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

2.1. Structural Elucidation of Three New Compounds (1, 2, and 3)

Stellerterpenoid A (1), yellow oil, had a molecular formula of C₁₉H₂₄O₈ deduced from the quasi-molecular ion peak at m/z 403.1344 [M + Na] ⁺ (calcd 403.1363) in the HRESIMS, with eight degrees of unsaturation. The ¹H NMR spectrum (Table 1) of 1 displayed the presence of three methyls at δ_H 2.19 (s, H-16), 1.78 (s, H-18) and 1.18 (d, J = 7.0 Hz, H-17), an oxymethines at δ_H 3.85 (s, H-19), a suite of methines including an olefinic proton at δ_H 7.48 (d, J = 1.8 Hz, H-1), and four methines at δ_H 4.31 (s, H-5), 3.57 (ddd, J = 10.6, 5.4, 2.4 Hz, H-8), 3.24 (d, J = 6.0 Hz, H-7), and 2.74 (m, H-11). Its ¹³C NMR and DEPT spectra (Table 2) revealed 19 carbon signals, including three methyls [δ_c 30.2 (C-16), 18.0 (C-17), and 10.0 (C-18)], three methylenes [δ_c 63.6 (C-19), 42.6 (C-14), and 38.9 (C-12)], six methines [δ_c 158.3 (C-1), 75.9 (C-5), 58.8 (C-7), 51.0 (C-10) and 40.3 (C-8)], and seven quaternary carbons consisting of two carbonyl [δ_C 209.8 (C-3), 209.2 (C-15)], one ester carbonyl group at δ_C 177.9 (C-13), one olefinic at δ_C 137.7 (C-2) and three oxygenated ones at δ_C 91.8 (C-9), 76.2 (C-4) and 66.0 (C-6).

The ¹H and ¹³C-NMR spectral data of 1 were similar to those of crotonianoid A [24] except for the occurrence of signals due to a methylene group (δ_C 42.6, C-14) and a ketone carbonyl (δ_C 209.2, C-15) in 1 instead of the ∆^14,15 double bond (δ_C 123.0, 136.5) in crotonianoid A. The phenomena suggested that the ∆^14,15 double bond in crotonianoid A was oxygenated, thus interpreting the presence of the methylene and ketone carbonyl in 1. In addition, the methylene of C-5 [δ_C 38.8, δ_H 2.41 (d, J = 18.6 Hz), 2.66 (d, J =18.6 Hz)] in crotonianoid A was replaced by one oxy methine [δ_C 75.9, δ_H 4.31 (s)] and the double bond was replaced with one epoxy structure at C-6 and C-7. The C-9 and C-13 via an oxygen atom to generate a five-membered lactone ring was demonstrated by signal offset are similar to crotonianoid A [24]. The HMBC cross-peaks from H-8 (δ_H 3.57) to C-14 (δ_C 42.6) and C-15 (δ_C 209.2) and from H-16 (δ_H 2.19) to C-14 (δ_C 42.6) and C-15 (δ_C 209.2) supported the location of the acetone unit at C-8. In addition, the HMBC correlations from H-11 (δ_H 2.74) to C-8 (δ_C 40.3), C-9 (δ_C 91.8), C-10 (δ_C 51.0), C-12 (δ_C 38.9) and C-13 (δ_C 177.9).

The relative configuration of 1 was established by the analysis of NOESY spectrum. The correlations of H-7/H-11, H-8/H-11and H-12β (Figure 3) allowed H-7, H-8, H-11, and C-9−C-11 bond were assigned β-directed, while the NOESY cross-peaks H-10/H-5 and H-17, and H-12α/H-17 indicated that H-5, H-10, and Me-17 were α-oriented, and H-10 and 4-OH were trans-oriented. The comparison of the calculated and experimental ECD spectra of 1 assigned its absolute configuration to be 4S, 5S, 6R, 7S, 8R, 10R, 11R (Figure 4a). Therefore, the structure of 1 was fully elucidated to be a rare 13, 14-seco nortigliane and named stellerterpenoid A. To be the best our knowledge, it is the second report of 13, 14-seco nortigliane diterpenoid besides crotonianoid A.

Stellerterpenoid B (2) was obtained as yellow oil with a molecular formula of C₁₅H₁₆O₃ according to its HRESIMS at m/z 267.0981 [M + Na] ⁺ (calcd 267.0992), suggesting eight degrees of unsaturation. The ¹H NMR spectrum (Table 1) of 2 exhibited the presence of a one double bond at δ_H 4.81 (s, H-12b) and 4.80 (s, H-12a) and three methyl peaks at δ_H 1.95 (s, H-14), 1.85 (s, H-15), and 1.80 (s, H-13). The ¹³C NMR spectrum (Table 2) in combination with the DEPT spectrum showed 15 carbon signals, including three methyls in the high magnetic field at δ_c 20.4 (C-13), 17.3(C-14), and 8.8 (C-15), an olefinic carbons at δ_c 111.3 (C-12), two methylene at δ_c 50.6 (C-7) and 36.3 (C-5), one methine at δ_c 40.3 (C-6) and eight quaternary carbons δ_c 204.3 (C-2), 202.6 (C-1,8), 165.1 (C-4), 148.9 (C-11), 146.1 (C-3), 145.9 (C-10) and 132.6 (C-9). The NMR feature indicated that 2 was a guaiacane type sesquiterpene.

The NMR spectral data of 2 were very similar to those of oleodaphnone [25] except for the replacement of one ketone carbonyl in 2 by one methylene in oleodaphnone. This observation suggested that one methylene in oleodaphnone was oxygeanted 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. The absolute configuration of 2 was determined by the ECD data. The calculated ECD spectrum agreed well with the experimental ECD spectrum of (6S)-2 (Figure 4b). Therefore, the structure of 2 was elucidated to be a guaiacane sesquiterpene with a unusual 1,2-diketone moiety and named as stellerterpenoid B.

Since 1,2-diketone fragment is rare in natural compounds, the biosynthetic pathway of 2 were proposed based on a biosynthetic precursor oleodaphnone (Scheme 1). The C-1 of oleodaphnone is oxidized by P450 enzyme to form hydroxyl intermediate i, which is further oxidized to form 2 [26].

Stellerterpenoid C (3) was obtained as white oil. Its HRESIMS exhibited a quasi-molecular ion peak at m/z 273.1454 [M + Na] ⁺ (calcd 273.1461), suggesting a molecular formula of C₁₅H₂₂O₃ with five degrees of unsaturation. The ¹H NMR spectroscopic data (Table 1) show the presence of three methyls at δ_H 1.80 (s, H-13), 1.64 (s, H-15) and 0.85 (d, J = 7.2 Hz, H-14), four methylenes at δ_H 4.79 (s, H-12a), δ_H 4.76 (s, H-12b), 2.57 (d, J =18.2 Hz, H-1a), 2.34 (d, J =18.2 Hz, H-1b) and 2.20 (ddd, J =13.8, 10.0, 3.5 Hz, H-8), 1.75 (ddd, J =13.8, 5.4, 3.5 Hz, H-8), and threes methines at δ_H 3.69 (m, H-7), 3.00 (m, H-6) and 2.29 (m, H-9).The ¹³C NMR spectrum showed 15 signals, which were ascribed to three methyls at δ_C 19.9 (C-13), 15.6 (C-14), and 7.9 (C-15), four methylenes at δ_C 112.8 (C-12), 50.3 (C-1), 32.2 (C-5), and 40.8 (C-8), three methylates at δ_C 70.5 (C-7), 51.3 (C-6), and 37.9 (C-9), and five quaternary carbons at δ_C 208.3 (C-2), 138.9 (C-3), 173.8 (C-4), 82.2 (C-10), and 149.0 (C-11) (Table 2).

The NMR spectra of 3 were extremely similar to those of known wikstronone C [27], except that the signals of one methylene in the referenced compound were replaced by 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 ¹H-¹H COSY spin system of H-6/H-7/H-8/H-9, indicated that a hydroxy was connected with C-7 in 3 to allow for the presence of oxymethine (Figure 2). In the NOESY spectrum, δ_H 0.85 (H₃-14) correlated with δ_H 2.57 (H-1β) suggested 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 β configuration because signal of C-10 (δ_C 82.2) is similar to C-10 (δ_C 83.2) in wikstronone C. The comparison of the calculated and experimental ECD spectra of 3 (Figure 4c) assigned its stereochemistry to be 6R, 7R, 9S, 10R. Hence, the structure of 3 was characterized and named stellerterpenoid C.

The known compounds were identified as prostratin (4) [28], Stelleraguaianone B (5) [12], Chamaejasnoid A (6) [29], auranticanol L (7) [30], wikstronone C (8) [27], and oleodaphnone (9) [25].

2.2. Biological Studies

Compounds 1-9 were evaluated for their inhibition on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages. The research results shows that compound 5 displayed the mild activity to inhibit NO production (IC₅₀ = 24.76 ± 0.4 μM) (Table 3).

The inhibitory rate of HepG2, A549 and HeLa cells growth of compounds 1–9 was measured by the MTT assay. Unfortunately, none of these nine compounds is active.

Furthermore, compounds 1–9 were also tested for anti-influenza virus activity against A/Puerto Rico/8/1934 (H1N1) virus, and nucleozin. However, none of the compounds showed inhibitory activity.

1.1. General Experimental Procedures

Optical rotations were recorded on a Jasco P-1020 polarimeter (JASCO Corporation, Tokyo, Japan). CD spectra were obtained on a Jasco J715 spectropo- larimeter. UV spectra were measured using a UV-8000 spectrophotometer (Shanghai Metash instruments Co.,Ltd, Shanghai, China). IR spectra were recorded on Bio-Rad FTS-135 spectrophotometer (Bio-rad Company, California, USA). NMR data were determined by Bruker Avance III HD-600 (Bruker BioSpin Group, Rheinstetten, Germany). ESI-MS and HR-ESI-MS were recorded on an APIQ star-Pulsar spectrometer (Applied Biosystems Corporation, Canada). The rotary evaporator is n-1300V-W type (Tokyo Physicochemical Co., LTD, Kyoto, Japan). Semipreparative HPLC was used Agilent 1260 (ZORABAX SB-C18, 4.6 mm × 250 mm, 1 ml/min; 9.4 mm × 250 mm, 3 ml/min.) (Agilent Technology Co., Ltd, California, USA) and NP7005-10C (CC: Nucifera C8M, 4.6 mm × 250 mm, 1 ml/min; 20× 250 mm,10 ml/min) (Hanbon Sci.& Tech, Jiangsu, China). Preparative HPLC used methanol and acetonitrile (pure reagents) were purchased from Merk Company (Merck, Darmstadt, Germany). Sephadex LH-20 (25-100 μm; Amersham Biosciences AB, Uppsala, Sweden), Thin layer silica gel plate (GF254) was purchased from Qingdao Marine Chemical Plant (Qingdao, China) and spots were visualized by heating silica gel plates sprayed with 5% H₂SO₄-EtOH.

3.3. Extraction and Isolation

Dried roots of Stellera chamaejasme (11.0 kg) was crushed and extracted with 70% acetone/H₂O 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 (15.0 L), and followed by successive partition with petroleum ether (3 × 15 L) and EtOAc (3 × 15 L). The EtOAc extract (3.35 kg) was separated by silica gel (2.5 × 160 cm) using a gradient of petroleum ether/EtOAc (5:1-1:1, v/v) and CHCl₃/MeOH (3:1–1:1, v/v) to give eight fractions (Fr. A~H).

Fr. C (45.75 g) was separated by silica gel (8 × 160 cm) using a gradient solvent petroleum ether/EtOAc (5:1–1:1, v/v) to afford six fractions (Fr. C1~C6). Fr. C3 (3.3 g) was repeatedly separated by silica gel (6 × 70 cm, CH₂Cl₂/MeOH, 10:1-1:1, v/v) to afford four fractions (Fr. C3-1~C3-4). Fr. C3-1 (198.4 mg) was purified by Sephadex LH-20 (MeOH) to yield 2 (8.3 mg), 5 (13.3 mg), 6 (3.7 mg), 7 (6.2 mg), and 8 (12.4 mg). Fr. C3-2 (1.2 g) was applied to a silica gel column (2 × 60 cm) washed with petroleum ether-acetone (15:1–1:1, v/v) to provide fractions C3-2-2 (63.7 mg), which was further separated by semi-preparative HPLC (MeOH/H₂O, 57:43, v/v) to afford 3 (3.7 mg) and 9 (3.8 mg). Fr. C4 (7.5 g) was subjected to silica gel (10 × 160 cm) using CHCl₃/MeOH (20:1–1:1, v/v) to give six fractions (Fr. C4-1~ C4-6). Fr. C4-2 (840 mg) was separated by silica gel column chromatography (1 × 60 cm, petroleum ether/CH₂Cl₂, 1:10-1:1, v/v) to afford fractions C4-2-2 (355.8 mg). Fr.C4-2-2 (355.8 mg) was purified by Sephadex LH-20 eluted with CHCl₃-MeOH (1:1, v/v) and then was used to silica gel (CHCl₃ / MeOH, 15:1, v/v, 2 × 70 cm) to yield 1 (39.3 mg) and 4 (105.3 mg).

3.4. Details of New Compounds

3.4.1. Stellerterpenoid A (1)

Yellow oil; [α${]}_D^{24}$−37.1 (c 0.24, MeOH); UV (MeOH) λ_max (log ε) 244 (1.88) nm; IR (KBr) ν_max 3449, 2918, 2920, 1769, 1703, 1631, 1424, 1361, 1292, 1220, 1164, 1089, 1040, 966, 930, 834, 800, 724, 676, 585, 530, 418 cm^-1; ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) data see Table 1 and Table 2; HRESIMS m/z 403.1344 [M + Na] ⁺ (calcd 403.1363).

3.4.2. Stellerterpenoid B (2)

Yellow oil; [α${]}_D^{24}$−11.0 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 207 (1.09) nm, 230 (1.01) nm and 301 (1.59) nm; IR (KBr) ν_max 3444, 2919, 2851, 1702, 1647, 1444, 1383, 1309, 1262, 1179, 1092, 896, 802 cm^-1; ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) data see Table 1 and Table 2; HRESIMS m/z 267.0981 [M + Na] ⁺ (calcd 267.0992).

3.4.3. Stellerterpenoid C (3)

Yellow oil; [α${]}_D^{24}$+91.8 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 239 (1.56) nm; IR (KBr) ν_max 3458, 3070, 2972, 2928, 2852, 1688, 1629, 1454, 1382, 1319, 1230, 1188, 1164, 1110, 1050, 978, 878, 807, 729, 655, 552, 463 cm^-1; ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) data see Table 1 and Table 2; HRESIMS m/z 273.1454 [M + Na] ⁺ (calcd 273.1461).

3.5. ECD Calculations for 1, 2 and 3

Based on the conformation determined from ROESY spectra and Chem 3D modeling, the low-energy conformers of model compound (4S,5S,6R,7S,8R,10R,11R) -1, (6R)-2, (6R,7R,9R,10R)-3 were generated within 10 kcal/mol energy window under MMFF94S via the software CONFLEX [31]. The selected conformers of each model compound with highest distribution (fuef63, fuef4 and fuef72-1 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 using TD-DFT-B3LYP/6-311G (+, 2d, p) of theory on optimized geometries through the CPCM model (in MeOH) [32]. The calculated ECD curves were generated using SpecDis 1.71 [33] and all the above calculations were carried out with the Gaussian 16 package of programs. All ECD curves were weighted by Boltzmann distribution after UV correction.

3.6. Determination of NO Production

Compounds 1-9 were evaluated for their anti-inflammatory activity using a LPS-induced NO production model. The NO production was calculated by measuring the content of nitrite in the supernatant of the culture by Griess reaction method. Cell viability was measured via MTT assay. The bioassays for NO production and cell viability of 1-9 were determined by a procedure described previously [34].

3.7. Anti-Influenza Virus Assay

Influenza strain A/Puerto Rico/8/1934 (H1N1) was used in this study. For the inhibitory activity assays, compounds 1-9 were dissolved in DMSO and Oseltamivir was used as positive control. MDCK cells (104/well) were inoculated in 96-well plates and cultured for 24 h, compounds of different concentrations (from 1.56 μM to 50 μM) were mixed with influenza virus (100 TCID₅₀) and added into the cells. IC₅₀ (50% virus inhibitory concentration) was calculated using the software CalcuSyn (Biosoft, Cambridge, UK) [35].

3.8. Anti-Tumor Assay

Cytotoxic activity assays against HepG2, A549 and HeLa cell lines were seeded on 96 well microplates and cultured in DMEM medium with 10% fetal bovine serum (FBS) at 37 ℃ and 5% CO₂ for 24 h.. Spetra-Max M2 (Molecular Devices, USA) was employed to record the optical density (λ = 490 nm). SPSS 21.0 software was used for data evaluation. IC₅₀ values were calculated based on the mean OD measured three times versus concentration curves of drugs. Adriamycin (10 mM, purity 99%, Solar bio Science&Technology Co. Ltd., Beijing, China) was used as the positive control [36].