Two undescribed benzopyran derivatives from Ranunculus sceleratus L. with their in vitro anti-inflammatory activity in lipopolysaccharide-activated RAW 264.7 cells

Abstract Previous studies on Ranunculus sceleratus L. have shown the existence of coumarins and their anti-inflammatory effect. Phytochemical work was conducted to investigate the bioactive compounds, leading to the isolation of two undescribed benzopyran derivatives, namely ranunsceleroside A (1) and B (3), together with two known coumarins (2, 4) from the whole plant of R. sceleratus L. All compounds were structurally identified by extensive spectroscopic analysis and then investigated for their inhibitory effect on nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) production induced by lipopolysaccharide (LPS) in RAW 264.7 murine macrophages, repectively. As a result, compound 1-4 presented inhibitory effects on the production of NO, TNF-α, IL-1β, and IL-6 in a concentration-dependent manner, which provides a potential chemical basis for the traditional use of R. sceleratus L. as an anti-inflammatory plant. Graphical Abstract


Introduction
Ranunculus sceleratus L. (Ranunculaceae) is widely distributed throughout the world.It was first recorded in the Shennong Traditional Herbal Scriptures about two thousand years ago.Traditionally, R. sceleratus was used for treating various diseases such as swelling, lump, malaria, and cholestatic hepatitis (Zhang et al. 2020).Modern pharmacological studies have shown that the extracts of R. sceleratus exhibit obvious anti-inflammatory effects (Prieto et al. 2003), antidermatophytic activity (Sharma et al. 2012), and the hydroalcoholic extracts of R. sceleratus can inhibit nitrite accumulation, which may be helpful in preventing inflammatory diseases mediated by excessive production of NO (Marrelli et al. 2022).In our previous study, the chemical constituents in R. sceleratus were analyzed using GC-MS and UHPLC-Q-Orbitrap HRMS (Zhang et al. 2020;Cao et al. 2022).However, the chemical profile in R. sceleratus has not been sufficiently clarified, except for several known steroids, coumarins, phenolic acids, and anthraquinone (Gao et al. 2005;Peng et al. 2011).In this study, we elaborate on a further chemical investigation on R. sceleratus, leading to the isolation and identification of two undescribed benzopyran derivatives (1, 3) and two known compounds (2, 4) (Figure 1), among which compound 4 was first isolated from this plant.Isoscopoletin (2), was reported to exhibit significance inhibitory effects on HBsAg and HBeAg secretion from HepG2 at the concentration of 100 µg/mL (Li et al. 2005), and strong inhibitory effect on three kinds of tumor cells including A549, MCF7 and HepG2, with the IC 50 values of 35.25 ± 1.62, 8.58 ± 1.34, and 4.76 ± 1.01 µM, respectively (Zhao et al. 2014).However, isoscopoletin-β-D-glucopyranoside (4) has not been reported for its activity.Then, we preliminarily evaluated the potential anti-inflammatory activities of the isolated compounds, aiming to explore the chemical basis of R. sceleratus as a worldwide anti-inflammatory herbal medicine.
The in vitro cytotoxicity of all isolated compounds on RAW264.7 cells were evaluated by CCK-8 assay.As shown in Table S2, compounds 1, 3, and 4 showed no cytotoxic activity toward RAW264.7 in the concentration range from 12.5 to 200 μM.Compound 2 showed very weak cytotoxic activity at 100 μM and moderate toxicity at 200 μM.Therefore, we selected the appropriate concentration of tested compounds with a cell survival rate greater than 90% for the anti-inflammatory assay.
LPS-activated Raw 264.7 macrophages are commonly used as an in vitro anti-inflammatory model (Gao et al. 2017;Daud et al. 2021).LPS can stimulate RAW264.7 cells to produce an acute inflammatory reaction and increase the level of NO in cells, directly participating in the occurrence and development of inflammation (Cha et al. 2016).
Compared with the control group, the levels of NO production in LPS-activated group increased significantly (p < 0.05), suggesting the LPS-stimulated RAW 264.7 cell model is successful.Compared with the LPS group, all the test compounds (1-4) exhibited moderate levels of NO inhibitory effect on the LPS-stimulated RAW 264.7 macrophages with IC 50 values ranging from 25.86 to 56.39 μM in a concentration-dependent manner (Table S3, Figure S20).The four isloated compounds 1-4 displayed lower IC 50 values than those reported (Riviere et al. 2014).
The levels of TNF-α and IL-1β increased at the initial stage of inflammatory reaction, triggering inflammatory cytokine cascade, so they directly reflect the intensity of inflammatory reaction (Duan et al. 2022;Huang et al. 2022).The elevated TNF-α can further stimulate the production of IL-6 and thus lead to the continuous aggravation of inflammatory reaction (Zelova and Hosek 2013).Therefore, regulating these pro-inflammatory cytokines production may be helpful to control inflammation (Janakiram and Rao 2012).Compared with the control group, the levels of TNF-α, IL-1 β and IL-6 in LPS-activated group increased significantly (p < 0.05).Compounds 2 and 3 exhibited strong inhibitory effects on the above pro-inflammatory factors at medium and high concentrations, suggesting that compounds 2 and 3 exhibit considerable anti-inflammatory activity in vitro (Figures S21-S23).

Plant material
The dried whole plant of R. sceleratus L. (10.0 kg) was collected from Feidong County, Hefei City, AnHui Province, China in March 2021 and identified by Dr. Jin Xie of Anhui Medical University.Its voucher specimen (No. 202103) has been deposited at the School of pharmacy, Anhui Medical University.

Extraction and isolation
The dried whole plant (10 kg) of R. sceleratus L. was refluxed with 95%EtOH (60 L) for 4 h at 60 °C, repeated four times, and then concentrated in vacuum to get the crude extract (2 kg).The extract was suspended with distilled water, and then successively partitioned three times between equal volumes of petroleum ether, ethyl acetate, and n-butanol.The solvent was removed and four fractions was obtained as petroleum ether extract (P, 237 g), EtOAc extract (E, 75.0 g), n-BuOH extract (B, 192 g) and water layer (W, 1198 g), respectively.

The effect on the viability of RAW 264.7 cells
RAW 264.7 cells were seeded into 96-well plates at a density of 1 × 10 4 cells/well.After incubation at 37 °C under a humidified 5% CO 2 atmosphere with compounds 1-4 at different concentrations of 6.25, 12.5, 25, 50, 100 μM for 24 h, cell viability was measured by a CCK-8 assay.Experiments were performed in triplicate, and data are expressed as the mean ± SD, and different letters indicate statistic significant difference at p < 0.05.

The effect on the production of NO, TNF-α, IL-1β, and IL-6 in LPS-activated RAW 264.7 cells
RAW 264.7 cells were planted into 96-well plates at a density of 1 × 10 4 cells/well, and activated by 1 μg/mL of LPS with or without test compounds for 24 h.The cell-free supernatant were collected.The nitrite concentration in the medium was measured at 540 nm as NO production using Griess reagent (Chen et al. 2020).The levels of TNF-α, IL-1β and IL-6 were detected by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer′s instructions and related references (Gao et al. 2017;Daud et al. 2021).Experiments were performed in triplicate, and data are expressed as the mean ± SD, and different letters indicate statistic significant difference at p < 0.05.

Conclusions
In summary, this study isolated four compounds from R. sceleratus L., including two previously undescribed benzopyran derivatives (1, 3).The four isolated compounds was evaluated for their in vitro anti-inflammatory activities.The compounds were tested for their ability to inhibit the production of NO and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in LPS-induced RAW 264.7 cells.
The results showed that all tested compounds exhibited moderate levels of NO inhibitory effect concentration-dependently, with IC 50 values ranging from 25.86 to 56.39 μM.Additionally, they significantly inhibited pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in LPS-induced RAW264.7 cells compared to the model group.Compounds 2 and 3 showed strong inhibitory effects on the three inflammatory factors at medium and high concentrations, suggesting considerable anti-inflammatory activity in vitro.Overall, this study provides a potential chemical basis for the traditional use of R. sceleratus L. as an anti-inflammatory plant and may lead to further discovery and development of new anti-inflammatory agents.

Disclosure statement
No potential conflict of interest was declared by authors.