Cellulose filter paper immobilized α-glucosidase as a target enzyme-oriented fishing tool for screening inhibitors from Cyclocarya paliurus leaves

2.1 Materials

α-Glucosidase (EC 3.2.1.20, G8823-1KU) was extracted from yeast and obtained from Solarbio Technology Co., Ltd. (Shanghai, China). p-Nitrophenyl-α-D-glucopyranoside (p-NPG) was supplied by YuanYe Biotechnology Co., Ltd. (Shanghai, China). Standards of (+)-catechin, ferulic acid and quercetin were purchased by National Institutes for Food and Drug Control (Beijing, China), and kaempferitrin and arjunolic acid were obtained from the Chengdu Herbpurify Co. Ltd. (Chengdu, China). Dopamine hydrochloride (PDA) and polyethyleneimine (PEI) were supplied by Macklin Biochemical Co., Ltd. (Shanghai, China). Tris (hydroxy-methyl) aminomethane (Tris, 99%) were obtained from Solarbio Technology Co., Ltd. (Shanghai, China). The medium-speed qualitative filter paper was purchased from Fushun Civil Affairs Filter Paper Factory (Liaoning, China). D101 macroporous resin was purchased from an industrial chemical company affiliated to Nan Kai University (Tianjin, China). Acetonitrile and formic acid were of HPLC grade and obtained from Millipore Co. (Merck, Darmstadt, Germany). Ultrapure water was prepared by a Milli‐Q water purification system (Millipore, Bedford, MA, USA).

2.2 Instruments and chromatographic method

The chromatographic analysis was carried out by an Agilent 1290 Ultra performance liquid chromatograph (UPLC) system coupled with quaternary solvent delivery system, auto-sampler, column oven and vacuum degasser. Chromatographic separation was performed on an Agilent ZORBAX Eclipse Plus C18 column (3.0 mm × 150 mm, 1.8 µm), and column temperature was set at 35℃. 0.1% formic acid in acetonitrile and 0.1% formic acid in water were selected as the mobile phase at a flow rate of 0.20 mL/min. For the analysis of the artificial model mixture, gradient elution program was as follows: 0–15 min, 20–80% A. For 70% eluent fraction of C. paliurus leaves, gradient elution program was as follows: 0–3 min, 20–40 % A; 3–5 min, 40–50 % A; 5–15 min, 50–75 % A; 15–45 min, 75–100 % A; 45–50 min, 100 % A (Li et al., 2022; He et al., 2022); Each solution was filtrated, and the volume of injection was 2 μL.

An Agilent 6545 quadrupole time-of-flight (QTOF) tandem mass spectrometer (Agilent Technologies, USA) equipped with a Dual AJS electrospray ionization source (ESI) was performed for MS analysis in negative ion mode. The acquisition parameters for MS/MS analysis were set as follows. Both the drying gas temperature and sheath gas temperature were maintained at 350 ℃. The flow rates of drying gas and sheath gas were 12.0 L/min and 10.0 L/min, respectively. The capillary voltage was set at 3.5 kV. The fragmentor voltage and skimmer voltage were 130 V and 65 V, respectively. The mass spectra were recorded at a scan range of m/z 100–1000, and the collision energies of MS² spectra were optimized from 10 to 50 eV.

2.3 Extraction and purification of C. paliurus leaves

Dried C. paliurus leaves were purchased from Zhangjiajie, Anhui Province, in China and identified by Prof. Ling Jin (College of Pharmacy, Gansu University of Chinese Medicine, Lanzhou, China). The extraction method of C. paliurus leaves was according to previous studies (He et al., 2022; Sun et al., 2020). Samples (50 g) were extracted twice by ultrasonication with 60% ethanol (450 mL and 350 mL) for 60 min each time. The extraction solutions were combined, filtrated and then concentrated to dryness by a rotary vacuum evaporator under reduce pressure. The residue was re-dissolved in 5% ethanol solution with the final concentration of 50 mg/mL. The obtained sample solution was transferred to column chromatography (30 × 3 cm) on D101 macroporous resin, and then eluted successively using water and 30, 50, 70 and 95% ethanol solutions. Five fractions were condensed and stored in a refrigerator at 4℃.

2.4 Preparation of α-glucosidase immobilized on CFP

α-Glucosidase immobilization was performed according to the method of our group (Li et al., 2021b). Briefly, the CFP was immersed into PDA and PEI (2 mg/mL each) dissolved 0.1 M tris buffer (pH 8.5), and coated for 7 h at 150 r/min and 30 °C to obtain PDA/PEI-modified CFP. Then, the modified CFPs (Diameter, 6 mm) were immersed into 0.05 mg/mL of α-glucosidase and shaken for 1 h at 150 r/min and 30 °C. The obtained CFP-immobilized α-glucosidase were washed several times with PBS (pH 6.0) and dried at 35 °C for subsequent screening.

2.5 Ligand fishing evaluation by an artificial model mixture

Artificial model compounds, which consisted of (+)-catechin (1) (Fu et al., 2021), ferulic acid (2) (Wang et al., 2018b) and quercetin (3) (Shen et al., 2020), were dissolved in 20 mM PBS (pH 8.0) containing 5 % DMSO to obtain the model solution (S₀, 1 mg/mL each). 6 discs of CFP-immobilized α-glucosidase were incubated with 2 mL of the model solution for 30 min at 70 °C. After incubation, the CFP discs were directly taken out by a tweezer and washed with 3 × 600 μL of PBS buffer to obtain the washing solutions (S₁-S₃). Then, the discs were eluted with 3 × 600 μL of 80% methanol for 10 min each time to obtain dissociation solutions (S₄-S₆). Solutions (S₀, S₁-S₃ and S₄-S₆) were analyzed by UPLC-QTOF-MS/MS.

2.6 Screening α-glucosidase inhibitors from C. paliurus leaves by ligand fishing

Both 70% eluent fraction of C. paliurus leaves and quercetin as the positive control were dissolved in PBS (20 mM, pH 8.0) containing 5 % DMSO to obtain the mixed solution with final concentrations of 2.0 mg/mL and 1 mg/mL, respectively. 2 mL of the mixed solution was incubated with 6 discs of CFP-immobilized α-glucosidase for 30 min at 70 °C. Active compounds, which exhibited specific affinity interactions with α-glucosidase, would form enzyme-inhibitor complexes, while inactive components with no or low affinity to α-glucosidase would keep free states. After incubation, the CFPs discs were instantaneously taken out and then washed three times with 600 μL of PBS to remove inactive components. Afterwards, the CFPs discs were immersed in 600 μL of 80% methanol for three times, 10 min each time, and then active compounds were dissociated from complexes. The dissociation solutions were collected and evaporated, and then the residue was re-dissolved in 80% methanol for UPLC-QTOF-MS/MS analysis.

2.7 α-Glucosidase inhibition assay

The inhibition activity of potential inhibitors on α-glucosidase was evaluated according to the method with some modification (Shen et al., 2020). α-Glucosidase and p-NPG were dissolved in 0.1 M PBS (pH 6.86) to obtain concentrations of 1 U/mL and 5.0 mM, respectively. 40 µL of α-glucosidase, 40 µL of potential inhibitors and 80 µL of PBS were pre-incubated for 10 min at 37 ℃. Then, 40 µL of p-NPG was added into the mixture to initiate the enzymatic reaction, and the mixture was incubated for 20 min. The absorbance of the mixture was measured at 405 nm. The inhibition rate (%) was calculated according to equation: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)=1-\frac{\left(\mathrm{A}\mathrm{i}-\mathrm{A}\mathrm{b}\mathrm{i}\right)}{(\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{b}\mathrm{c})}\times 100\%$$

Where A_c, A_bc, A_i and A_bi present absorbances of the mixture (enzyme without inhibitor, no enzyme and inhibitor, enzyme and inhibitor, and inhibitor without enzyme), respectively.

2.8 Molecular docking

Molecular docking was carried out to further investigate affinity interactions between potential inhibitors and α-glucosidase by Autodock 4.2 software. According to the previous studies (Ning et al., 2019; Shen et al., 2020), α-glucosidase (PDB ID: 3A4A) was utilized to perform the docking assay, and its crystal structure was obtained from the Protein Data Bank ( https://www.rcsb.org/pdb). The original ligands and water of α-glucosidase were removed by PyMOL software. The hydrogen atoms and gasteiger charges were added to α-glucosidase, and then the file was saved as receptor.pdbqt by AutoDockTools. The 3D structures of potential inhibitors were acquired by ChemBio3D Ultra 14.0, and then their torsions along with rotatable bonds were assigned and the files were saved as ligand.pdbqt by AutoDockTools, respectively. The semi-flexible docking was performed by Autodock Vina (Hu et al., 2021; Li et al., 2022). The grid parameters were shown as follows: x  = 25.38, y = -2.75 Å, z = 18.186, and the dimensions 52 × 68 × 62 Å (Hu et al., 2021). The docking result with the lowest binding energy and the highest scoring was selected to be the most optimal docking model of the α-glucosidase-inhibitor complex. The binding interactions between potential inhibitors and α-glucosidase were visualized using Discovery Studio 4.5 and PyMOL software.

3.1 Ligand fishing evaluation by an artificial model mixture

The artificial model mixture, including (+)-catechin (1), ferulic acid (2) and quercetin (3), was used to investigate the feasibility of ligand fishing assay. Quercetin and (+)-Catechin as two known α-glucosidase inhibitors were employed as the positive controls (Wan et al., 2021; Shen et al., 2020; Fu et al., 2021), while ferulic acid as the negative control had a weak affinity to α-glucosidase (Wang et al., 2018b). The 6 discs of CFP-immobilized α-glucosidase were incubated with 600 μL of model solution (S₀) to form enzyme-inhibitor complexes for 30 min at 70℃. After incubation, the CFP discs were instantaneously taken out by a tweezer. The discs were washed three times with 600 μL of PBS buffer to remove weak affinity to α-glucosidase from enzyme-inhibitor complexes (S₁-S₃), and then immersed in 600 μL of 80% methanol to denature α-glucosidase for the dissociation of strong α-glucosidase inhibitors (S₄-S₆). As shown in Fig. 2, quercetin and (+)-catechin as the positive control were successfully fished out from S₀ and could be determined in dissociation solutions S₄-S₆. Quercetin could have the stronger affinity than (+)-catechin due to the amount of quercetin was higher than (+)-catechin which were released by denaturing CFP-immobilized α-glucosidase in S₄-S₆. However, ferulic acid as the negative control was not detected in S₄-S₆ at all, because it was washed gradually in washing solution S₁-S₃ due to its weak affinity to α-glucosidase. The result was just as we expected and similar with the literatures (Wang et al., 2018b; Shen et al., 2020), indicating that the affinity screening strategy based on ligand fishing and UPLC-QTOF-MS/MS was effective and reliable to screen natural α-glucosidase inhibitors in the artificial model mixture.

Close

Fig. 2

Extract ion chromatograms of the artificial model mixture (S₀), PBS washing solutions (S1-S3) and dissociation solutions (S4-S6) in the feasibility evaluation of ligand fishing assay.

Export to PPT

3.2 Screening α-glucosidase inhibitors from C. paliurus leaves by ligand fishing assay

Enzyme inhibition assay in vitro was used to estimate inhibitory activities of five fractions of the crude extract from C. paliurus leaves on α-glucosidase. The results showed that the 70% ethanol fraction of C. paliurus leaves showed significant inhibitory activity against α-glucosidase, compared with the other four fractions. It was obvious that both 70% ethanol fraction and quercetin (as the positive control) inhibited α-glucosidase activity in a concentration-dependent manner and their IC₅₀ values were calculated to be 17.81 ± 0.18 µg/mL and 4.15 ± 0.04 µg/mL, respectively. Results suggested that 70% ethanol fraction might contain active compounds against α-glucosidase.

The 70% ethanol fraction containing quercetin was incubated with CFP-immobilized α-glucosidase for 30 min at 70℃. Quercetin was used as the positive control to further verify reliability and practicality of the proposed ligand fishing method in the extract from C. paliurus leaves. After incubation, potential inhibitors would form α-glucosidase-inhibitor complexes, and then complexes were separated from the reaction solution in virtue of the instantaneously-separated characteristic of CFP. The discs were washed with PBS buffer (3 × 600 μL), and then active components would be released from the complexes by adding 80% methanol (3 × 600 μL). As shown in Fig. 3, a total of 37 peaks were determined in the chromatogram, implying that these compounds could have binding abilities to α-glucosidase (Fig. 2 B). In addition, quercetin was successfully captured and detected at 10.30 min (Fig. 2 C), suggesting that the ligand fishing assay is reliable and applicable for screening natural α-glucosidase from complex matrixes.

Close

Fig. 3

Total ion chromatograms of 70% ethanol fraction of Cyclocarya paliurus leaves (A), screened active compounds by CFP-immobilized α-glucosidase (B), active components and quercetin (as positive control) fished out by CFP-immobilized α-glucosidase (C).

Export to PPT

3.3 Identification of potential α-glucosidase inhibitors

Dammarane triterpenoids are characteristic indicators and active compounds of C. paliurus, and exhibited anti-diabetic, anti-hyperglycemia, anti-oxidative and anti-inflammatory effects (Li et al., 2021a; Sun et al., 2020; Zhou et al., 2021; Zhao et al., 2019). 3,4-seco-dammarane triterpenoids of C. paliurus are rare in natural plants, characterized by the breaking of the chemical bond between C-3 and C-4 of dammarane triterpenoids. The differences in the chemical structures of triterpenoids were mainly due to the existence of a variety of aglycones, and types of glycosyl moieties and their connecting positions on the aglycones. In the negative ion mode, dammarane triterpenoids, except for 3,4-seco-dammarane triterpenoids, often form addition ions [M + HCOO]⁻ due to the addition of formic acid in the mobile phase. Retention time (Rt), calculated and measured molecular mass, adduct ion, mass error, MS/MS fragment ions, molecular formula, identification and references are summarized in Table 1. Chemical structures of potential α-glucosidase inhibitors are shown in Fig. 4.

Close

Fig. 4

Chemical structures of screened potential α-glucosidase inhibitors from Cyclocarya paliurus leaves.

Export to PPT

Compound 5, 17, 20, 22 and 26 exhibited same adduct ions at m/z 841.49 [M + HCOO]⁻ and molecular ions at m/z 795.49 [M−H]⁻, which produced fragment ions m/z 753.47 [M−H−42 Da]⁻, m/z 735.46 [M−H−42 Da-18 Da]⁻, 621.43 [M−H−42 Da-132 Da]⁻, m/z 607.41 [M−H−42 Da-146 Da]⁻, and m/z 131.03 [Ara-H₂O-H]⁻, Hence, they were cyclocarioside A/H, compound 2 and cypaliuruside V isomers (Li et al., 2012; Yan et al., 2021; Zhu et al., 2021), and the MS/MS spectrum and fragmentation pattern of cyclocarioside H are shown in Fig. 5. Compound 16 had the adduct ion at m/z 827.47 [M + HCOO]⁻ and molecular ion at m/z 781.47 [M−H]⁻. It gave fragment ions at m/z 739.46 [M−H−42 Da]⁻, m/z 721.45 [M−H−42 Da-18 Da]⁻, m/z 607.42 [M−H−42 Da-132 Da]⁻, m/z 131.03 [Ara-H₂O-H]⁻ and m/z 101.02 [Ara-H₂O-CH₂O-H]⁻, which was identified as cyclocarioside C (Jiang et al., 2006), and its MS/MS spectrum and fragmentation pattern are shown in Fig. S1.

Close

Fig. 5

MS/MS spectrum and fragmentation pattern of cyclocarioside H of C. paliurus leaves in negative ion mode.

Export to PPT

Compound 12 gave the molecular ion at m/z 635.41 [M−H]⁻, which were identified as pterocaryoside A based on fragment ions at m/z 535.32 [M−H−100 Da]⁻, m/z 489.35 [M−H−146 Da]⁻, m/z 471.34 [M−H−146 Da-18 Da]⁻, m/z 389.26 [M−H−146 Da-18 Da-82 Da]⁻, m/z 371.25 [M−H−146 Da-18 Da-82 Da-18 Da]⁻, and m/z 101.02 (Kennelly et al., 1995). Its MS/MS spectrum and fragmentation pattern are shown in Fig.S2. Compound 29, 32 and 37 exhibited same molecular ions at m/z 663.44 [M−H]⁻, which were identified as cypaliuruside K/S and cyclocarioside Y isomers based on fragment ions at m/z 535.32 [M−H−28 Da-100 Da]⁻, m/z 517.38 [M−H−146 Da]⁻, m/z 499.37 [M−H−146 Da-18 Da]⁻, m/z 389.26 [M−H−28 Da-100 Da-146 Da]⁻, m/z 371.25 [M−H−28 Da-100 Da-146 Da-18 Da]⁻ and m/z 101.02 (Zhou, et al., 2021; Liu et al., 2020; Zhu et al., 2021). The MS/MS spectrum and fragmentation pattern are shown in Fig. 6. Compound 25 and 27 exhibited same molecular ions at m/z 603.38 [M−H]⁻, and further produced fragment ions at m/z 521.31 [M−H−82 Da]⁻, m/z 471.34 [M−H−132 Da]⁻, m/z 453.33[M-H-132 Da-18 Da]⁻, m/z 389.26 [M−H−82 Da-132 Da]⁻, m/z 371.25 [M−H−82 Da-132 Da-18 Da]⁻ and m/z 101.02. Hence, they are identified as cypaliuruside A and cyclocarioside I isomers (Cui et al., 2015; Zhou et al., 2021) and the MS/MS spectrum and fragmentation pattern are shown in Fig.S3. Compound 18 had molecular ion m/z 633.39 [M−H]⁻, which generated fragment ions at m/z 551.32 [M−H−82 Da]⁻, m/z 471.34 [M−H−162 Da]⁻, m/z 453.33 [M−H−162 Da-18 Da]⁻, m/z 389.26 [M−H−82 Da-162 Da]⁻, m/z 371.25 [M−H−82 Da-162 Da-18 Da]⁻, m/z 161.04 [Glu-H]⁻, and m/z 101.02. Therefore, it was identified as cypaliuruside J (Zhou et al., 2021), and the MS/MS spectrum and fragmentation pattern are shown in Fig. S4.

Close

Fig. 6

MS/MS spectrum and fragmentation pattern of cypaliuruside K of C. paliurus leaves in negative ion mode.

Export to PPT

3.4 α-Glucosidase inhibition assay in vitro

To further validate the reliability of ligand fishing assay, inhibitory activities of active compounds against α-glucosidase were evaluated by enzyme inhibition assay in vitro. However, it is worth mentioning that commercially available standards from C. paliurus are few. IC₅₀ values of kaempferitrin and arjunolic acid were measured, and they are 1.12 mM and 339.02 μM, respectively. In addition to, cypaliuruside J, cypaliuruside D, cypaliuruside I, cypaliuruside K, cyclocarioside Z9, cyclocarioside Z13 and cyclocarioside Z14 showed remarkable inhibitory activities against α-glucosidase with IC₅₀ values of 2.22 µM, 24.25 µM, 65.97 µM, 175.31 µM, 282.23 µM, 330.29 µM and 369.54 µM, respectively (Li et al., 2021a; Zhou et al., 2021). Sun et al., (2020) found that cyclocarioside Z5 and cyclocarioside Z7 significantly increased glucose consumption in 3 T3-L1 adipocytes, which could be active components for the anti-diabetes effect (Sun et al., 2020). The result and literature reports demonstrated that the ligand fishing strategy based on CFP-immobilized α-glucosidase coupled with UPLC-QTOF-MS/MS could successfully screen the potential α-glucosidase inhibitors from C. paliurus leaves.

3.5 Molecular docking

Molecular docking is an effective method to visualize interactions between ligands and receptors, and predict the possible binding sites and steric conformations of ligands (Álvarez-Chimal et al., 2022; Du et al., 2022). To further elucidate binding interactions of screened potential inhibitors and α-glucosidase, molecular docking was performed. As shown in Table 2 and Fig. 7, Fig. 8, Fig. S5, S6, S7 and S8, cyclocarioside A, cypaliuruside K, cypaliuruside J, cyclocarioside C, cyclocarioside I and pterocaryoside A could smoothly enter the active site pocket of α-glucosidase and interacted with major amino acid residues. From the point view of the affinity, cyclocarioside A, cypaliuruside K, cypaliuruside J exhibited significantly lower binding energy (-9.4, −8.9 and −8.8 kcal/mol, respectively) than the positive control quercetin (-8.5 kcal/mol). Cyclocarioside A interacted with four amino acid residues Gly160, Arg176, Ser162, and Pro151 by forming four hydrogen bonds with the distances ranging from 2.3 Å to 3.7 Å, and formed hydrophobic forces with Pro151, Lys148, Pro149, Trp238 and Phe173 (Fig. 7). Cypaliuruside K formed five hydrogen bonds with amino acid residues Thr310, Arg315, Leu313, Ser240 and His 280, and interacted with Val232, Leu313, Lys156, Arg315, Val216, Phe178 and Tyr158 by hydrophobic forces. The H-bond distances ranged from 2.4 Å to 3.7 Å (Fig. 8). Cypaliuruside J interacted with amino acid residues Ser240, Arg315, Asp307, Pro312, Thr310 and Leu313 by forming seven hydrogen bonds with the average distance of 2.6 Å, and formed hydrophobic bindings with Tyr158, Arg315 and Val216, Phe159 and phe178 (Fig. S5). Although cyclocarioside C, cyclocarioside I and pterocaryoside A had slightly high binding energy (-8.2, −8.1 and 8.0 kcal/mol, respectively), which was close to the affinity of acarbose (-8.1 kcal/mol). Cypaliuruside C might inhibit the α-glucosidase activity by forming four hydrogen bonds with amino acid residues Asp363, Asp362, Gly361 and Ser544, and forming hydrophobic bindings with Lys523, Lys524, Leu323 and Phe321. The H-bond distances ranged from 2.0 Å to 3.5 Å (Fig. S6). As shown in Fig. S7 and Fig. S8, cyclocarioside I and pterocaryoside A formed four hydrogen bonds with amino acid residues Asp363, Asp362 and Phe321, and three hydrogen bonds with Ser241 and Asp242, and their average H-bonds distances were 2.15 Å and 2.33 Å, respectively. They interacted with amino acid residues Leu439 and Phe321 (cyclocarioside I), and with Tyr158, Phe314, and Lys156 (pterocaryoside A) by hydrophobic forces, respectively. Meanwhile, pterocaryoside A interacted with Tyr158 by the pi-sigma interaction. Some studies have reported that amino acid residues Arg 315, Pro312, Tyr158, Pro312, Asp242, Lys156, Ser241, Leu 313, His280, Val216, and Phe178 of α-glucosidase played critical roles in in substrate catalysis and binding mechanism (Xie et al., 2021; Shen et al., 2020; Hu et al., 2021). The results suggested that hydrogen bonds, hydrophobic forces, and Van der Waals were key forces in the binding interactions between potential inhibitors and α-glucosidase. Previous studies demonstrated that hydrogen bonds and hydrophobic interactions between ligands and enzymes could be beneficial to enhance the stability of the inhibitor-enzyme complex, and then exerted essential effects on inhibitory activity toward α-glucosidase (Du et al., 2022; Liu et al., 2021b; Xie et al., 2021).

Close

Fig. 7

Molecular docking study of potential inhibitor cyclocarioside A interacting with amino acid residues in the active site of α-glucosidase.

Export to PPT

Close

Fig. 8

Molecular docking study of the potential inhibitor cypaliuruside K interacting with amino acid residues in the active site of α-glucosidase.

Export to PPT