Two new sesquiterpenoids from the roots of Sarcandra glabra

Abstract Two new sesquiterpenoids, sarglanoids G and H (1 and 2), together with two known analogues (3–4) were isolated from the roots of Sarcandra glabra. Compound 2 was a rare elemene-type sesquiterpenoid with a furan ring fragment, which is mostly lactone ring in its analogues. The structures of new compounds were determined on the basis of extensive spectroscopic methods including HR-ESI-MS, 1D and 2D NMR, and the absolute configuration of 1 was established by ECD calculations. Their anti-inflammatory activities were also evaluated.


Introduction
The genus Sarcandra (family Chloranthaceae) is widely distributed in the over south of China, Cambodia, Indian, Japan and Korea (Wing and Geoffrey 1996). Sarcandra glabra (Thunb.) Nakai is an evergreen subshrub, and the whole plant is used for medicinal purposes as a traditional Chinese medicine for the treatment of heat-toxic and traumatic injury . Modern pharmacological studies suggest the extraction of the roots of S. glabra has important effects such as anti-inflammatory (Yaermaimaiti et al. 2016) and antitumor activity . Up to now, more than 200 compounds were isolated and determined from S. glabra, such as sesquiterpenoids, flavonoids and coumarins (Hu et al. 2013;Wang et al. 2016;Zeng et al. 2021). Recent research confirmed that sesquiterpenoids were important bioactive components in S. glabra (Wang et al. 2021). The sesquiterpenoids in S. glabra are mainly of lindenanetype, and a small amount of aromadendrane-type, eduesmane-type, valeriane-type and elemene-type compounds is also included. These types of sesquiterpenoids also have significant biological activity (Yang 2017;Chen et al. 2022;Wang et al. 2022). In order to find further pharmacological activities and to explore structural diversity, we investigated the sequiterpenoids from the roots of S. glabra. As result, a new aromadendrane-type sesquiterpenoid 1 and a new elemene-type sesquiterpenoid 2, together with two known analogues (3-4) were isolated from the dichloromethane extract of S. glabra. Herein, the details of the isolation, structure elucidation, and anti-inflammatory activities of these sesquiterpenoids were described.

Result and discussion
Compound 1 was obtained as a colorless oil and had a molecular formula of C 15 H 22 O 3 as determined from the positive ESI-HRMS ion peak at m/z 273.1461 [M þ Na] þ (calcd for C 15 H 22 O 3 Na þ 273.1461), which indicated five degrees of unsaturation. The IR absorption peaks ( Figure S4.8) at 3420 and 1640 cm À1 denoted the presence of hydroxy and conjugated ketone carbonyl. The 1 H NMR (Table S1.1) and HSQC spectra ( Figure S4.3) of 1 exhibited three methyl groups (d H 1.40, 1.25, 1.10, each 3H, s), and an oxygenated methylene (d H 4.34, d, J ¼ 12.2 Hz; 4.27, d, J ¼ 12.2 Hz). According to the 13 C NMR data (Table S1.1), a conjugated ketone carbonyl (d C 202.3), a double bond (d C 168.1 and 134.6), two oxygenated carbons (d C 81.3 and 61.6), along with 10 other aliphatic carbons were present in 1. The aforementioned spectroscopic features indicated that compound 1 was an analogue of 14-hydroxyaromadendr-1(10)-en-9-one (Dwight et al. 2001), a known aromadendrane-type sesquiterpenoid isolated from Hyptis verticillata. The only difference was the presence of an oxygenated tertiary carbon (d C 81.3) in 1, while the tertiary carbon in 14-hydroxyaromadendr-1(10)-en-9-one is found at d C 36.7. In HMBC spectrum, the correlations from Me-15 (1.40, s) to C-3 (d C 38.7), C-4 (d C 81.3) and C-5 (d C 54.2) suggested that the oxygenated tertiary carbon should be assigned to C-4 in 1. The presence of a hydroxyl group at C-4 was further determined by the same unsaturation of compound 1 and 14-hydroxyaromadendr-1(10)-en-9-one. Meanwhile, the larger coupling splitting between the oxygenated methylene hydrogen protons (12.2 Hz) can be interpreted as the formation of intramolecular hydrogen bond between OH-14 and carbonyl group (C-9), so that the C14-C10 bond could not rotate freely (Li et al. 2006;Shen et al. 2017). Therefore, the planar structure of compound 1 was elucidated as shown in Figure 1.
The relative configuration ( Figure S1.1) of 1 was deduced from the ROESY spectrum ( Figure S4.6). The ROESY cross-peaks of H-6 (H-7)/Me-13 and H-6 (H-7)/Me-15 indicated that H-6, H-7, Me-13 and Me-15 were co-facial and were randomly assigned as a-oriented. The H-5 could be assigned as b-orientation through the ROESY correlation from Me-12 to H-5. The absolute configuration of compound 1 was further determined as 4S,5S,6R,7R by ECD calculation with time-dependent density functional theory (TD-DFT) ( Figure S1.2). Accordingly, compound 1 was ascertained as an aromadendrane-type sesquiterpenoid, and based on our previous research (Wang et al. 2022), the compound 1 was named as sarglanoid G.
Compound 2 was obtained as a colorless oil and showed a molecular formula of C 15 H 18 O 3 , as determined by the ESI-HRMS ion peaks at m/z 269.1150 [M þ Na] þ (cald. For 269.1148), suggesting 7 degrees of unsaturation. The NMR data of 2 (Table S1.1) are showing high similarities with curzeren (Zhou et al. 2011) with the major difference being the presence of an extra ketone carbonyl (d C 188.4) and a primary alcohol moiety (d H 3.97, 2H, s; d C 66.5) in 2. Analysis of 2 D NMR spectra of 2 ( Figure S1.1) indicated that the ketone carbonyl and hydroxy group were located at C-9 and C-15, respectively, which was confirmed by HBMCs from H-1 (d H 5.91, dd, J ¼ 17.3, 10.8 Hz) and Me-14 (d H 1.26, s) to C-9 (d C 188.4), and from H-15 (d H 3.97, s) to C-3 (d C 113.1), C-4 (d C 148.4), and C-5 (d C 48.6). In the ROESY spectrum, no correlation signal between H-5 and Me-14 was observed, so the relative configurations of H-5 and Me-14 cannot be determined. To determine the relative configuration of 2, the 13 C chemical shifts of two diastereomers anti-2 (5S Ã , 10S Ã ) and syn-2 (5 R Ã , 10S Ã ) were calculated by the TD-DFT gage-independent atomic orbital (GIAO) method at the mPW1PW91/6-311 þ G (d,p) level of theory. The calculation results and DP4þ analysis (Grimblat et al. 2015) show that the configuration of 2 is anti (5S Ã , 10S Ã ) ( Figure S1.3). Unfortunately, the absolute configuration of 2 cannot be determined because there is no Cotton effect in its ECD spectrum. Finally, the structure of compound 2 was established as shown in Figure 1 and named sarglanoid H.
Anti-inflammatory effect is a widely studied pharmacological activity of S. glabra (Zeng et al. 2021;Chen et al. 2022). In our previous studies, a large number of sesquiterpenoids with significant anti-inflammatory activity were found from S. glabra (Wang et al. 2015). Thus, anti-inflammatory effects of 1-4 were also measured in this research. Unfortunately, the results indicated that none of these compounds have anti-inflammatory activity, showing IC 50 values over 50 lM.

General experimental procedures
The 1D and 2D NMR spectra were carried out on a Bruker Avance III-500 or AV-600 NMR Instrument in CDCl 3 . Optical rotations were recorded in MeOH with a JASCO P- 1020 polarimeter at room temperature. IR spectra were measured on a Bruker Tensor-27 infrared spectrophotometer. ECD spectra were acquired by a JASCO J-810 spectrometer. UV spectra were recorded by a Shimadzu UV-2450 UV/vis spectrophotometer. ESI-HRMS data were measured by an Agilent 6520B UPLC-Q-TOF instrument. Preparative HPLC was carried out on a Shimadzu LC-20AR system equipped with an Ultimate XB-C18 column (250 mm Â 20 mm i.d., 5 lm) with a flow rate of 10.0 mL/min, using a UV detector (SPD-20A). Analytical HPLC was carried out on an Agilent 1100 system outfitted with a DAD-UV detector. ODS (40-63 lm, FuJi, Japan), Sephadex LH-20 (40-70 lm; Amersham, Pharmacia Biotech AB, Uppsala, Sweden), and TLC analysis (precoated silica gel GF254 plates) were employed in separation of crude and monitoring of column chromatography.

Plant material
The roots of S. glabra (Thunb.) Nakai were collected from the Guangxi Province in China in September 2016 and were identified by Professor Mian Zhang of China Pharmaceutical University. A voucher specimen (no. SG201609) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.

Anti-inflammatory activity
NO production was quantified by measuring the accumulation of nitrite in the cell culture supernatant with Griese reagent . Briefly, RAW 264.7 cells (6 Â 10 6 cells/mL) were seeded in 96-well plates and pretreatment with compounds for 1 h before LPS (1 lg/mL) stimulation. The isolated culture supernatant was mixed with Griese reagent (Beyotime Biotechnology, China). NaNO 2 was used to generate a standard curve, and the absorbance of the mixture was measured at 540 nm. In the experiment, monomethylarginine monoacetate (L-NMMA) was used as a positive control.

Conclusion
Studies on the structural diversity of the CH 2 Cl 2 extract of S. glabra roots led to the isolation of four sesquiterpenoids (1-4) including two new compounds, sarglanoids G (1) and H (2). Sarglanoid H (2) was identified as a rare elemene-type sesquiterpenoid with a furan ring fragment (Lou et al. 2010;Zhang et al. 2015;Salihila et al. 2019;Ulises et al. 2019). Elemene is a second-line broad-spectrum anti-tumour drug that has been used in China for more than two decades (Bai et al. 2022). The structural analogue, sarglanoid H (2), may have potential in antitumor activity, although it did not show biological activity in experiments of anti-inflammatory activity based on the traditional application of S. glabra. Therefore, the biological activities of these compounds need further investigation.

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