Polycyclic Polyprenylated Acylphloroglucinol Derivatives from Hypericum acmosepalum

Hypericum acmosepalum belongs to the Hypericum genus of the Guttiferae family. The characteristic components in Hypericum are mainly a series of polycyclic polyprenylated acylphloroglucinols (PPAPs), flavonoids, and xanthones. Among them, the PPAPs have received much attention due to their novel structures and diverse pharmacological activities and have become hot spots in organic chemistry and medicinal chemistry. However, there are few reports about the chemical constituents of Hypericum acmosepalum at present, especially the PPAPs. This research is dedicated to the study of the air-dried aerial parts of Hypericum acmosepalum, which were extracted with 95% EtOH under reflux, then suspended and successively partitioned with petroleum ether and ethyl acetate. Five PPAP derivatives were obtained using various chromatographic techniques, and their structures were determined by NMR spectroscopic data, including two new phloroglucinol derivatives, hyperacmosin A (1) and hyperacmosin B (2). Those compounds were evaluated for their neuroprotective effect using two models.

Hypericum acmosepalum N. Robson, commonly known as Xiangzhen tree in Yunnan Province, belongs to the Hypericum genus [16]. It mostly grows in Yunnan, Guizhou, and Sichuan Province, and is used in folk medicine to treat inflammation and hepatitis [17]. Currently, there are few studies on the chemical constituents of H. acmosepalum [18]. As a part of an ongoing research program aimed at the isolation, structural characterization, and pharmacological evaluation of bioactive PPAPs from the Hypericum genus [6,19,20], a preliminary study was carried out on the petroleum ether-soluble part of the 95% EtOH extract from the air-dried aerial parts of H. acmosepalum, and obtained five PPAP new compounds were elucidated by the analysis of spectroscopic data and the known compounds were concluded by comparison of the NMR data with the published literature.
3. The Figure 2 should be changed to: 4. The Figure 5 should be changed to:   Figure S16), with ten degrees of unsaturation. Its IR (see Figure S18) absorptions implied the presence of hydroxy (3467 cm −1 ) and carbonyl groups (1726 and 1618 cm −1 ). The 1 H-NMR and 13  Thus, the relative configuration was determined as shown in Figure 5. In the ECD spectra, the chirality of C-1, 5, 7 in the skeleton structure of 2 was consistent with compound 1 because the spectra of 2 had a high degree of similarity to the spectra of 1 ( Figure 6). Furthermore, the ROESY correlation between H-20 (δ H 4.62) and H-29 (δ H 2.21) indicated that the orientation of the H-20 was α-oriented. Therefore, the absolute configuration of 2 was determined to be (1R, 5S, 7S, 20S), and was named hyperacmosin B.    1.38 and 1.36, 1: δH 1.36 and 1.38), 23 and 35 (2: δH 1.25 and 1.28, 1: δH 1.27 and 1.25). In the     Furthermore, the ROESY correlation between H-20 (δH 4.62) and H-29 (δH 2.21) indicated that the orientation of the H-20 was α-oriented. Therefore, the absolute configuration of 2 was determined to be (1R, 5S, 7S, 20S), and was named hyperacmosin B.
HMBC ROESY   The five compounds were evaluated for neuroprotective effect in two models: L-glutamic acid (L-Glu) and oxygen-glucose deprivation (OGD) by the MTT method [26]. Donepezil and PHPB (potassium 2-(1-hydroxypentyl)-benzoate) were used as the positive controls. L-glutamic acid and sodium hyposulfite were used as the damage agents. In the model of L-Glu, the cell survival rate of the positive control groups had increased compared with the control group. In experiment groups, hyperacmosin A (1) and sampsonione O (4) increased the survival rates of the SK-N-SH cells to 68.20%, 80.90% compared with the control group respectively (Table 2), which showed remarkable neuroprotection activity. In the model of OGD, PHPB increased the survival rates of the SK-N-SH cells to 80.30% compared with the control group (Table 2). While the cell survival rate of hyperacmosin B, oxepahyperforin and sampsonione L had increased, those compounds did not exhibit obvious neuroprotective activity against OGD induced-injury on SK-N-SH cells compared with the control group.

General Information
Optical rotations were measured on a JASCO P-2000 polarimeter (Jasco Inc., Tokyo, Japan) using methanol as a solvent. UV spectra were determined with a JASCO V-650 spectrophotometer (Jasco Inc., Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). ECD and NMR spectra were obtained on Varian Inova-500 spectrometer

Plant Material
The air-dried aerial parts of H. acmosepalum were collected from the Wenshan, Yunnan Province, China, in July 2016. The plant was identified by Prof. Lin Ma. A voucher specimen (No. ID-S-2764) was deposited in the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

Extraction and Isolation
Air-dried and powdered aerial parts of H. acmosepalum (17.0 kg) were extracted three times, with 95% EtOH (170 L per extraction) under reflux. The 95% EtOH extracts were concentrated in vacuo to provide a crude extract (1.8 kg), which was suspended in water and then partitioned with petroleum ether and EtOAc successively. The petroleum ether soluble part (500.0 g) was separated by silica gel column chromatography (200−300 mesh) in reduced pressure and eluted with a gradient of petroleum ether-EtOAc (1:0 to 0:1) to obtain 11 fractions (Fr. 1-Fr. 11).

Neuroprotection Bioassays
The neuroprotection of compounds was determined by the MTT method in SK-N-SH cells, grown in Dulbeco's modified containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL). Cell cultures were incubated at 37 • C under a 5% CO 2 atmosphere. The compounds and positive control were dissolved with DMSO (10 µmol/L) respectively. The cells were seeded onto 96-well microplates at a density of 1 × 10 5 cells/well. After incubation at 37 • C for 24 h under a 5% CO 2 atmosphere, the cells were pre-incubated with compounds and positive controls respectively for 1 h. In the L-Glu, the DMEM (Dulbecco's modified Eagle's medium) without L-Glu was added to the cells of the normal group, the DMEM containing L-Glu (the final concentration of 27 mM) was added to the cells of other groups. After 4 h of co-incubation, 100 µL of MTT (0.5 mg/mL) were added to each well after the withdrawal of the culture medium and were incubated for an additional 4 h at 37 • C. The resulting formazan crystals were dissolved in 150 µL of DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 570 nm. In the OGD, the L-DMEM (low-sugar Dulbecco's modified Eagle's medium) without sodium hyposulfite was added to the cells of the normal group, the L-DMEM containing sodium hyposulfite (the final concentration of 3.5 mM) was added to the cells of other groups, and then incubated for 24 h. After 24 h, MTT solution (0.5 mg/mL) was added for 4 h at 37 • C. Finally, the formazan crystals were solubilized by DMSO and were spectrophotometrically measured at 570 nm. All data presented in our study were obtained from three independent experiments. Survival rate (%) was obtained by the following formula: Improved survival rate (%) = (survival rate of the experimental group-survival rate of the control group)/Survival rate of the control group.

Conclusions
In this study, five phloroglucinols were isolated from the petroleum ether soluble part of the 95% EtOH extract from H. acmosepalum, including two new phloroglucinols and three known compounds. In the neuroprotection screening, the compound 1 (hyperacmosin A) and compound 4 (sampsonione O) showed significant neuroprotective activity in L-Glu induced-injury on human neuroblastoma SK-N-SH cells. However, those compounds did not exhibit neuroprotective activity in the OGD.
In summary, PPAPs research is currently in focus, but the low polarity and instability of the PPAPs greatly increase the difficulty in their separation and structure determination. Additionally, this work provides preliminary evidence for the neuroprotective function of these compounds, but further investigations of this neuroprotective function and other activities are warranted. By elucidating how these molecules interact, this work provides the necessary data to explore the relationship between neuroprotective activity and molecular structure.