(±)-Sarcanan A, a pair of new enantiomeric dihydrobenzofuran neolignans from the aerial parts of Sarcandra glabra

Abstract (+)-Sarcanan A (1a) and (−)-Sarcanan A (1b), a pair of new dihydrobenzofuran neolignan enantiomers, together with six known compounds (2–7), were isolated from the aerial parts of Sarcandra glabra. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of 1a and 1b were determined by analyses of the electronic circular dichroism (ECD) data. All compounds were evaluated for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 cells, and compounds 2–4 exhibited moderate inhibition against NO production. Graphical Abstract


Introduction
Sarcandra glabra (Thunb.) Nakai (Chloranthaceae), an evergreen subshrub, is widely distributed in many southern provinces of China and other Southeast Asian countries (Editor Committee for Flora of China 1982). The whole plants of S. glabra have long been used as a traditional Chinese medicine (TCM) to treat inflammation, tumor, and traumatic injuries (National Pharmacopeia Committee 2015). Previous chemical investigations of this plant have resulted in the isolation of a number of sesquiterpenoids (Li et al. 2006;He et al. 2010;Hu et al. 2013;Chi et al. 2019;Wei et al. 2019; Wang et al. 2021), triterpenoids (Luo et al. 2005), coumarins (Feng et al. 2010;) and flavonoids (Liu et al. 2021), and some of them possessed extensive biological properties, such as cytotoxic, anti-inflammatory, and autophagy-inducing activities. In our screening program aimed at the discovery of new bioactive metabolites from medicinal plants (Peng et al. 2017;Liu et al. 2018;Bai et al. 2019), we undertook a phytochemical study of S. glabra, from which (±)-sarcanan A (1a/1b), a pair of new dihydrobenzofuran neolignan enantiomers, together with six known compounds (2-7) were isolated. Their structures were elucidated on the basis of extensive spectroscopic data and ECD analysis. Bioassay verified that compounds 2-4 showed certain inhibitory activity on NO production in RAW264.7 cells. Herein, we reported the isolation, structural elucidation, and NO inhibitory activity of these compounds ( Figure 1).  (d C 158.7, 146.9, 142.0, 134.8, 133.6, 132.8, 128.9 Â 2, 116.3 Â 2, 114.1, and 113.5), a disubstituted double bond (d C 132.4 and 123.4), two sp 3 methines (one oxygenated at d C 94.5), and two methyls. The above-mentioned NMR data implied that compound 1 possessed a dihydrobenzofuran neolignan feature (Tang et al. 2015).
Compound 1 was primarily obtained with a small specific rotation value and no Cotton effect in its ECD spectrum, suggesting a near racemic nature. Subsequent chiral-phase separation afforded the enantiomers 1a and 1b, which showed opposite specific rotation values ([a] 25 D þ43 for 1a; [a] 25 D À41 for 1b) and ECD curves ( Figure  S2). The absolute configurations of 1a and 1b were determined by analysis of their ECD data. The ECD data of 1a and 1b were similar to those of (þ)-conocarpan and (À)-conocarpan, respectively, which suggested (7S,8S) and (7R,8R) configurations for 1a and 1b, respectively (Tang et al. 2015).
All isolated compounds (1a/1b and 2-7) were evaluated for their inhibitory effects on nitric oxide (NO) production of LPS-induced RAW264.7 cells using the Griess assay. The results showed that compounds 2-4 exhibited comparable inhibitory activities with IC 50 values of 22.8, 17.6, and 6.8 mM, respectively, to the positive control quercetin (IC 50 ¼ 18.8 mM), while other compounds were inactive (IC 50 > 50 mM).

Plant material
The aerial parts of Sarcandra glabra were collected from Jiangxi Province, China, in December 2020, and were authenticated by one of the author (D.-p. Yang). A voucher specimen (accession number: CSH-202012) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University, China.

Cell culture and viability assay
The mouse macrophage cell line RAW264.7 was cultured in Dulbecco's modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA) which was supplemented with 10% FBS, 100 units/mL penicillin and 100 mg/mL streptomycin under humidified 95% air-5% CO 2 atmosphere at 37 C. The cell viability assay was detected by MTT method. RAW264.7 cells were plated in 96-well plates (5 Â 10 3 /100 mL/well) and incubated for 24 h. Following the drug or control was applied for 24 h, 20 mL MTT solution was added and incubated for 4 h. The crystals were dissolved in 100 mL DMSO after removal of the culture medium. Finally, absorbance (A) was measured at 490 nm.
3.5. Measurement of NO production RAW 264.7cells were plated at density of 5 Â 10 4 /100 mL/well in 96-well plates for 24 h and treated with various concentrations of the compounds or the fractions in the presence of 500 ng/mL LPS. After 24 h, the 50 mL medium was collected and mixed with the same volume of Griess reagent and the absorbance (A) was measured at 540 nm. No obvious cytotoxic effects were observed in the selected concentration range.

Conclusion
In summary, phytochemical investigation of the aerial parts of S. glabra led to the isolation of a pair of new enantiomeric dihydrobenzofuran neolignans (1a/1b) and six known compounds, including two lignans (2 and 7), two chalcones (3 and 4), and two dihydrochalcones (5 and 6). The structures were determined by combined spectroscopic and ECD methods. All compounds were examined for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW264.7 cells, and compounds 2-4 exhibited comparable inhibition on NO production to the positive control. Studies toward their mechanisms are in progress.

Disclosure statement
No potential conflict of interest was reported by the authors.