Neuroprotective Norsesquiterpenoids and Triterpenoids from Populus euphratica Resins

Two new octanorlanostane-type triterpenes, euphraticanoids A and B (1 and 2), two new trinorsesquiterpenoids, euphraticanoids C and D (3 and 4), and eight known triterpenoids (5, 6, 8–13) along with one steroid (7) were isolated from Populus euphratica resins. The structures of these new compounds, including their absolute configurations, were characterized by spectrocsopic, chemical, and computational methods. Biological evaluation revealed that compounds 4, 7–9, 12, and 13 display neuroprotective activities in H2O2-induced HT-22 cells with 4, 8, and 9 occurring in a concentration-dependent manner and 7, 12, and 13 reaching the maximum effects at 20 μM. Meanwhile, the neuroprotective properties of all isolates were accessed using glutamate-induced SH-SY5Y cells and disclosed that compounds 3, 4, 8, and 9 could dose-dependently protect neural cell injury in a concentration range of 10–40 μM. Finally, a brief structure–activity relationship was briefly discussed.


Introduction
Populus euphratica, a plant of the family Salicaceae, spreads over the world in places such as China, Russia, Mongolia, India, and Iran. In China, the tree is mainly distributed in the west of China with abiotic surroundings exemplified as desert or saline and alkaline lands [1]. The resins secreted by the tree, known by the elegant name "the tears of poplar," have been used to treat tuberculous adenitis, throat, and duodenal ulcer swelling in China [2]. Previous studies revealed the presence of salicin derivatives, volatile oils, and phenolics in the resins of P. euphratica [3,4]. In recent years, we have become interested in chemical investigations of medicinal plant resins. As a result, an increasing number of structurally novel terpenoids have been characterized [5][6][7]. In a continuous study on medicinal resins, the title material was investigated, resulting in the isolation of diterpenoids with cytotoxic and potent wound-healing promotion properties [8][9][10]. The current work is an in-depth investigation on P. euphratica resins, which led to the characterization of 12 terpenoids and one steroid with euphraticanoid compounds A-D (1-4) being new ones ( Figure 1). To get an insight into the biological profiling of these secondary metabolites, neuroprotective properties of all the isolates were evaluated in either H 2 O 2 or glutamate-induced neural or human neuroblastoma cells. In this paper, we describe the isolation, structure characterization, and neuroprotective activities of all the isolated compounds.  in MeOH). Its molecular formula was deduced as C 22 H 34 O on the basis of its HRESIMS (high resolution electrospray ionization mass spectroscopy), 13 C NMR, and DEPT spectra. The 1 H NMR spectrum of 1 shows signals for five methyl singlets (δ H 1.05, 0.98, 0.84, 0.84, and 0.64). The 13 C NMR and DEPT spectra (Table 1) indicate 22 carbons ascribed to five methyl, six methylene, six methine with three sp 2 ones, and five non-protonated carbons (four aliphatic and one olefinic). A comparison NMR data of 1 with those of commiphorane G2 [11] revealed their resemblance except for a 13  , and H-9/Hb-2 suggest that these protons are at α-oriented. Finally, we examined the relative configuration of C-3 using the nuclear Overhauser effect (NOE) irradiation. A NOE enhancement was observed between 3-OH and H 3 -20 ( Figure S14), evidently indicating the α-orientation of 3-OH in 1 contrary to that of commiphorane G2. Further NMR data comparison of ring A of 1 with the counterpart of epimansumbinol (6) [12] which bears a 3α-OH found their accordance, also securing the above conclusion. Thus, the planar structure and the relative configuration of 1 was deduced as shown in Figure 1. To clarify the absolute configuration of 1, electronic circular dichroism (ECD) calculation was carried out at B3LYP/6-311 + G(d) l level. The results show that the calculated ECD spectrum of (3R,5R,8R,9R,10R,14R)-1 ( Figure 4) agrees well with the experimental one, indicating the absolute configuration of 1 is 3R,5R,8R,9R,10R,14R. In this way, the structure of 1 was identified and named as euphraticanoid A.

Biological Evaluation
Neuroexcitotoxicity and oxidative stress have been implicated as playing a dominant role in neurodegenerative disorders such as Alzheimer's disease (AD), ischemic stroke, as well as Parkinson's disease (PD) [22][23][24][25][26]. In this study, all the compounds isolated from P. euphratica resin were applied to detect neuroprotective bioactivities against glutamate-induced excitotoxicity in SH-SY5Y cells and to examine their antioxidative effects against H 2 O 2 in HT-22 cells. In primary screening, HT-22 cells were pretreated with 20 µM of different compounds, following by 600 µM H 2 O 2 stimulation for 24 h. Our results show that eight out of thirteen compounds could significantly prevent H 2 O 2 -induced oxidative stress with compounds 4, 7-9, 12, and 13 are more potent ( Figure 6A). Therefore, compounds 4, 7-9, 12, and 13 were submitted to a dose-dependent response experiment. The results show that 4, 8, and 9 could dose-dependently protect neural cells from H 2 O 2 -induced oxidative stress injury ( Figure 6B,D,E), and 7, 12, and 13 possess neuroprotective property against oxidative stress at lower concentrations (10 µM and 20 µM) (Figure 6C,F,G). Of note, neuroprotective effects of 7, 12, and 13 reach the maximum at 20 µM and decline at 40 µM. Last but not the least, all the compounds except 8 show no cytotoxicity toward HT-22 cells even at 40 µM ( Figure 6D). Although 8 is cytotoxicity against HT-22 cells at 40 µM, it appears that the cytotoxic effect might be negligible. Interestingly, compounds 1, 2, 5, 6, 8, and 9 are all octanortriterpenoids. However, the fact that 8 and 9 are active and the other analogs are inactive indicate the importance of α, β-unsaturated ketone to keep the activity. In addition, it was observed that compounds 12 and 13 are active, while in contrast 10 and 11 are inactive, implying that ursane-type rather than oleanane-type triterpenoids might contribute to neuroprotection. Boldly, it was found that compounds 4, 7-9, and 13 all bear a common α, β-unsaturated ketone, whether such a functional group is essential for keeping the activity needs further exploration. The neuroprotective properties of all the compounds were also observed in glutamate-induced SH-SY5Y cells. Cells were pretreated with 20 μM of different compounds followed by 10 mM glutamate stimulation for 24 h. The results show that 3, 4, 8-10 could significantly prevent glutamateinduced excitotoxicity ( Figure 7A). Thereafter, 3, 4, 8, and 9 were assessed for a dose-dependent response and revealed that the neuro protection of 4, 8, and 9 against glutamate-induced The neuroprotective properties of all the compounds were also observed in glutamate-induced SH-SY5Y cells. Cells were pretreated with 20 µM of different compounds followed by 10 mM glutamate stimulation for 24 h. The results show that 3, 4, 8-10 could significantly prevent glutamate-induced excitotoxicity ( Figure 7A). Thereafter, 3, 4, 8, and 9 were assessed for a dose-dependent response and revealed that the neuro protection of 4, 8, and 9 against glutamate-induced excitotoxicity is dose-dependent ( Figure 7B−E). Despite that octanortriterpenoids 8 and 9 are different types of compounds from 3 and 4, their neuroprotective activities against glutamate-induced excitotoxicity might imply that the presence of an α,β-unsaturated ketone is pivotal for keeping neuroprotection.

Plant Material
The resins of P. euphratica were collected by Ming-Yang Zong from Bayin, Xinjiang Autonomous Region, in November 2011. A voucher specimen (CHYX0573) identified by Prof. Bin Qiu at Yunnan University of Traditional Chinese Medicine is deposited at School of Pharmaceutical Sciences, Shenzhen University, China.

ECD Calculation for Compounds 1-4
Conformation search using molecular mechanics calculations was performed in CONFLEX version 7.0 with MMFF force field with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then the predominant conformers were optimized at B3LYP/6-311+G(d) level in Gaussian 09 [27]. The optimized conformation geometries and thermodynamic parameters of all selected conformations were provided. The optimized conformers of 1-4 were used for the ECD calculation, which were performed with Gaussian 09 (B3LYP/6-311+G(d)). The solvent effects were taken into account by the polarizable-conductor calculation model (PCM, methanol as the solvent). Percentages for each conformation are shown in Tables S34-S37.

MTPA Esterification of 4
Compound 4 (1 mg) was dissolved in 600 µL of anhydrous deuteration pyridine, which was divided into two equal portions in NMR sample tube. To each portion was added 1.5 µL of either R-MTPA-Cl or S-MTPA-Cl, and then the mixtures were kept at room temperature for 2 h. Finally, the 1 H NMR data were collected using the mixtures without purification.

Bioactivity Assay
Mouse hippocampus cell line (HT-22 cells) was purchased from iCell company in Shanghai, China, and human neuroblastoma cell line (SH-SY5Y) was purchased from Cellcook Company in Guangzhou, China. The experimental procedures of cell culture and treatments were performed as described previously [28]. Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) with the addition of 10% fetal bovine serum (FBS, Gibco, Australia), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco, Thornton, Australia) at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 incubator. SH-SY5Y cells were plated at a density of 2 × 10 4/ well of a 96-well plate for 24 h. Thereafter, SH-SY5Y cells were pretreated with compounds (10, 20 and 40 µM) or PBS, and followed by a 24 h stimulation of either 10 mM glutamate (Sigma) or PBS (HyClone). HT-22 cells were pretreated with compounds (10, 20 and 40 µM) or PBS and followed by a 24 h stimulation of either 600 µM H 2 O 2 (Sigma) or PBS. After all treatments, 10 µL/well of CCK-8 solution was added into each well and incubated for 1 h. The absorbance was determined at 450 nm by using a microplate reader (BioTek, Winooski, VT, USA).
All quantified biological data are expressed as means ± standard error of the mean (SEM) of n independent experiments. Statistical analyses were performed by one-way analysis of variance (ANOVA) following by a post-hoc multiple-comparison Tukey test whereby p < 0.05 (** p < 0.01, *** p < 0.001) was considered significant.

Conclusions
To conclude, the current study led to the characterization of two new nortriterpenoids (1 and 2), two new norsesquiterpenoids (3 and 4), and nine known compounds (5-13) from P. euphratica resins. Biological evaluation revealed that 4, 7-9, 12, and 13 are neuroprotective agents and the presence of α,β-unsaturated ketone in the structure might be crucial for keeping the activity. This study might shed light on further structure modification for developing new generation of neuroprotective drugs.