Hyperacmosin R, a New Decarbonyl Prenylphloroglucinol with Unusual Spiroketal Subunit from Hypericum acmosepalum

Two previously undescribed polycyclic polyprenylated acylphloroglucinols, hyperacmosins R-S (1–2), were obtained from the aerial parts of Hypericum acmosepalum. Their structures were elucidated by extensive spectroscopic analysis and electronic circular dichroism calculation (ECD). Compound 1 featured an unprecedented 5,8-spiroketal subunit as well as the loss of C-2′ carbonyl in the phloroglucinol ring. In addition, compounds 1 and 4 showed weak hepatoprotective activity against paracetamol-induced HepG2 cell damage at 10 μm. The plausible biosynthetic pathway of 1 was proposed via a retro-Clasisen reaction and decarboxylation.

Hypericum acmosepalum is distributed in Guangxi, Yunnan, Sichuan, and Guizhou provinces in China. As a kind of traditional Chinese medicine, it has been used to treat hepatitis and relieve swelling and inflammation [11]. Our previous chemical investigations into this plant resulted in the isolation of many bioactive PPAPs with diverse carbon scaffolds, and some of them showed hepatoprotective and neuroprotective activities [12][13][14][15][16]. In order to obtain more of this type of molecules, our continuing study of this plant led to the identification of two new PPAPs, hyperacmosins R-S (1-2), as well as nine known ones ( Figure 1). It is worth mentioning that compound 1 possesses a rare 5,8-spiroketal subunit, together with the loss of C-2 carbonyl in the phloroglucinol ring. Herein, the details of the isolation, structural elucidation, and the plausible biosynthetic ways of compound 1 are reported. 2

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Molecules 2022, 27, x FOR PEER REVIEW 2 of 10 the details of the isolation, structural elucidation, and the plausible biosynthetic ways of compound 1 are reported.

Results and Discussion
All of the isolated compounds were evaluated for their hepatoprotective activities against paracetamol-induced HepG2 cell damage, and glutathione was used as the positive control. As shown in Table 2, hyperacmosin R (1) and furoadhyperforin isomer B (4) exhibited weak hepatoprotective activity at 10 µm. a Results are expressed as the means ± SD (for samples, n = 3; for normal and control, n = 6; b positive control (20 µm); *** p < 0.01 vs. normal; # p < 0.05, ## p < 0.01 vs. control.

General Experimental Procedures
Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO Inc. Tokyo, Japan). UV spectra were measured on a JASCO V650 spectrophotometer (JASCO Inc.  Figure 5. Plausible biosynthetic pathway for 1.

Hepatoprotection Bioassays (In Vitro)
The hepatoprotective effects of compounds 1-11 were determined by a (MTT) colorimetric assay in HepG2 cells. Each cell suspension of 2 × 10 4 cells in 200 µL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) was placed in a 96-well microplate and precultured for 24 h at 37°C under 5% CO 2 atmosphere. Fresh medium (100 µL) containing bicyclol and test samples was added, respectively, and the cells were cultured for 1 h. The cultured cells were exposed to 16 mM paracetamol for 24 h. Then, 100 µL of 0.5 mg/mL MTT was added to each well, after the withdrawal of the culture medium, and incubated for additional 4 h. The resulting formazan was dissolved in 150 µL DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 570 nm. Percentage inhibition was calculated as: inhibition (%) = [OD (sample) − OD (control)]/[OD (normal) − OD (control)] × 100%.

Conclusions
In summary, a detailed chemistry investigation of H. acmosepalum led to the identification of 11 PPAPs, including 2 previously undescribed ones, hyperacmosins R-S (1-2). Especially, hyperacmosin R (1) possesses a rare 5,8-spiroketal subunit, together with the loss of C-2 carbonyl in the phloroglucinol ring. All the isolates were evaluated for their hepatoprotective activities. Among them, hyperacmosin R (1) and furoadhyperforin isomer B (4) exhibited weak capabilities against paracetamol-induced HepG2 cell damage at 10 µm. Furthermore, the plausible biosynthetic pathway of hyperacmosins R (1) was proposed. This study enriched the members and the structural diversity of PPAPs from H. acmosepalum.