New Cyclic Diarylheptanoids from Platycarya strobilacea

Five new cyclic diarylheptanoids (platycary A–E, compounds 1–5) and three previously identified analogues (i.e., phttyearynol (compound 6), myricatomentogenin (compound 7), and juglanin D (compound 8)) were isolated from the stem bark of Platycarya strobilacea. The structures of these compounds were determined using NMR, HRESIMS, and electronic circular dichroism (ECD) data. The cytotoxicity of compounds 1–5 and their ability to inhibit nitric oxide (NO) production, as well as protect against the corticosterone-induced apoptosis of Pheochromocytoma (PC12) cells, were evaluated in vitro using the appropriate bioassays. Compounds 1 and 2 significantly inhibited the corticosterone-induced apoptosis of PC12 cells at a concentration of 20 μΜ.


Introduction
More than 500 diarylheptanoids, an important class of plant secondary metabolites with a aryl-C7-aryl skeleton (linear or cyclic type), have been isolated from a number of plant families, including the Aceraceae, Actinidiaceae, Betulaceae, Burseraceae, Casuarinaceae, Zingiberaceae Leguminosae, Myricaceae, and Juglandaceae [1]. Cyclic diarylheptanoids are further divided into meta,meta-bridged biphenyls and meta,para-bridged diphenyl ether heptanoids, many of which have beneficial biological activities such as anti-tumor, anti-inflammatory, estrogenic, anti-amyloidogenic, and anti-emetic activity [1,2]. Hence, the discovery of new natural diarylheptanoids and their phytochemical characterization is of considerable potential benefit to medical science. Platycarya strobilacea Sieb. et Zucc. (Juglandaceae) is a deciduous shrubby tree distributed in East Asia [3]. Previous phytochemical studies have found that this species contains large amounts of ellagitannins [4], flavonoids [5], and sesquiterpenoids [6,7], but apparently only one cyclic diarylheptanoid (i.e., platycarynol) [8]. Since a number of cyclic diarylheptanoids have been isolated from many species of Juglandaceae, in this paper we described the results of an extensive investigation of the stem bark of P. strobilacea in which eight cyclic diarylheptanoids, including five new ones (compounds 1-5), were isolated and identified. Furthermore, the anti-tumor cytotoxic activity of the five new isolates, and their ability to inhibit nitric oxide (NO) production and the corticosterone-induced apoptosis of PC12 cells, was evaluated in vitro.
Compound 4 has the molecular formula C 20 H 20 O 4, determined from HRESIMS and NMR data. Its 1 H and 13 C-NMR data (Tables 1 and 2) are quite similar to that of compound 3, except for an additional methoxyl moiety (δ C 56.1), and slight differences in the chemical shifts of C-3 , C-4 , and C-5 that hint to the linkage site of the methoxyl group. The HMBC correlations between -OCH 3 and C-4 further verified that the methoxyl group was located at C-4 . Therefore, the structure of compound 4 was proposed and named platycary D.  3 3.91 (s) 3"-OCH 3 3.72 (s, 3H) a Data were measured in CD 3 OD; b Data were measured in CDCl 3 .
Compound 5 was isolated as white needle-shaped crystals. Its molecular formula was deduced to be C 20 H 20 O 5 using HRESIMS (m/z 339.1232 [M − H] − , calcd for 339.1232). The 1 H and 13 C-NMR spectra of compound 5 (Tables 1 and 2) exhibited characteristic signals of a carbonyl group, a methoxyl group, and two 1,3,4-trisubstituted phenyl groups (as were also apparent for compound 4), while the difference between compound 5 and compound 4 was the heptanoid chain. Detailed analysis of the NMR data of compound 5 revealed two new signals at δ C 55.9 and 56.2 ppm, which are typical indicators of epoxide functionality [13,14], whereas two olefinic carbons (δ C 131.6 and 131.9) were present in compound 4. The expected 6,7-epoxy-heptan-3-one moiety was established using 1 H-1 H COSY and HMBC correlations. In addition, the linkage site of the methoxyl group was assigned to C-3" by the HMBC correlation of OCH 3 /C-3". The relative stereochemistry of the epoxide was determined as cis based on the coupling constant between H-6 and H-7 (J = 4.3 Hz) (13,14), which was confirmed by a strong cross peak between H-6 and H-7 in the NOESY spectrum. Therefore, the structure of compound 5 was proposed and named platycary E. Previously, Jahng and Park (2018) summarized series of cyclic diaryl ether heptanoids and their biological properties. Some of these compounds showed cytotoxic activities against human cancer cell lines, nitric oxide (NO) production inhibitory activity, neuroprotective activity, radical scavenging activity, and osteogenic activity [2]. In view of the valuable pharmacological activities of cyclic diarylheptanoids, we tested the cytotoxic activity of compounds 1-5 against human cancer cell lines (including HL-60, A549, SMMC-7721, MCF-7, and SW480), and their ability to inhibit NO production in Lipopolysaccharide (LPS) activated murine macrophage RAW 264.7 cells and protective effect of corticosterone-induced apoptosis in Pheochromocytoma (PC12) cells. The results indicated that none of the five compounds showed cytotoxic activities (at 40 µM) or inhibited NO production (at 50 µM). However, compounds 1 and 2 significantly inhibited the corticosterone-induced apoptosis of PC12 cells at the concentration of 20 µM (Figure 4). The PC12 cell line is the most commonly used neuronal cell line for neuroscience research in vitro studies. A previous study indicated that corticosterone (CORT) at high concentrations induces cell injury and cell death in PC12 cells, and desipramine (DIM) can antagonize the neurotoxicity caused by CORT [15]. Our report indicated that compounds 1 and 2 are two potential cytoprotective agents for neurological diseases associated with corticosterone-induced neurotoxicity.

Plant Material
The stem bark of P. strobilacea was collected from Cili county, Hunan Province, P.R. China (29°42' N and 111°13' E) in August 2018, and identified by Professor Zhang Dai-gui (Key Laboratory of Plant Resources Conservation and Utilization, Jishou University). A voucher specimen (zdg20180809) was deposited at the College of Plant Protection, Hunan Agricultural University.

Extraction and Isolation
Dried barks of P. strobilacea (12.5 kg) were powdered and extracted three times with EtOH (95% v/v) at room temperature, then concentrated under reduced pressure to obtain a crude residue (1.8 kg). The residue was suspended in H2O (4 L) and then extracted with petroleum ether (PE) and EtOAc successively, to yield a PE-soluble fraction (35.

Plant Material
The stem bark of P. strobilacea was collected from Cili county, Hunan Province, P.R. China (29 • 42 N and 111 • 13 E) in August 2018, and identified by Professor Zhang Dai-gui (Key Laboratory of Plant Resources Conservation and Utilization, Jishou University). A voucher specimen (zdg20180809) was deposited at the College of Plant Protection, Hunan Agricultural University.

Extraction and Isolation
Dried barks of P. strobilacea (12.5 kg) were powdered and extracted three times with EtOH (95% v/v) at room temperature, then concentrated under reduced pressure to obtain a crude residue (1.8 kg). The residue was suspended in H 2 O (4 L) and then extracted with petroleum ether (PE) and EtOAc successively, to yield a PE-soluble fraction (35.0 g) and an EtOAc-soluble fraction (95.0 g). The EtOAc-soluble fraction was further subjected to silica gel CC (100-200 mesh) with elution of CHCl 3 -MeOH (100:0 → 60:40, v/v) to give seven fractions (Fr. A-G). Fr.B (5.7 g) was decolorized on MCI gel (eluted with 90 % MeOH) and then chromatographed on an ODS-C18 column that eluted with acetone-H 2 O (3:7→10:0, v/v) to afford 10 sub-fractions (B1−B10). After TLC detection, sub-fractions B5, MTS solution was added to each well. An absorbance was measured at 492 nm using a Thermo Multiskan FC. All data was expressed as mean ± SD and paired t-tests implemented in SPSS 13.0 were used to evaluate the statistical significance of the differences between separate treatment groups and the control.
The above biological activities tests were conducted at the Natural Drug Activity Screening Center, Kunming Institute of Botany, Chinese Academy of Sciences.

Conclusions
Phytochemical investigation of the stem bark of P. strobilacea afforded five new cyclic diarylheptanoids (i.e., platycary A−E (compounds 1-5)) and three known ones: phttyearynol (compound 6), myricatomentogenin (compound 7), and juglanin D (compound 8). Biological activities of compounds 1-5 were evaluated using cytotoxicity, NO, and corticosterone-induced apoptosis assays. Compounds 1 and 2 exhibited significant protective effects against the corticosterone-induced apoptosis of PC12 cells at the concentration of 20 µM, indicating that compounds 1 and 2 are two potential cytoprotective agents for neurological diseases. The identification of these new compounds significantly increases both the number of diarylheptanoids produced by P. strobilacea and the number of diarylheptanoids with potential medical value.