Enantiomeric Isoflavones with neuroprotective activities from the Fruits of Maclura tricuspidata

Seven pairs of enantiomeric isoflavones (1a/1b–7a/7b) were obtained from the ethyl acetate extract of the fruits of Maclura tricuspidata (syn. Cudrania tricuspidata), and successfully separated by chiral high-pressure liquid chromatography (HPLC). The structures and absolute configurations of the enantiomeric isoflavones were established on the basic of comprehensive spectroscopic analyses and quantum chemical calculation methods. Compounds 1, 1a, and 1b exhibited neuroprotective activities against oxygen-glucose deprivation/reoxygenation (ODG/R)-induced SH-SY5Y cells death with EC50 values of 5.5 µM, 4.0 µM, and 10.0 µM, respectively. Furthermore, 1, 1a, and 1b inhibited OGD/R-induced reactive oxygen species generation in SH-5Y5Y cells with IC50 values of 6.9 µM, 4.5 µM, and 9.5 µM, respectively.

, 1 was supposed to be an optically pure compound. Therefore, a modified Mosher's experiment was carried out to establish the absolute configurations at the C-2′′ and C-2′′′ positions 20 . Interestingly, when the (R) and (S)-MTPA esters of 1 were subjected to RP-C 18 23), which exhibited the mirror image-like ECD curves (Fig. 2).
The molecular formula of compound 2 was C 25 H 26 O 7 (HRESIMS, m/z 439.1741 [M + H] + ). The analyzing 1D and 2D NMR data of 2 indicated that 2 was a stereoisomer of 1. Although CE curves were detected in the ECD spectrum of 2 ( Fig. 2) along with a measurable optical rotation ([α] 24 D +2.1), its racemic nature was demonstrated based on chiral HPLC analysis. Further enantiomer separation using chiral HPLC resulted in the isolation of enantiomers 2a (t R 23). In order to determine the absolute configurations of the enantiomers 1a and 1b, as well as 2a and 2b, quantum chemical ECD calculations were carried out and the results were compared with the experimental data. Four possible stereoisomers based on differences at the C-2′′ and C-2′′′ positions of the gross structure were built and separately subjected to a Merck molecular force field (MMFF) conformation search, followed by geometry   optimization in density functional methods. The ECD data of the selected conformers were calculated using the time-dependent DFT (TDDFT) method. As shown in Fig. 2, the calculated ECD spectra for the (2′′S,2′′′R) and (2′′R,2′′′S)-isomers were well matched with the experimental spectra of 1a and 1b, respectively, and the simulated spectra for the (2′′R,2′′′R) and (2′′S,2′′′S)-isomers were highly consistent with the experimental spectra of 2a and 2b, respectively. Besides, in order to further confirm the results, the additional ECD calculations were carried out using the CAM-B3LYP and WB97XD functionals, which yielded consistent ECD results (Fig. 2). On this basis, the absolute configurations of 1a, 1b, 2a, and 2b were assigned as depicted, which were named as (2′′S,2′′′R)-cudraisoflavone U, (2′′R,2′′′S)-cu draisoflavone U, (2′′R,2′′′R)-cudraisoflavone U, and (2′′S,2′′′S)-cudraisoflavone U, respectively.
The racemic compounds 1-7 were evaluated for neuroprotective activity against oxygen-glucose deprivation/ reoxygenation (ODG/R)-induced neuronal cell death in SH-SY5Y cells. Of these, 1 exhibited a significant protective effect with an EC 50 value of 5.5 µM (carnosine was used as a positive control, EC 50 13.4 µM) ( Table 2) 21 . The rest of the compounds were inactive (EC 50 > 20 µM). Accordingly, enantiomers 1a and 1b were further separately examined for their neuroprotective potential and both were found to attenuate ODG/R-induced neurotoxicity with EC 50 values of 4.0 µM and 10.0 µM, respectively (Table 2).
Moreover, although the causes of neurodegenerative diseases have not been clearly elucidated, many experimental evidences suggested that oxidative stress resulting in the generation of reactive oxygen species (ROS) plays a pivotal role in neurodegenerative diseases 16,17,22 . Furthermore, recent biological studies indicate that several isoflavones are beneficial for reducing oxidative stress in neurons and protecting against neurodegenerative diseases [22][23][24][25] . Consequently, the inhibitory effect of 1, 1a, and 1b on the ODG/R-induced intracellular  ROS generation in SH-5Y5Y cells was assessed. As shown in Table 2, 1, 1a, and 1b inhibited ROS generation in ODG/R-induced SH-5Y5Y cells with IC 50 values of 6.9 µM, 4.5 µM, and 9.5 µM, respectively. Interestingly, 2 did not inhibit ODG/R-induced neuronal cell death although 2 has the same gross structure as that of 1. On these grounds, it is suggested that the variety of stereochemistry has an apparent effect on the neuroprotective potential of these isoflavones. Recent study demonstrated that isoflavones from M. tricuspidata exerted neuroprotective activity via induction of Nox4-targeting miRNAs and inhibition of the MAPK signal cascade in in vitro and in vivo models of cerebral ischemia 26 .
Besides, recently studies indicated that ingested flavonoids are mostly metabolized in the small and large intestines, and liver, then enter the bloodstream and can reach the central nervous system (CNS) by transporting across the blood brain barrier (BBB) [27][28][29] . However, to date, the knowledge about their capacity of reaching the CNS remain insufficient and inconsistent. The degree to which flavonoids can enter the CNS is still a disagreement, in spite of several studies indicated their presence in brain tissue after oral administration 28,29 . Therefore, the knowledge regarding flavonoids transport across BBB and how this is regulated is crucial. Recent study reported that flavonoids might pass through the BBB by transmembrane diffusion, which is dependent on the degree of their lipophilicity 27,30,31 . Furthermore, the evaluations of transmembrane transport of different flavonoids such as genistein, (+)-catechin, hesperidin, and quercetin via blood-brain barrier cells models indicated that after treatment for 3 h, the obtained concentrations of these flavonoids were 3-10 µM, which was sufficient concentration to have beneficial effects 30,[32][33][34] . In present study, isolated compounds from M. tricuspidata were genistein-based flavonoids, suggesting they may possess the ability to pass through BBB and reach the sufficient concentration.
Consequently, the isolated compounds from M. tricuspidata could be promising candidates for the treatment of cerebral ischemia and more investigations are needed to understand their cellular mechanisms of action in the brain for fully exploring their neuroprotective potential.

Plant materials.
The collection of fruits of Maclura tricuspidata and deposition of voucher specimen (KH1-5-090904) were carried out as previously described 7 . Extraction and Isolation. Fresh fruits of M. tricuspidata (10.7 kg) were extracted in 100% MeOH (3 × 10 L) at room temperature over the course of ten days. The extracts were concentrated under vacuum to afford a residue (TH1-1-1, 630.9 g), which was further extracted with n-hexane (48.43 g) and EtOAc (27.8 g).

Measurement of cell viability and intracellular ROS and statistical analysis. The protective effects
against ODG/R-induced cell death and intracellular ROS generation in SH-SY5Y cells of test compounds and statistical analysis were carried out as previously described 35 . All experimental data are expressed as the mean value ± standard deviation from three replicates for each experiment. Statistical significance between multiple groups was determined by one-way ANOVA (PRISM Graph Pad, San Diego, CA, USA). When the ANOVA showed a significant difference, Bonferroni's multiple comparison post hoc test was conducted. P values less than 0.05 were regarded as statistically significant.