measuring the amount of nitrite in the cell culture supernatant using Griess reagent, the activity of iNOS (Sigma-Aldrich, St. Louis, MO) was calculated in terms of NO concentration. Cell viability was determined with MTT reduction assay [12]. Absorbances were measured at 540 nm using a microplate reader. The positive control was parthenolide. analyzed. Comparing retention times of the peaks with those of the standard phenolics allowed for qualitative determination, whereas peak area measurement allowed for quantitative determination.

Molecular Docking Analysis
Molecular docking study was performed on the isolated compounds to study the mode of their interaction in the active site of the yeast isomaltase from Saccharomyces cerevisiae (Pdb id:3A4A) with 84% similarity to S. cerevisiae α-glucosidase, human angiotensin-converting enzyme (ACE) (Pdb id: 2XYD), and nitric oxide synthase (NOS) enzyme (Pdbid: 3E7G) [13][14][15] using Auto Dock Tools (version 1.5.6). These structures were then prepared for molecular dynamics (MD) studies using UCSF Chimera [16]. Using PROPKA, pH was fixed and optimized to 7.5 [17]. The structures of compounds (P1-P6) were drawn using ChemBioDraw Ultra 12.1 [18]. For αglucosidase, ACE and NOS all prepared systems were subjected to 20 ns MD simulations, as detailed in the simulation section.

Molecular dynamic (MD) simulations
The application of molecular dynamic (MD) simulations in the study of biological systems allows for the exploration of physical motions of atoms and molecules that are not easily accessible by other techniques [19]. The understanding gained from running this simulation provides a detailed look into the dynamical evolution of biological systems, such as conformational changes and molecule interaction [19]. The MD simulations of all systems were carried out using the GPU version of the PMEMD engine included in the AMBER 18 package [20].
The partial atomic charge of each compound was calculated with ANTECHAMBER's General Amber Force Field (GAFF) technique [21]. The Leap module of the AMBER 18 package implicitly solvated each system within an orthorhombic box of TIP3P water molecules within 10 Å of any box edge. The Leap module was used to neutralize each system by incorporating Na + and Clcounter ions. A 2000-step initial minimization of each system was carried out in the presence of a 500 kcal/mol applied restraint potential, followed by a 1000-step full minimization using the conjugate gradient algorithm without restraints.
During the MD simulation, each system was gradually heated from 0K to 300K over 500ps, ensuring that all systems had fixed number of atoms and fixed volume. The system's solutes were subjected to a 10kcal/mol potential harmonic constraint and a 1ps collision frequency. Following that, each system was heated and equilibrated for 500ps at a constant temperature of 300K. To simulate an isobaric-isothermal (NPT) ensemble, the number of atoms and pressure kept constant within each system for each production simulation, with a stable system's pressure at 1 bar using the Berendsen barostat [22].
For 20 ns, each system was MD simulated. The SHAKE method was used to constrain the hydrogen bond atoms in each simulation. Each simulation used a 2fs step size and integrated an SPFP precision model. An isobaric-isothermal ensemble (NPT) with randomized seeding, constant pressure of 1 bar, a pressure-coupling constant of 2ps, a temperature of 300K, and a Langevin thermostat with a collision frequency of 1ps was used in the simulations.

Post-MD Analysis
After saving the trajectories obtained by MD simulations every 1 ps, the trajectories were analyzed using the AMBER18 suite's CPPTRAJ [23] module. The Origin [24] data analysis program and Chimera [16] were used to create all graphs and visualizations.
The Poisson-Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA and MM/GBSA) approach has been found to be useful in the estimation of ligand-binding affinities [25][26][27]. The Protein-Ligand complex molecular simulations used by MM/GBSA and MM/PBSA compute rigorous statistical-mechanical binding free energy within a defined force field. Binding free energy averaged over 200 snapshots extracted from the entire 20 ns trajectory. The estimation of the change in binding free energy (ΔG) for each molecular species (complex, ligand, and receptor) can be represented as follows [28]: ∆G bind = E gas + G sol − TS (2) The terms Egas, Eint, Eele, and Evdw symbolize the gas-phase energy, internal energy, Coulomb energy, and van der Waals energy. The Egas was directly assessed from the FF14SB force field terms. Solvation free energy (Gsol) was evaluated from the energy involvement from the polar states (GGB) and non-polar states (G). The non-polar solvation free energy (GSA) was determined from the Solvent Accessible Surface Area (SASA) [29,30] using a water probe radius of 1.4 Å.
In contrast, solving the GB equation assessed the polar solvation (GGB) contribution. Items S and T symbolize the total entropy of the solute and temperature, respectively.

Statistical analysis
All determinations were performed in triplicate and were represented as mean ± standard deviation.
Microsoft Excel® was used to analyze all IC50 data, and GraphPad Prism 8® was used to calculate the IC50 values by converting the concentrations to their logarithmic value and then choosing non-linear inhibitor regression equation (log inhibitor) vs normalized response-variable slope equation.
One-way ANOVA was used for the statistical analysis, followed by Tukey's test and p values <0.05 were considered significant.

Identification of the critical residues responsible for ligands binding
The total energy involved when rutin, isoquercitrin, isorhamnetin-  Table S1. TPC, TFC and antioxidant activity using ABTS, FRAP and ORAC methods of P.