Sinoflavonoids NJ and NK, anti-inflammatory prenylated flavonoids from the fruits of Podophyllum hexandrum Royle

Abstract Two new prenylated flavonoids named sinoflavonoids NJ and NK (1–2), along with ten known compounds were isolated from the fruits of Podophyllum hexandrum Royle. The chemical structures were determined through NMR spectroscopic data and MS analysis. Sinoflavonoid NJ (1) with an unusual 5,11-dioxabenzo[b]fluoren-10-one skeleton was firstly reported from Berberidaceae. The isolated flavonoids were tested with LPS-induced RAW 264.7 mouse macrophages model for their anti-inflammatory activity. Sinoflavonoid NJ (1) showed the most potent inhibition on nitric oxide production with IC50 value as 0.06 μM. GRAPHICAL ABSTRACT


Introduction
Prenylated flavonoids, characterized by a flavonoid skeleton with lipophilic prenyl side-chains, were mainly reported as plant secondary metabolites with diverse biological activities (Shi et al. 2021;Xu et al. 2022).Among them, icaritin from Epimedii Herba represented the first natural prenylated flavonoid which has been investigated on a phase III clinical trial in China for the treatment of hepatocellular cancer (Fan et al. 2019).It has been revealed that prenylation might increase the bioactivities of Sinoflavonoid; prenylated flavonoid; Podophyllum hexandrum; structure elucidation; anti-inflammatory SHORT COMMUNICATION flavonoid backbone (Mukai 2018).Therefore, prenylated flavonoid is a good resource in the drug discovery for the treatment of multiple diseases.As a traditional Tibetan medicine, the fruit of Podophyllum hexandrum Royle (previously known as Sinopodophyllum hexandrum, Berberidaceae) has been used for thousands of years for the treatment of amenorrhea, dead fetus, and placental retaining (Kong et al. 2010).Modern phytochemical studies on P. hexandrum have resulted in the isolation of more than 60 flavonoids with diverse activities (Sun et al. 2014(Sun et al. , 2015;;Wang et al. 2017;Sun et al. 2019Sun et al. , 2020Sun et al. , 2021)).Most of them had prenylated side-chains included prenyl and its variations after hydroxylation, cyclization and oxidation.As part of our ongoing efforts towards exploring novel natural products from medicinal plant resources (Zhao et al. 2020;Ding et al. 2021;Hu et al. 2021;Zhu et al. 2022), two new prenylated flavonoids (1 and 2) and ten known natural products were obtained from the fruits of P. hexandrum (Figures S1).Herein we report the extraction, isolation, structural elucidation and preliminary anti-inflammatory activity of these compounds.
The isolated flavonoids 1-8 was tested by measuring the inhibitory effects on the NO production in LPS-induced RAW 264.7 macrophage cells.Although these compounds showed more or less cytotoxic activities to macrophages at concentrations ranging from 1 to 25 μM (Table S3), they inhibited the production of NO in a dose-dependent manner below 5 μM in cells stimulated with lipopolysaccharides (LPS).The IC 50 values at 0.06-1.09μM for compounds 1-8 (Table S4) indicated they were anti-inflammatory at low dosages.

Conclusion
The phytochemical investigation on the fruits of P. hexandrum led to discovery of two new prenylated flavonoids and ten known compounds.Among them, sinoflavonoid NJ (1) with a rare 5,11-dioxabenzo[b]fluoren-10-one skeleton was firstly reported from Berberidaceae.This notably expands the structural diversity of prenylated flavonoids reported to date.Except for the cytotoxic lignans all isolated flavonoids 1-8 were tested for their anti-inflammatory activity by using LPS-induced RAW 264.7 mouse macrophages model.All tested compounds displayed strong inhibition on NO production, and sinoflavonoid NJ (1) with the unique 5,11-dioxabenzo[b]fluoren-10-one core skeleton showed the most potent activity with IC 50 value of 0.06 μM, which indicated the cyclization of C-3 and C-6′ help to improve the anti-inflammatory activity.

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

Figure 1 .
Figure 1.structures of compounds 1 and 2 isolated from the fruits of P. hexandrum.