A new hemiacetal chromone racemate and α-glucosidase inhibitors from Ficus tikoua Bur

Abstract From the petroleum ether and ethyl acetate portions of the 95% ethanol extract of Ficus tikoua Bur., a new hemiacetal chromone racemate, named (±)-ficunomone (1), together with twenty-two known flavonoids (2-23) were isolated. The new structure was elucidated by NMR, HRESIMS, and X-ray single-crystal diffraction analysis, and the known structures were determined by comparison of spectroscopic data with those reported from literatures. All the compounds were assayed for their inhibitory activities against yeast α-glucosidase, seven flavonoids could inhibit α-glucosidase, among which compounds 22 and 23 exhibited the highest inhibitory activity, with IC50 values at 5.12 ± 0.10 and 3.43 ± 0.15 μM respectively. Preliminary structure and relationship activity of all the compounds was analysed. Kinetic analysis of compounds 22 and 23 indicated that they are both uncompetitive inhibitors. Molecular docking studies revealed that they bound to amino acid residues of the α-glucosidase activity pocket. Graphical Abstract


Introduction
Diabetes mellitus (DM), which comprises Type 1 and Type 2, is a metabolic disease associated with insulin dysfunction and characterised by hyperglycemia. Type 2 DM is more common, accounting for about 90% of diabetic patients. Continuous postprandial hyperglycemia can cause multiple metabolic abnormalities and vascular diseases, leading to the occurrence and deterioration of various complications (Tahergorabi and Khazaei 2012). Therefore, lowering postprandial blood glucose is one of the important measures to prevent diabetes and reduce its complications and mortality. a-Glucosidase is a decisive enzyme for regulating postprandial blood sugar, thus is a critical target for the treatment of diabetes (Chiba 1997). Currently, many kinds of a-glucosidase inhibitors involving flavonoids, terpenoids, alkaloids, phenols, organic acids have been reported (Jiang et al. 2020), providing valuable clues for the search of novel antidiabetic drugs.
Ficus plants are widely distributed in tropical and subtropical regions. They can be used both for food and medicine (Shi et al. 2018). Pharmacological studies have found that most of Ficus plants exhibit anti-tumor, hypoglycemic, anti-inflammatory and analgesic effects . Ficus tikoua Bur. is traditionally used in China to treat traumatic injury, dysentery and dyspepsia (Editorial Committee of Flora of China 1998). It is documented that the fruit of F. tikoua Bur. has the effect of treating diabetes (Si et al. 2015). Previous phytochemical investigations on the plant led to the isolation of flavonoids, steroids, triterpenoids, coumarins (Xu et al. 2011;Wei et al. 2012a;2012b;Fu et al. 2018). In order to verify the antidiabetic potential of F. tikoua Bur. and its active constituents, we investigated the chemical constituents of the aerial parts of F. tikoua Bur., and screened a-glucosidase inhibitory activities of all the compounds isolated. As a result, a new racemic hemiacetal chromone, named as (±) ficunomone (1), and twenty two known compounds were obtained from the F. tikoua Bur. ( Figure  1). a-Glucosidase inhibitory activity assay was used to test the bioactivity of all the compounds. Ficusin A (22) and 6-[(1R Ã ,6R Ã )-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl] 5, 7, 4 0 trihydroxyiso-flavone (23) exhibited the highest a-glucosidase inhibitory activity (with IC 50 values at 5.12 ± 0.10 and 3.43 ± 0.15 lM respectively, the positive control acarbose at 33.93 ± 0.02 lM). Herein, we report the isolation, structural elucidation and inhibitory activities on a-glucosidase (Saccharomyces cerevisiae) of the compounds, preliminary structure and activity relationship, and the mechanisms underlying the interaction of the most active compounds with a-glucosidase.

Results and discussion
The  Figure S6) showed the presence of two hydroxyl groups [d:12.29 (1H, s), 12.05 (1H, s)], the remaining signal peaks on the spectrum appeared in pairs. On 13 C NMR spectrum ( Figure S7), the signals also appeared in pairs, and the optical rotation of compound 1 was 0 , all the information convinced us that compound 1 might be a racemic mixture made up of a pair of enantiomers, this speculation led us to try to separate the enantiomers with chiral columns, which ended up in failure, thus we further continued structural interpretation of the racemic mixture with spectra. The signals on 1 H and 13 C NMR spectra corresponding to one enantiomer were in highly similarity with those of spatheliachromen (Bohlmann et al. 1980) except the addition of a hydroxyl group [d H :7.20/7.17 (1H, s)] and a methyl group [d H :1.64/1.59 (3H, s)] at C-2, the HMBC correlations ( Figure S9) from H-3 [d H : 3.16/3.12 (1H, d, J ¼ 8.0 Hz), 2.69/2.65 (1H, s)] to 2-CH 3 (d C 28.4/28.2), from 2-CH 3 [d H : 1.64/1.59 (3H, s)] to C-3 (d C 47.6/47.5), from 2-OH [d H :7.20/7.17 (1H, s)] to C-2 (d C 102.0/101.9), C-3 (d C 47.6/47.5) and 2-CH 3 (d C 28.4/28.2) evidenced that the hydroxyl and methyl group were at C-2. That compound 1 was a racemic mixture made up of two enantiomers and its structure were further confirmed by X-ray single crystal diffraction, and a trivial name was given as (±)-ficunomone ( Figure 1).
Compounds 1 À 23 were tested in vitro against a-glucosidase (S. cerevisiae). Compounds 4,8,11,17,19,22 and 23 showed obvious inhibition on a-glucosidase at the concentration of 2 mM, and thus, their dose-response relationships were further studied to provide their IC 50 values. The result (Table S1) showed compounds 22 and 23 were most active, with IC 50 values at 5.12 ± 0.10 and 3.43 ± 0.15 lM, and the IC 50 value of the positive control (acarbose) was 33.93 ± 0.02 lM.
The structures of all the compounds and their a-glucosidase inhibitory activities demonstrated that the oxidation of bond between C-2 and C-3 of the flavonoids increased the activity, whereas glycosylation decreased the activity, which was in correspondence with that reported from literature (Tang et al. 2020). In addition, the position of the C-ring (at C-2 or C-3), the isopentenyl substituent and its position on the A ring and whether it was formed into a ring with its vicinal group all could affect the a-glucosidase inhibitory activity. In general, isoflavones with isopentenyl on their A ring had better activity (17,19,(22)(23). For flavones (4, 6-7, 9-10, 12) and flavanones (2-3, 5, 8, 11, 13-15), the isopentenyl at C-8 weakened the activity (4 and 6; 5 and 15), while the isopentenyl at C-6 enhanced the activity (5 and 11). For flavanones, the addition of 3-OH was harmful to the activity, whereas 3 0 -OH was conducive. However, when the isopentenyl group was present, 3-OH could also be beneficial to the activity (11 and 14). For isoflavones (16-23), basically, the presence of isopentenyl could improve the activity, but when it was at C-6 and formed into a pyran ring with its vicinal hydroxy group, the activity decreased (19 and 20).
Kinetic analysis of compounds 22 and 23 on a-glucosidase revealed that they both inhibited a-glucosidase in an uncompetitive mode, because the regression lines did not intersect on the y-axis in the Lineweaver-Burk plot but paralleled each other ( Figure S1).
Molecular docking results of compounds 22 and 23 showed ( Figure S2) that compound 23 performed best in terms of binding to the target, it could be clearly seen binding to the amino acid residues of the protein pocket. Compound 23 contains multiple six-membered rings and has strong hydrophobicity. It can form a strong hydrophobic interaction with amino acids (TYR-709, TRP-710, ASP-568, HIS-561, LEU-563, PHE-444, PHE-385) of the active site, playing an important role in stabilising the molecules in the protein cavity. In addition, 7-OH and 4 0 -OH can form stable hydrogen bonds with amino acid residues of LYS439 and ASP568, by which effectively anchoring small molecules into protein pockets. Compound 22 has the similar binding mode as compound 23.

General experimental procedures
Silica gel GF 254 plates (Yantai Jiangyou Silicon Development Company, Yantai, China) were used for Thin-layer chromatography (TLC) analysis, and spots were detected under UV light or by heating after being sprayed with 5% H 2 SO 4 in EtOH. Chromatographic silica gel (200 $ 300 mesh) was purchased from Qingdao PuKe Silicon Development Company (Qingdao, China). Sephadex LH-20 gel for chromatography was bought from Pharmacia Fine Chemical Co., Ltd. (Pharmacia, Germany). MCI was bought from Fisher Wharton Company (America), RP C18 was bought from Sigma Aldrich company (America), HPLC purification was achieved on a Shimadzu HPLC system equipped with LC-20AR pumps and a model SPD-M20A UV detector (Shimadzu, Kyoto, Japan). UV spectra were obtained in MeOH on a Perkin-Elmer Lambda 650 UV/ vis spectrophotometer. IR spectra were obtained on Fourier transform infrared Spectrometer Ltd. Tianjing,China). HRESIMS data were recorded on a VG-Autospec-3000 mass spectrometer (Beckman Coulter, Inc. America). 1 D and 2 D NMR spectra were performed on an Advance Neo-400 spectrometer (Bruker, Bremerhaven, Germany). X-ray single-crystal were determined by D8 Quest X-ray single-crystal diffractometer. Absorbance was obtained on an ELX800 microplate reader (BioTek, USA).

Plant material
The whole grass of F. tikoua Bur. were collected from Huishui County, Guizhou Province of China, which were authenticated by associate Prof. Qin-De Long (Department of Pharmacognosy, Guizhou Medical University), a voucher specimen (No. 20180915) was deposited at the Herbarium of School of Pharmaceutical Sciences, Guizhou Medical University.

In vitro assay for a-glucosidase inhibitors
The a-glucosidase inhibition assay was conducted according to the reported protocols (Hashim et al 2015;Li et al 2019). Mixtures containing 40 lL of a-glucosidase enzyme (0.6 U/mL in PBS) and 8 lL of compounds 1-23 (dissolved in DMSO) or acarbose (positive control, dissolved in DMSO) were preincubated at 37 C for 30 min. The reaction was initiated with 20 lL of substrate solution (pNPG, 5 mM in PBS) and incubated for another 30 min, and then the reaction was quenched by adding 80 lL of Na 2 CO 3 (0.2 M). The absorbance was recorded at 405 nm by a microplate reader. Blank was prepared by adding PBS instead of a-glucosidase, and control was prepared by adding DMSO instead of the samples. Compounds showing the inhibition rate higher than 50% were further assayed for their dose-response relationships using six different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mmol/L). The inhibitory activity expressed as IC 50 was determined using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). All samples were assayed in triplicate.

Enzyme kinetics of compounds 22 and 23 on a-glucosidase inhibition
The types of inhibition of compounds 22 and 23 against a-glucosidase were determined using the Lineweaver-Burk equation of enzyme kinetics. Different concentrations of compounds 22 and 23 (0, 5.75, 11.5, 23, 46 lM) and different concentrations of pNPG (0.625, 1.25, 2.5, 5, 10 mmol/L) were prepared, detailed experimental procedures are described in 3.4.

Molecular docking
Schr€ odinger Maestro software was used for molecular docking, a-glucosidase crystal structure was extracted from RCSB protein data bank (PDB ID: 4J5T). The 3 D structures of compounds 22 and 23 were constructed using ChemBio3D HotLink Window in ChemBioDraw ultra12.0 software. The protein was processed with Schrodinger's Protein Preparation Wizard, the crystal water was removed, the missing hydrogen atoms were added, the missing peptides were repaired, and finally the protein energy was minimised, the geometry of structures was optimised (Rajeswari, 2014;Fazi et al, 2015), docking was done through the Glide module, the results simulated the interaction forces and binding mode of compounds 22 and 23 with a-glucosidase.

Conclusion
The investigation on the chemical constituents of F. tikoua Bur. led to the purification of a new hemiacetal chromone racemate, (±)-ficunomone (1) (first hemiacetal chromone isolated in this plant), as well as twenty-two known flavonoids (2-23). The a-glucosidase inhibitory activities of seven flavonoids (4,8,11,17,19,22,23, most of which are isoflavones or with isopentenyl moiety) were strong. Preliminary analysis of structure and activity relationship showed that double bond between C2 and C3 increased the activity, whereas glycosylation decreased the activity. In addition, the skeleton of the flavonoids, the isopentenyl substituent and its position and whether it was formed into a ring with its vicinal group all affected the a-glucosidase inhibitory activity. The inhibition type of compounds 22 and 23 (strongest inhibitors against a-glucosidase) was verified as both uncompetitive by Lineweaver-Burk Plot. Uncompetitive inhibitors, compared with the more commonly seen competitive and noncompetitive inhibitors, are regarded to be better for drug development (Ha et al. 2022). Molecular docking studies of compounds 22 and 23 revealed that they might exert the activity by binding to amino acid residues of the a-glucosidase activity pocket.
This is the first report on compounds 8, 11, and 19 inhibiting a-glucosidase. Flavonoids especially compounds 22 and 23 from F. tikoua Bur. could be considered as potential antidiabetic medicines. This investigation provided valuable clues for expanding the application of F. tikoua Bur. and searching for new a-glucosidase inhibitors from natural sources.