Sinensiols H–J, three new lignan derivatives from Selaginella sinensis (Desv.) Spring

Sinensiol H (1) was isolated as a pale yellow amorphous powder. The negative HRESIMS [M − H]− at m/z 371.1133 (calcd for 371.1136) suggested its molecular formula to be C20H20O7, corresponding to 11 degrees of unsaturation. The IR spectrum showed absorption bands characteristic of hydroxy group (3450 cm−1), carbonyl (1765 cm−1), and aromatic system (1608, 1516, 1490 cm−1). Analysis of its 1H NMR (DMSO-d6) data (Table 1) revealed the presence of two ABX benzene rings [δH 6.92 (d, J = 1.2 Hz, 1H, H-2), 6.83 (d, J = 7.9 Hz, 1H, H-5) and 6.79 (dd, J = 7.9, 1.2 Hz, 1H, H-6); 6.59 (d, J = 1.5 Hz, 1H, H-2′), 6.62 (d, J = 8.0 Hz, 1H, H-5′), and 6.47 (dd, J = 8.0, 1.5 Hz, 1H, H-6′)]. The 13C NMR (Table 1) and HSQC data showed signals due to twelve aromatic carbons, three methylenes (one oxygenated), one oxygenated tertiary carbon, one ester group, one methylenedioxy group (δC 100.7), one methoxy group (δC 55.4), and one methine. The chemical shift values of the 1D NMR of 1 were similar to those of the known compound 8′β-hydroxyhinokinin [14], the major difference being the absence of signals for a methylenedioxy (δH 5.93, δC 101.2) and the presence of signals for a methoxy group (δH 3.67, δC 55.4) in 1. The HMBC correlations (Figure 2) from 3′-OCH3 (δH 3.67, s, 3H) to C-3′ indicated the methoxy group was located at C-3′. In the ROESY spectrum, the correlations of 8′-OH/H2-7 and H-8/H2-7′ (Figure 3a) suggested a trans orientation of H-8 and 8′-OH. The experimental ECD spectrum of 1 (Figure S16 in Supporting Information File 1) showed two positive Cotton effects (CEs) at 204 and 231 nm, which matched well with those in the calculated ECD curve for the (8S,8′R)-stereoisomer (Figure 3b). Consequently, the structure of 1 was determined as shown in Figure 1, and named sinensiol H.

Table 1: 1H NMR and 13C NMR data of compounds 13 (δ in ppm and J in Hz).

No. 1a 1b 2c 3c δH δC δH δC δH δC δH δC 1   132.9   133.8   138.2   140.6 2 6.85 (d, 1.5, 1H) 109.8 6.92 (d, 1.2, 1H) 109.6 6.34 (d, 1.8, 1H) 105.2 6.84 (s, 1H) 107.4 3   148.0   147.1   154.4   149.1 4   146.4   145.5   135.9   148.1 5 6.76 (d, 7.9, 1H) 108.5 6.83 (d, 7.9, 1H) 108.0   151.4 6.81–6.77 (m, 1H) 108.7 6 6.80 (dd, 7.9, 1.5, 1H) 122.4 6.79 (dd, 7.9, 1.2, 1H) 122.1 6.35 (d, 1.8, 1H) 110.2 6.78–6.74 (m, 1H) 120.5 7 3.13 (dd, 14.5, 5.0, 1H)
2.95 (dd, 14.5, 8.8, 1H) 30.1 2.76–2.71 (m, 2H) 29.1 3.23–3.25 (m, 2H) 39.8 4.54 (t, 6.4, 1H) 74.9 8 2.70 (dd, 8.8, 5.0, 1H) 50.3 2.83–2.78 (m, 1H) 49.9 5.67–5.57 (m, 1H) 131.6 7.82–1.71 (m, 1H)
1.70–1.60 (1H, overlapped) 39.6 9   177.4   178.0         1′   126.5   127.1   138.2   139.8 2′ 6.48 (d, 1.9, 1H) 112.2 6.59 (d, 1.5, 1H) 114.0 6.34 (d, 1.8, 1H) 105.2 6.31 (s, 1H) 105.1 3′   146.9   147.1   154.4   154.3 4′   145.3   145.1   135.9   135.8 5′ 6.84 (d, 8.1, 1H) 115.0 6.62 (d, 8.0, 1H) 115.1   151.4   151.2 6′ 6.53 (dd, 8.1, 1.9, 1H) 122.7 6.47 (dd, 8.0, 1.5, 1H) 122.4 6.35 (d, 1.8, 1H) 110.2 6.30 (s, 1H) 110.1 7′ 2.62 (br s, 2H) 43.1 2.64–2.59 (m, 2H) 41.7 3.23–3.25 (m, 2H) 39.8 2.50 (t, 7.1, 2H) 36.7 8′   78.4   78.0 5.67–5.57 (m, 1H) 131.6 1.70–1.60 (1H, overlapped)
1.60–1.44 (m, 1H) 28.7 9′ 4.18 (d, 10.0, 1H)
3.91 (d, 10.0, 1H) 77.0 4.14 (d, 9.4, 1H)
3.81 (d, 9.4, 1H) 75.5         3-OCH3         3.78 (s, 3H) 56.4     3′-OCH3 3.84 (s, 3H) 56.1 3.67 (s, 3H) 55.4 3.78 (s, 3H) 56.4 3.81 (s, 3H) 61.0 4-OCH3         3.76 (s, 3H) 61.0     4′-OCH3         3.76 (s, 3H) 61.0 3.76 (s, 3H) 56.3 –OCH2O– 5.94 (s, 2H) 101.1 5.96 (s, 2H) 100.7     5.93 (s, 2H) 102.2 4′-OH     8.78 (s, 1H)           8′-OH     5.38 (s, 1H)          

aRecorded at 600/150 MHz for 1H/13C in CDCl3; brecorded at 600/150 MHz for 1H/13C in DMSO-d6; crecorded at 600/150 MHz for 1H/13C in MeOH-d4.

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Figure 2: Key HMBC and 1H-1H COSY correlations of 13.

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Figure 3: (a) Key ROESY correlations of compound 1. (b) Experimental and calculated ECD spectra of 1.

Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C20H24O6 by the HRESIMS peak at m/z 359.1497 [M − H]− (calcd for 359.1500). The IR spectrum of 2 showed the presence of hydroxy (3417 cm−1) and aromatic (1593, 1509 cm−1) groups. The 1H NMR spectrum recorded in MeOH-d4 (Table 1) of compound 2 displayed signals for two aromatic protons at δH 6.35 (d, J = 1.8 Hz, H-6) and δH 6.34 (d, J = 1.8 Hz, H-2), one methine at δH 5.67–5.57 (m, H-8), one methylene at δH 3.23–3.25 (2H, m, H2-7) and two methyl groups at δH 3.78 (3H, s, 3-OCH3 and δH 3.76 (3H, s, 4-OCH3). The 13C NMR spectrum of 2 (Table 1) revealed 10 carbon signals for a benzene, one olefinic carbon, one methylene and two methoxy groups. The above mentioned 1D NMR data of 2 in combination with its molecular formula indicated that the compound must be a symmetrical dimeric benzene derivative. Further analysis of NMR data suggested that the structure of 2 was quite similar to that of (E)-5,5′-(but-2-ene-1,4-diyl)bis(3-methoxybenzene-1,2-diol) [15]. The main difference was that the hydroxy group at C-4 and C-4′ in (E)-5,5′-(but-2-ene-1,4-diyl)bis(3-methoxybenzene-1,2-diol) was substituted by a methoxy group in 2, which was confirmed by the HMBC correlation (Figure 2) from δH 3.76 (4-OCH3, 4′-OCH3) to δC 135.9 (C-4, C-4′). The absorption band near 999 cm−1 in the IR spectrum (Figure S26 in Supporting Information File 1) indicated that the double bond has an E configuration [16-19]. Therefore, the structure of compound 2 was established as shown in Figure 1, and named as sinensiol I.

Sinensiol J (3) was isolated as a white amorphous powder. Its HRESIMS showed [M + HCOO]− at m/z 391.1394 (calcd for 391.1398), consistent with the molecular formula of C19H22O6. The 1H and 13C NMR data (Table 1) of 3 were extremely similar to those of the rac-1-(benzo[d][1,3]dioxol-5-yl)-4-(3,4,5-trimethoxyphenyl)butan-1-ol [20], the significant difference being the absence of signals for a methoxy group in the 1H and 13C NMR spectra. The flat ECD curve (Figure S38 in Supporting Information File 1) and nearly zero optical rotation of 3 ([Graphic 1] −1.34, c 0.28, MeOH) suggested that 3 was possibly a racemic mixture. Enantioseparation of 3 by HPLC using a chiral-pak IA column provided the enantiomers with a ratio about 3:2 (Figure S28, Supporting Information File 1) suggested its mixture feature. Unfortunately, the limited amount available of this compound did not allow the elucidation of its absolute configuration.

The remaining known compounds were identified as (+)-pinoresinol di-O-β-ᴅ-glucopyranoside (4) [21], dehydrodiconiferyl alcohol-4-O-β-ᴅ-glucopyranoside (5) [22], and lariciresinol-4-O-β-ᴅ-glucopyranoside (6) [23] (Figure 1) by comparing their physiochemical properties and spectral data with those reported in the literature.

Biological activity

The isolated compounds were screened for their inhibitory effects on the LPS-induced NO production in RAW 264.7 macrophages. NG-Monomethyl-ʟ-arginine monoacetate salt (ʟ-NMMA, Sigma) was used as the positive control. As a result, compounds 1, 2, 4, and 5 showed mild inhibitory activities with inhibition rates in the range of 9.47–18.75%, compound 3 showed moderate activity with an inhibition rate of 42.06 ± 2.02% at a concentration of 50 μM (ʟ-NMMA, 59.31 ± 2.19%, Table 2).

Table 2: Inhibitory effects of compounds 16 on LPS-stimulated NO production.

sample concentration (μM) inhibition (%) 1 50 18.75 ± 2.13 2 50 69.16 ± 0.81 (cytotoxicity)   12.5 15.93 ± 1.37 3 50 42.06 ± 2.02 4 50 9.47 ± 2.38 5 50 11.40 ± 0.81 6 50 3.36 ± 2.38 ʟ-NMMAa 50 59.31 ± 2.19

aPositive control.

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