Chemical constituents isolated from Hosta ensata and their anti-inflammatory activities

Abstract A new phenol derivative, hostaphenol A (1), along with 16 known ones (2–17) were isolated from an ethanolic extract of the whole plants of Hosta ensata F. Maek. Their structures were elucidated by HRMS and NMR data as well as comparison with those reported in literature. The report of the first cyclopeptide and compounds 5, 6, 8, 10, 12–15, and 17 in the Asparagaceae family. Compound 2, as well as compounds 3, 4, 7, 9, 11, and 16 were reported for the first time from the Hosta genus and this plant, respectively. All compounds significantly reduced nitric oxide (NO) production at a concentration of 40 μM with no toxicity in RAW 264.7 cells stimulated by lipopolysaccharide. Among them, compounds 2–5 (40 μM) exerted obvious NO inhibitory activities, and their inhibition rate was exceeded 50%. Graphical Abstract

As a part of our continuing research to discover more phytochemicals with anti-inflammatory activities that had been derived from the Hosta genus.In this study, chemical constituents isolated from an ethanolic extract of the whole plants of H. ensata and their anti-inflammatory in vitro were performed.As a result, a new phenol derivative, hostaphenol A (1), along with 16 known ones (2-17) (Figure 1) were isolated by repeated silica gel column chromatography from H. ensata.Herein, we reported the isolation and structure elucidation, as well as their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Anti-inflammatory activities of compounds 1-15 in LPS-induced RAW 264.7 cells
As the whole plant of H. ensata was an important traditional Chinese medicine that has been widely used to treat inflammation-related diseases (Yang and He 2021).Therefore, all isolated compounds, except for 16 and 17 due to their insolubility, were evaluated their anti-inflammatory in LPS-induced RAW 264.7 cells.
As shown in Table 1, compounds 1-15 at a concentration of 40 μM had no significant cytotoxicity for RAW 264.7 cells (p > 0.05).Subsequently, inhibitory effects of compounds 1-15 at a concentration of 40 μM against NO production in LPS-stimulated RAW 264.7 cells were performed.As a result, all compounds obviously reduced NO level (Figure 2) compared with only treated with the LPS.Among them, compounds 2 − 5 exerted remarkable NO inhibitory activities with inhibition rates of 54.8 ± 12.8%, 60.2 ± 5.1%, 50.9 ± 15.1%, and 59.7 ± 6.6%, respectively (Table 2).

Plant material
The whole plants of H. ensata were collected in August 2019 from Changchun city, Jilin Province, China.This plant (voucher specimen No. He-201908) was authenticated  by Professor Guoyue Zhong (one of the corresponding author of this paper), and deposited at the last corresponding author's laboratory.

Extraction and isolation
The dried powders of H. ensata (40 kg) were extracted with 95% ethanol-water (200 L × 4 times), followed by 50% ethanol-water (200 L × 4 times) by maceration at room temperature.The ethanolic extract of H. ensata (He, 10.7 kg) was obtained after filtration, mergence, and evaporation.Subsequently, 10.0 kg of He was suspended in water and successfully partitioned with petroleum ether, ethyl acetate, and n-butanol to afford the corresponding fractions.

Cell culture and cell viability assay
The RAW 264.7 cells were incubated in dulbecco's modified eagle's medium, which was described in our previous work (Fang et al. 2021(Fang et al. , 2022;;Yang and He 2022).The CCK-8 kit was selected to evaluate the cell viability of isolated compound at a concentration of 40 μM as described previously (Fang et al. 2021).All data were performed in independent triplicate tests and were presented as the mean ± standard deviation (Sd).A p < 0.05 was labelled as statistically significant.

NO inhibitory assay
The NO inhibitory activity of isolated compound at a concentration of 40 μM was determined by the Griess reaction method, and the protocol was detailed described previously (Fang et al. 2021).All data were performed in independent triplicate tests and were presented as the mean ± standard deviation (Sd).A p < 0.05 was labelled as statistically significant.

Conclusions
In summary, a new phenol derivative (1) and 16 known ones (2-17) were isolated from an ethanolic extract of the whole plant of H. ensata.The report of the first cyclopeptides (12-15) in the Asparagaceae family.In addition, compounds 5, 6, 8, 10, 12-15, and 17, as well as 2, together with 3, 4, 7, 9, 11, and 16 were reported for the first time from the Asparagaceae family, Hosta genus, and this plant, respectively.Further, in vitro assay suggested that compounds 2-5 (40 μM) exerted obvious NO inhibitory activities in LPS-induced RAW264.7 cells, and their inhibition rate was exceeded 50%.The findings support the traditional use of H. ensata.

Figure 2 .
Figure 2. effects of compounds 1 − 15 on No production in lPs-stimulated raW 264.7 macrophages.data are represented as the mean ± sd of three experiments.## p < 0.01, compared with the normal cells; **p < 0.01, compared with the lPs-treated cells.
aValues are mean ± sd of three independent experiments at a concentration of 40 μM.

Table 2 .
effects of compounds 1 − 15 on No inhibition rates in lPs-stimulated raW 264.7 macrophages.