Abietane diterpenoids with neuroprotective activities from Phlegmariurus carinatus

Abstract Two new abietane diterpenoids, phlecarinatone A (1) and phlecarinatone B (2), along with two known analogues (3 and 4), were isolated from Phlegmariurus carinatus. The structures of 1 − 4 were unambiguously elucidated by comprehensive spectroscopic analyses. Abietane diterpenoids were isolated from the plant for the first time. All isolates were tested for their neuroprotective activities against H2O2-induced SH-SY5Y cells injury, and compound 2 showed moderate effect at the concentrations ranging from 5 ∼ 20 μM in vitro assay. Graphical Abstract


Introduction
Phlegmariurus carinatus (Desv.) Ching, belonging to the family Huperziaceae, is an epiphytic club moss mainly distributed in tropical and subtropical regions (Ching 1982). For thousands of years, the plant has been used as folk medicine for the treatment of rheumatism, pain and swelling (Wu and Song 1998;Zhang and Zhang 2004). To date, more than 600 Lycopodium alkaloids have been reported. Especially, the well-known compound huperzine A, a high selectivity acetylcholinesterase (AChE) inhibitor obtained from Huperzia serrata, has been used for the treatment of myasthenia gravis (MG) and Alzheimer's disease (AD) in China (Liu et al. 1986;Kozikowski and T€ uckmantel 1999;Bai et al. 2000;Cheng et al. 1996). Hence, phytochemical investigations of Huperziaceae family have generally focused on Lycopodium alkaloids. However, the AChE inhibitory activity of these alkaloids was lower than that of huperzine A (Ying et al. 2014;Xiong et al. 2019;Vallejo et al. 2020;Zhu et al. 2020). Abietane diterpenoids, a large family of naturally occurring diterpenoids, were found to exhibit potent cytotoxic, anti-microbial and anti-inflammatory activities (Sun andLi 2012；Wu et al. 2014;Ngo et al. 2021;Ulubelen et al. 2001).
Compound 2 was isolated as a white powder. It was assigned the molecular formula C 21 H 28 O 5 based on HRESIMS (m/z 361.2019 [M þ H] þ , calcd for C 21 H 29 O 5 þ , 361.2015) data analyses. A detailed comparison of the 1 D NMR data of 2 and 3 revealed that they are similar compounds. The 1 D NMR data of 2 closely resembled those of 3, except that a hydroxy group at C-11 (d C 138.6) and a methoxy group at C-12 (d C 152.2) in 3 was replaced by a methylenedioxy group (d C 100.5) in 2, which was confirmed by the HMBC correlations from H 2 -21 (d H 5.83) to C-11 (d C 135.8) and C-12 (d C 153.5). In addition, the methylene at d C 35.9 (C-6) in 3 was replaced by an oxygenated methine carbon (d C 72.6) in 2 which was confirmed by HMBC correlations of H-6 (d H 4.49) with C-5 (d C 54.7), C-7 (d C 203.1) and C-10 (d C 39.4) . The NOESY correlations of H-6 (d H 4.49)/H 3 -19 (d H 1.11) and H-6/H 3 -20 (d H 1.32) indicated a a-orientation of 6-OH. Hence, compound 2 was determined as 6,14-dihydroxy-11,12-methylenedioxyabieta-8,11,13-triene-7-dione and named phlecarinatone B . Additionally, two known compounds, inuroyleanol (3) (Frontana et al. 1994) and 7dehydroabietanone (4) (Su et al. 1994), were identified by comparison of their reported 1 D NMR data.

Neuroprotective activity assay
The neuroprotective effects of all the isolates on H 2 O 2 -induced decrease in cell viability were evaluated in human SH-SY5Y neuroblastoma cells. Compound 2 showed moderate neuroprotective activity ( Figure S3). The rest of the compounds exerted weak protective effects (Table S2).

Plant material
Whole herbs of P. carinatus were collected in the county of Jingzhou, Hunan Province, People's Republic of China, in the winter of 2021. The plant was authenticated by Prof. Gui-Shan Tan (Central South University, Changsha, China). A voucher specimen (No. 20210108) was deposited in the Key Laboratory of Prevention andTreatment of Cardiovascular and Cerebrovascular Diseases, Ministry of Education, Gannan Medical University.

Bioassay
SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and with 100 units/mL penicillin and 100 lg/mL streptomycin at at 37 C in a 5% CO 2 humidified incubator. Cells were plated at a density of 5000 cells per well in 96-well plates and cultured for 24 h. All compounds tested were dissolved in dimethylsulfoxide (DMSO) and then diluted to corresponding concentrations with cell culture medium. The antioxidant N-acetylcysteine (NAC, 100 mM) was used as a positive control for H 2 O 2 -induced cell damage models. Cells were pretreated with the test compounds 2 h then incubated with 100 lM H 2 O 2 for another 24 h without changing the culture medium. Then cells were incubated with the 10 mL of the MTT solution (5 mg/mL) for 2 h, and then 100 mL of DMSO to solubilize the formed formazan. The optical density (OD) levels were measured at 490 nm using a microplate reader.

Conclusions
In conclusion, four compounds, including two new abietane diterpenoids (1 and 2) and two known compounds (3 and 4), were isolated from the whole plant of P. carinatus. All compounds were tested for their neuroprotective activities toward damaged SH-SY5Y cells induced by H 2 O 2 . The bioassays suggested that only compound 2 significantly attenuated H 2 O 2 -induced cell damage at concentrations of 10 and 20 mM.