Terpenoids from the Roots of Stellera chamaejasme (L.) and Their Bioactivities

An undescribed diterpene, stellerterpenoid A (1), and two undescribed sesquiterpenoids, stellerterpenoids B and C (2–3), together with six known compounds, prostratin (4) stelleraguaianone B (5), chamaejasnoid A (6), auranticanol L (7), wikstronone C (8), and oleodaphnone (9), were isolated from the roots of Stellera chamaejasme L. Their structures were elucidated by extensive spectroscopic data (1D, 2D NMR, IR, UV, and HR-ESI-MS). The absolute configuration of 1–3 was elucidated based on ECD calculation. Among them, stellerterpenoid A was a rare 13, 14-seco nortigliane diterpenoid and stellerterpenoid B was a guaiacane-type sesquiterpenoid with an unusual 1, 2-diketone moiety. The known stelleraguaianone B (5) exhibited moderate activity for suppressing NO production in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages cells with an IC50 value of 24.76 ± 0.4 μM. None of the compounds showed anti-influenza virus or anti-tumor activity in vitro.


Introduction
The genus Stellera is comprised of 10-12 species, two of which are distributed in China (Stellera chamaejasme Linn.and Stellera formosana Hayata ex Li) [1].S. chamaejasme L, also known as 'Lang Du', is a toxic plant that is widely distributed in north and southwest China [2,3].The issue of S. chamaejasme spreading is increasingly serious as it consumes soil moisture and nutrients [4].The coverage of forage plants and grassland yield were greatly decreased due to the uncontrolled spread of S. chamaejasme, which competes with other plants for nutrient space and weakens the normal growth of herbage [5].The roots of S. chamaejasme can be used to extract industrial alcohol and make paper due to its high content of wolfsbane fiber [6].Although the whole plant of S. chamaejasme L is poisonous, it has certain medicinal values.S. chamaejasme was widely used in traditional Chinese medicine (TCM) for the effects of clearing heat, detoxification, detumescence, reducing inflammation, stopping ulcers, and removing saprophytic muscle [7][8][9][10][11].Many studies have been conducted on its chemical composition and pharmacological activities in recent decades.A series of compounds have been reported, including highly oxidized daphnane-type diterpenes [12,13], guaiane-type sesquiterpenoids [14 -18], unusual C-3/C-3 -biflavanones [19][20][21][22], lignans [14], and coumarins [20].Some compounds from S. chamaejasme have displayed many different and interesting biological activities in modern pharmacological research.For example, gnidimacrin, a 1-alkyldaphnanetype diterpene, was found to have strong anti-cancer activity with a broad anti-cancer spectrum [23].Daphnane diterpenes, stelleralides D-J, exhibited more potent anti-HIV activity (EC 50 = 0.73~0.98nM) than zidovudine (positive controls, EC 50 = 32 nM) [24].Moreover, chamechromone can significantly inhibit the expression of proinflammatory cytokines in RAW 264.7 cells [25].According to the above literature, the diterpenoids 0.73~0.98nM) than zidovudine (positive controls, EC50 = 32 nM) [24].Moreover, chamechromone can significantly inhibit the expression of proinflammatory cytokines in RAW 264.7 cells [25].According to the above literature, the diterpenoids and sesquiterpenoids are possibly the active components in this plant owing to the anti-inflammatory, anti-viral, and anti-tumor activities.
In order to identify the natural compounds with anti-inflammatory, anti-viral, and anti-tumor activities from S. chamaejasme, the methanol extract of the roots were phytochemically investigated in our continuous research work.As a result, three new terpenoids, including stellerterpenoid A (1) with a rare 13, 14-seco nortigliane skeleton, stellerterpenoid B (2) with an unusual 1, 2-diketone moiety, and a guaiacane sesquiterpenoid stellerterpenoid C (3), together with six known terpenoids (4-9), were identified from the ethyl acetate extract (Figure 1).In addition, the anti-inflammatory, anti-influenza virus, and anti-tumor activities of the nine compounds were tested.Herein, the isolation, identification, structural characterization, and biological assessment of these compounds are reported.

Results and Discussion
The relative configuration of 1 was elucidated by the analysis of NOESY spectrum.The NOESY correlations of H-7/H-11, H-8/H-11, and H-8/H-12β (Figure 3) allowed the H-7, H-8, H-11, and C-9-C-11 to be assigned the β-orientation, while the NOESY cross-peaks of H-10/H-5, H-5/H-17, and H-12α/H-17 indicated that H-5, H-10, and Me-17 were αoriented.The negative cotton effect at 250 nm in the experimental ECD curve of 1 assigned its absolute configuration as 4S, 5S, 6R, 7S, 8R, 10R, and 11R (Figure 4a).Therefore, the structure of 1 was fully elucidated to be a rare 13, 14-seco nortigliane diterpenoid and named stellerterpenoid A. To the best our knowledge, it is the second report of 13, 14-seco nortigliane diterpenoid other than crotonianoid A.   Stellerterpenoid B (2) was obtained as a yellow oil with a molecular formula of C15H16O3 according to its HR-ESI-MS at m/z 267.0981 [M + Na] + (calcd 267.0992) (Figure S14), suggesting eight degrees of unsaturation.The 1 H NMR spectrum (Table 1) of 2 exhibited one pair of double bonds at δH 4.81 (s, H-12b) and 4.80 (s, H-12a) and three methyl peaks at δH 1.95 (s, H-14), 1.85 (s, H-15), and 1.80 (s, H-13).The 13 C NMR spectrum (Table 2), in combination with the DEPT spectrum, showed 15   The plausible biosynthetic pathways of 1 were proposed in Scheme 1. Crotonianoid A originated from prostratin through the intermediate products i, ii, iii, and iv [26].Furthermore, the C-14 and C-15 double bond in crotonianoid A were rearranged to C-15 and C-16 by claisen rearrangement to obtain intermediate vi, which were oxidized to obtain vii, and HCHO was then removed to obtain compound 1.The NMR spectral data of 2 were very similar to those of oleodaphnone [27] except for the replacement of one ketone carbonyl in 2 by one methylene in oleodaphnone.This observation suggested that one methylene in oleodaphnone was oxygenated into one ketone carbonyl in 2. The HMBC correlation from H-14 (δ H 1.95) to C-1 (δ C 202.6) verified that the ketone was located at C-1 (Figure 2).The experimental ECD spectrum of 2 demonstrated the negative cotton effect at 230 nm and the positive cotton effect at 327 nm, which agreed well with the calculated ECD spectrum of (6S)-2 (Figure 4b).Therefore, the structure of 2 was elucidated to be a guaiacane sesquiterpene with an unusual 1,2-diketone moiety and named stellerterpenoid B.
Since a 1,2-diketone fragment is rare in natural compounds, the biosynthetic pathway of 2 was proposed based on oleodaphnone (Scheme 2).The C-1 of oleodaphnone is oxidized by P450 enzyme to form hydroxyl intermediate i, which is further oxidized to form 2 [28].2).
The NMR spectra of 3 were extremely similar to those of known wikstronone C [29], except that the signals of one methylene were replaced by one oxy-methine in 3. The above-mentioned NMR spectral feature suggested that one methylene was oxygenated into one oxy-methine in 3. The HMBC correlations from H-7 (δH 3.69) to C-8 (δC 40.8) and C-6 (δC 51.3), together with the 1 H-1 H COSY spin system of H-6/H-7/H-8/H-9, indicated that a hydroxy was connected with C-7 in 3 (Figure 2).In the NOESY spectrum, CH3-14 (δH 0.85) correlated with H-1β (δH 2.57), suggesting that CH3-14 is β-oriented.The NOESY correlations of H-7/H-9 and H-6/H-9 suggested the α-orientation of H-6 and H-7 (Figure 3).The 10-OH is defined as the β-configuration because the chemical shift of C-10 (δC 82.2) is similar to that (δC 83.2) of wikstronone C. The positive cotton effect at 216 nm and the negative cotton effect at 244 nm in experimental ECD spectrum of 3 (Figure 4c) assigned its stereochemistry as 6R, 7R, 9S, and 10R.Hence, the structure of 3 was characterized and named stellerterpenoid C.
The NMR spectra of 3 were extremely similar to those of known wikstronone C [29], except that the signals of one methylene were replaced by one oxy-methine in 3. The above-mentioned NMR spectral feature suggested that one methylene was oxygenated into one oxy-methine in 3. The HMBC correlations from H-7 (δ H 3.69) to C-8 (δ C 40.8) and C-6 (δ C 51.3), together with the 1 H-1 H COSY spin system of H-6/H-7/H-8/H-9, indicated that a hydroxy was connected with C-7 in 3 (Figure 2).In the NOESY spectrum, CH 3 -14 (δ H 0.85) correlated with H-1β (δ H 2.57), suggesting that CH 3 -14 is β-oriented.The NOESY correlations of H-7/H-9 and H-6/H-9 suggested the α-orientation of H-6 and H-7 (Figure 3).The 10-OH is defined as the β-configuration because the chemical shift of C-10 (δ C 82.2) is similar to that (δ C 83.2) of wikstronone C. The positive cotton effect at 216 nm and the negative cotton effect at 244 nm in experimental ECD spectrum of 3 (Figure 4c) assigned its stereochemistry as 6R, 7R, 9S, and 10R.Hence, the structure of 3 was characterized and named stellerterpenoid C.
The inhibitory rate of HepG2, A549, and HeLa cell growth of compounds 1-9 was measured by the MTT assay.Unfortunately, none of these nine compounds were active (IC 50 > 50 µM).

Plant, Virus Strain, and Cancer Cell Materials
The fresh roots of S. chamaejasme L were collected in Dali city, Yunnan Province, People's Republic of China, on 12 August 2021, and identified by Dr Zhijun Zhang, Kunming University of Science and Technology.A voucher specimen (KUMST20211007) has been deposited at the Key Laboratory of Phytochemistry, Kunming University of Science and Technology.The RAW 264.7 was purchased from Conservation Genetics CAS Kunming Cell Bank (KCB200603YJ).The A/PR/8/34 (HIN1) virus strain was donated by Professor Yang Zifeng from Guangzhou Medical University, China.The A549 (human lung cancer cells), HepG2 (liver cancer cells), and HeLa (cervical cancer cells) were donated by Nanjing KGI Biotechnology Company (Nanjing, China).

Extraction and Isolation
The fresh roots of S. chamaejasme L were air-dried.Dried roots of Stellera chamaejasme (11.0 kg) were crushed and extracted with 70% acetone/H 2 O three times (3 × 50 L) at room temperature to give a crude extract (10.1 kg) under reduced pressure distillation.The extract was mixed with water (15.0L), followed by successive partitioning with petroleum ether (3 × 15 L) and EtOAc (3 × 15 L).The EtOAc extract (3.35 kg) was separated by silica gel column chromatography (25 × 200 cm) using a gradient of petroleum ether/EtOAc (5:1-1:1, v/v) and CHCl 3 /MeOH (3:1-1:1, v/v) as the eluents to give eight fractions (Fr.A~H).highest distribution (fuef63, fuef4, and fuef72-1) of conformers for compounds 1-3, respectively, were further optimized by the density functional theory method at the B3LYP/6-31G (d) level.The ECD calculations were performed using level TD-DFT-B3LYP/6-311G (+, 2d, p) of the theory on optimized geometries through the CPCM model (in MeOH) [34].The calculated ECD curves were generated using SpecDis 1.71 [35] and all of the above calculations were carried out with the Gaussian 16 package of programs.All ECD curves were weighted by Boltzmann distribution after UV correction.

Determination of NO Production
The murine macrophage cell line RAW 264.7 was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (10,000 U/mL)-streptomycin (10,000 µg/mL) at 37 • C in a humidified incubator containing 5% CO 2 .The RAW 264.7 (8 × 10 4 /well) cells were left to acclimate for 24 h before any treatments and were seeded onto 96-well plates and pretreated by compounds 1-9 (3.125-50 µM) 1 h prior to treatment with 1 µg/mL LPS.Afterward, co-stimulation for 24 h at 37 • C was carried out in an incubator under 5% CO 2 .Then, Griess reagents I and II (100 µL) were mixed with cell culture medium (70 µL) and incubated at room temperature for 10 min with horizontal shaking, after which the absorbance was measured at 540 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).N(G)monomethyl-Larginine, monoacetate salt (L-NMMA) and DMSO were used as positive and negative controls, respectively [36].

Anti-Influenza Virus Assay
Influenza strain A/Puerto Rico/8/1934 (H1N1) was used in this study.MDCK cells (1 × 10 4 /well) were inoculated in 96-well plates and cultured for 24 h before viral infection.The infection medium was MEM (0.1 mM non-essential amino acid, 1% FBS, 1 mM sodium pyruvate, 1 µg/mL trypsin (Sigma-Aldrich, Beijing, China), and 100 U/mL penicillin/streptomycin).The cells were treated with various concentrations (from 3.125 µM to 50 µM) of tested compounds 1-9 and detected by Promega (Madison, WI, USA) CellTiter-Glo ® reagent following the protocol provided by the supplier.The emitted luminescence (RLU) was quantified with a Promega Victor III plate reader.Oseltamivir was used as the positive control [37].

Anti-Tumor Assay
HepG2, A549, and HeLa cell lines were used for cytotoxic activity.Cells were cultured in DMEM medium at 37 • C in 5% CO 2 .The medium was composed of 10% FBS, 1% HEPES, 1% mixed penicillin (10,000 U/mL), and streptomycin (10,000 µg/mL) fluid, as well as 10% fetal bovine serum.The cells (1 × 10 5 per well) were seeded on 96-well microplates and allowed to adhere for 12 h before drug addition.Each cell line was treated with compounds 1-9 at different concentrations (3.125-50 µM) in triplicate for 48 h.Spectra-Max M2 (Molecular Devices Inc, San Jose, CA, USA) was employed to record the optical density (λ = 490 nm).IC 50 values were calculated based on the mean OD values measured three times versus the concentration curves of drugs.Adriamycin (10 mM, purity 99%, Solar bio Science and Technology Co., Ltd., Beijing, China) and DMSO were used as the positive and negative control [38].

Statistical Analysis
All biological experiments were repeated at least three times and the results were obtained using Statistical Package for Social Sciences (SPSS Version 21.0) software [36].

Table 3 .
Inhibitory effects of compounds 1-9 on LPS-induced NO production in RAW 264.7 macrophages.

Table 3 .
Inhibitory effects of compounds 1-9 on LPS-induced NO production in RAW 264.7 macrophages.