Undescribed Cyclohexene and Benzofuran Alkenyl Derivatives from Choerospondias axillaris, a Potential Hypoglycemic Fruit

The fruit of Choerospondias axillaris (Anacardiaceae), known as south wild jujube in China, has been consumed widely in several regions of the world to produce fruit pastille and leathers, juice, jam, and candy. A comprehensive chemical study on the fresh fruits led to the isolation and identification of 18 compounds, including 7 new (1–7) and 11 known (8–18) comprised of 5 alkenyl (cyclohexenols and cyclohexenones) derivatives (1–5), 3 benzofuran derivatives (6–8), 6 flavonoids (9–14) and 4 lignans (15–18). Their structures were elucidated by extensive spectroscopic analysis. The known lignans 15–18 were isolated from the genus Choerospondias for the first time. Most of the isolates exhibited significant inhibitory activity on α-glucosidase with IC50 values from 2.26 ± 0.06 to 43.9 ± 0.96 μM. Molecular docking experiments strongly supported the potent α-glucosidase inhibitory activity. The results indicated that C. axillaris fruits could be an excellent source of functional foods that acquire potential hypoglycemic bioactive components.


Introduction
Choerospondias axillaris (Roxb.)B.L. Burtt.& A.W. Hill (Anacardiaceae), a tall deciduous tree bearing edible fruits, is distributed in slopes, hills or valleys at altitudes ranging from 300 to 2000 m in India, China, Japan, Bhutan, Laos, Vietnam, and Thailand [1][2][3].The fruit, known as south wild jujube in China, with a light yellow flesh and sour-sweet taste, is valued for its substantial nutritional and therapeutic values in several regions of the world [4,5].In Traditional Chinese Medicine (TCM), the fruit of C. axillaris has been historically utilized for its potential therapeutic properties to improve cardiac function as well as to regulate blood sugar levels and alleviate symptoms linked to diabetes [1,[3][4][5][6][7].Furthermore, it is used widely in China's food industry for producing fruit pastilles and juice [8,9] and is utilized in Nepal for processing local products, e.g., pickles, fruit leathers, jam, and candy [1,10].Regardless of insufficient reports on global sales data and socio-economic purposes, the market value of C. axillaris was found to be approximately USD 0.65 million in Kathmandu, Nepal [1,8,11].C. axillaris as a functional food provides health benefits beyond basic nutrition due to its physiologically active components, which play an active role in promoting overall health and regulating disease progression [1][2][3][4][5][6][7][8][9][10].
So far, 26 compounds consisting of flavonoids, phenolic acids, organic acids, fatty acids, and sterols have been isolated from C. axillaris fruits [12][13][14][15], among which flavonoids represent a class of the most important and effective components that proven to have various

Quantum Chemistry ECD Calculation
Conformational analysis was performed with the MMFF forcefield (Merck Molecular forcefield) using the Monte Carlo algorithm implemented in Spartan'10 software (Wavefunction, Irvine, CA, USA, 2018).ECD calculations were carried out, following optimizing the conformers at B3LYP/6-31+g (d, p) level in MeOH using the CPCM polarizable conductor calculation model, the conformers with a Boltzmann population of over 5% were chosen.The theoretical calculation of ECD was conducted in MeOH using time-dependent density functional theory (TD-DFT) at the B3LYP/6-311+g (d, p) level for all conformers of compound 18b.Rotatory strengths for a total of 50 excited states were calculated.ECD spectra were generated using the program.SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California, San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

α-Glucosidase Inhibitory Assay
The α-glucosidase inhibitory activity was evaluated according to a previous method [33].In brief, α-glucosidase solution (0.025 U/mL, dissolved in 0.1 M phosphate-buffered saline (PBS) solution with a pH of 6.8), was mixed with 50 µM inhibitor and incubated at 37 • C for 10 min.pNPG solution (1 µM, dissolved in 0.1 M PBS solution) was added to the mixed solution and incubated at 37 • C for 40 min.Sodium carbonate (1 M) solution was added to terminate the reaction.Quercetin and acarbose were used as a positive control.Quercetin is selected as a reference in α-glucosidase assay because of its efficacy as a therapeutic inhibitor; it binds strongly within the binding region (active site) of the protein and forms a compact structure, whereas acarbose is a common medication utilized in clinical practice to manage postprandial hyperglycemia [30,34,35].The absorbance was recorded at 405 nm usin a microplate reader.The α-glucosidase inhibitory rate was calculated as follows: Inhibitory rate% = [(A0 − A1)/A0)] × 100%, where: A0 and A1 represent the absorbance of the blank group (containing enzyme, pNPG, PBS buffer, and DMSO), the sample group (containing enzyme, pNPG, PBS buffer, and samples), respectively.IC 50 values were computed based on the Reed-Muench method [36].

Molecular Docking Analysis
The X-ray crystallographic structures of α-glucosidase from Saccharomyces cerevisiae (PDB ID: 3A4A) at a resolution of 1.6 Å were downloaded from the RCSB Protein Data Bank (https://www.rcsb.org(accessed on 7 February 2024)); prior to docking, water molecules and hetero atoms of the protein were removed, missing atoms were repaired, polar hydrogens and kollman charges were added.The 3D structure of the ligands was prepared in Chem3D by energy minimization using the MM2 force field, saved in Sybyl Mol2 format and converted to AutoDock PDBQT format with Open Babel 3.1.1chemical toolbox [37].Molecular docking simulations were performed using AutoDockTools-1.5.7 with Lamarckian genetic algorithm 4.2 to understand the interactions and binding of isolated compounds with selected proteins following previously described protocols with minor modification [38,39].The docking results were visualized and analyzed using the PyMOL 2.6.0 molecular graphics system and open-source visualization tools [40][41][42].

Statistical Analysis
All data were analyzed and presented as mean values ± SD from three independent assays (n = 3).The IC 50 values were determined by Graph Pad Prism version 5 (University of California, San Diego, CA, USA).

Molecular Docking Studies
To rationalize the significant in vitro α-glucosidase inhibition results achieved, molecular docking studies were designed [58][59][60][61], thereby illustrating the docking affinities and the docking conformations of the isolated compounds with α-glucosidase from Saccharomyces cerevisiae isomaltase (PDB ID: 3A4A), a principal diabetes-related enzyme located in the small intestine, which catalyzes the cleavage of α-glucopyranoside bond in oligosaccharides and disaccharides, leading to increases in blood glucose concentration.
The molecular docking results, including the binding affinities of ligands (6, 7, 8 and quercetin) that exhibited potent α-glucosidase inhibition and their interactions (hydrogen bonds and hydrophobic interactions), are shown in Figure 4.The highest docking score indicates greater affinity of the compound towards the protein which finds well fit inside the binding pocket of the protein.This is consistent with the results of the α-glucosidase inhibitory assay.
Compound 6 demonstrated the highest binding affinity of −9.77 kcal/mol which is attributed to the hydrogen bonding interactions observed with Arg 442A (2.8 Å), Asp 215A (2.8 Å), and His112A (3.The positive control quercetin showed a binding affinity of −7.73 kcal/mol, which is consistent with the previous study by Qin et al. [28], who proved the reason why it possesses less α-glucosidase inhibition compared to compounds 6, 7 and 8. Notably, it formed a hydrogen bond interaction with amino acid residues Asp 352A, Arg 315A, Asp 215A and Gln 279.

Structural Activity Relationship
The new compounds possess analogous alkenyl side chains; nonetheless, the inhibitory effect significantly changed when a heterocyclic aromatic ring was attached instead of a non-aromatic cyclic ring.The results showed that the compounds with benzofuran arene moiety tend to exert remarkable α-glucosidase inhibitory activity than alkenyl cyclohexenol and cyclohexenone derivatives.These significant differences in activity indicate that the presence of a heterocyclic aromatic ring undoubtedly plays a crucial role in the compounds exerting α-glucosidase inhibitory activity, which is attributed to the high docking score and hydrogen, hydrophobic interactions in the docking study.
The flavanonols dihydroquercetin (10), dihydrokaempferol (11) and flavanones eriodictyol (12) and naringenin (13) possess carbonyl groups; however, they differ in the number of OH groups, resulting in the observed differences in inhibition of α-glucosidase.Flavan-3-ol (9), with the absence of the carbonyl group, displayed a notable α-glucosidase inhibition; furthermore, the presence of the rhamnosyl group at C-3 on quercitrin (14) altered the enzyme inhibition activity, which is consistent with previous research findings by Li et al.,[29].

Conclusions
In this study, chemical investigation of the fresh fruits of C. axillaris led to the isolation and structural elucidation of 18 compounds including 7 new (1-7) and 11 known (8-18),
a Data expressed as means ±