1. Introduction

Selaginella involven Spring is abundantly distributed in south China. It has been used as a folk herbal medicine for its hemostasis, antitumor,antibacterial,anti-inflammation,antiviral activities and treatment of cardiovascular diseases ^{[1, 2, 3]}. A number of biflavonoids, lignans,alkaloids and anthraquinones have been identified from genus Selaginella ^{[4, 5, 6]}. However,few terpenoids were reported ^[7]. In our study on chemical constituents of the genus Selaginella involven Spring,here we report the isolation and structural elucidation of two new compounds named as 3β,12,16- trihydroxy-6,8,11,13-abietatrien (1),(8R,8'S)-4,4',8-trihydroxyl- 3,3'-dimethoxyl-9'-lignanolide (2) and a new natural product 4,4'-dihydroxyl-3,3',5,5'-dimethoxyldiphenyl diketone (3). Abietane diterpenoid is also isolated for the first time from genus Selaginella. 2. Experimental

Herbs of Selaginella involven Spring were collected from Mao’er Mountain,Guangxi Zhuang Autonomous Region,China,in June 2009,and authenticated by Prof. Zhenji Li (Xia Men University,Xia Men,China). A voucher specimen (No. 20090617) was deposited in School of Pharmaceutical Sciences,Central South University.

The whole herbs of Selaginella involven Spring (10.0 kg) were refluxed in 70% EtOH twice (100 L,80 L,2 h each time). After the removal of the solvent under reduced pressure,the extract (0.8 kg) was subjected to polyamide with a gradient elution of H₂O,30%, 50%,70%,95% EtOH to afford five fractions. The 50% EtOH portion was subjected to silica gel column chromatography with a gradient elution of CHCl₃/acetone (100:0-100:10) to obtain eight subfractions (S1-S8). Separation of sub-fraction S5 on Sephadex LH-20 (MeOH/H₂O in gradient) and Pre-HPLC (YMC-Pack ODS-A; 250 mm × 10 mm,10 μm; 3 mL/min; 35% ACN/H₂O) yielded compound 1 (10 mg),compound 2 (20 mg) and compound 3 (30 mg),respectively.

The high differentiated PC-12 cells were seeded at constant density (1 × 10⁴/cm²) and cultured in DMEM,supplemented respectively with 10% fetal bovine serum,penicillin (100 IU/mL), streptomycin (100 mg/mL) and L-glutamine (2 mmol/L). To establish the H/R model,PC-12 cells were subjected to 5 h of hypoxia (N₂/CO₂,95:5) in pre-conditioned hypoxic medium (serum free DMEM without glucose and sodium pyruvate,which was incubated under hypoxic condition for 2 h) followed by 20 h of reoxygenation. Hypoxic medium was replaced with fresh medium upon switching to reoxygenation. The compounds extracted were added into media to explore its effect on H/R-induced cellular injury. At the end of experiments,cells were collected for analysis of cellular apoptosis. 3. Results and discussion

Compound 1 was obtained as an amorphous yellow powder. Its molecular formula was determined as C₂₀H₂₈O₃ by HREIMS (m/z 316.2033; cacld. 316.2033). The IR spectrum revealed the presence of hydroxyl (3405 cm^-1),aromatic (1600,1560 and 1457 cm^-1) groups. The absorption band at 278 nm in the UV spectrum suggested the presence of an aromatic ring. The 1H NMR spectrum of compound 1 (Table 1) displayed the presence of four tertiary methyl groups (δ_H 0.89,0.89,0.97,1.14),three methylene groups at δ 3.14,3.51 (m,2H),1.97-2.00 (m,1H),1.66-1.69 (m,1H),1.58- 1.63 (m,2H),three methine groups at δ 3.74 (m,1H),3.34 (dd,1H, J = 13.2 Hz,6.4 Hz) and 1.90 (d,1H,J = 3.0 Hz),two cis olefinic protons at δ 5.78 (dd,1H,J = 8.8,2.4 Hz) and 6.47(dd,1H,J = 8.8, 2.4 Hz),two singlet aromatic protons at δ 6.60 and 6.78. The ¹³C NMR and DEPT spectra suggested that compound 1 (Table 1) is a diterpenoid derivative with a total of twenty carbons ^{[8, 9, 10]}, consisting of four methyls (δ_C 17.1,20.6,20.6,28.1),three methylenes (δ_C 27.8,34.2,66.2),seven methines (δ_C 34.8, 50.4,76.8,109.2,125.7,125.9,128.0),two sp³ quaternary carbons (δ_C 37.2,38.2),four sp² quaternary carbons (δ_C 124.2,127.7,146.6, 154.8). The HMBC spectrum allowed us to construct compound 1 as an abietane-type derivative with trihydroxy group at C-3,C-12 and C-16. The structure is very close to that of 6-dehyohinokiol ^{[8, 9]},but markedly different in C-16 with a hydroxyl group. The correlations from H-15,H-16,and Me-17 to C-13 suggested that C- 15 is in the middle of the three-carbon chain fragment C-16-C- 15-C-17. The correlations from Me-18 and Me-19 to the C-3 carbonyl carbon establsihed the linkages of the C-5,C-4 and C-3. H- 3 appeared doublets of doublets and had coupling constants of 6.4 and 13.2 Hz. These facts indicated a typical 3β-hydroxyl substituted in A ring ^{[10, 11]}. In the modified Mosher’s method,since the biogenetically related diterpnoids have a trans-chair conformation, the A and B rings,the C-20 methyl group,and the C-5 proton were presumed to be trans-diaxial ^[10]. From these data,the structure of compound 1 (Fig. 1) was determined as 3β,12,16-trihydroxy-6,8,11,13-abietatrien. The full assignment of ¹H NMR and ¹³C NMR spectra was accomplished with the aid of DEPT,HSQC,¹H-¹H COSY and HMBC experiments (Fig. 2).

Table 1

Table 1 1H NMR (400 MHz) and 13C NMR(100MHz) data of compound 1-3 in DMSO-d6 (δ in ppm,J in Hz). a Signal patterns are unclear due to overlapping, assignments were based on HSQC and HMBC experiments.

Table 1 1H NMR (400 MHz) and 13C NMR(100MHz) data of compound 1-3 in DMSO-d6 (δ in ppm,J in Hz).

Fig. 1

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Fig. 1.The structure of compound 1.

Fig. 2

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Fig. 2.Key 1H-1H COSY and HMBC correlations of compound 1.

Compound 2 was obtained as a colorless solid. Its molecular formula was determined as C₂₀H₂₂O₇ by HREIMS (m/z 374.1350; cacld. 374.1360). The IR spectrum revealed the presence of hydroxyl (3325 cm^-1),aromatic (1612,1560 and 1512 cm^-1) and carbonyl (1748 cm^-1) groups. Its UV spectrum showed the maximum absorption bands at 228 nm and 282 nm. The ¹H NMR spectrum of compound 2 (Table 1) showed six aromatic protons at δ 6.91 (d,1H,J = 1.6 Hz),6.74 (dd,1H,J = 8.0 Hz,1.6 Hz),6.69 (d,1H, J = 8.0 Hz),6.63 (d,1H,J = 8.0 Hz),6.54 (d,1H,J = 1.6 Hz),6.40 (dd, 1H,J = 8.0 Hz,1.6 Hz),as two ABX systems. Additionally,three CH2 groups at δ 4.15 (d,1H,J = 9.6 Hz) and δ 3.82 (d,1H,J = 9.6 Hz),d 2.77 (d,2H,J = 2.4 Hz) and δ 2.61 (d,2H,J = 4.0 Hz),and a CH δ 2.77 (m,1H),two aromatic methoxy groups at δ 3.76 (s,3H) and δ 3.65 (s,3H),were observed. The ¹³C NMR and DEPT,HSQC spectrum showed the presence of a lactone C=O at δ 178.3 (C-9'),three CH₂ groups at δ 78.3 (C-9),41.9 (C-7),29.2 (C-7'),and a CH groups at δ 49.9 (C-8'),two methoxy carbons at δ 55.7 and δ 55.9,as well as carbon signals of two aromatic rings (δ 113.6-147.6). From the analysis of HMBC and ¹H-¹H COSY,compound 2 is structurally very close to wikstromol ^[12],which was also isolated by us from Selaginella involven Spring. There was a marked difference in C-9 Chemical shift in the two compounds. In wikstromol this signal was at δ 70.3 (C-9),while in compound 2 it was at δ 78.3 (C-9). In the HMBC spectrum,C-1',C-9',C-7 showed correlations with H-8', which indicated that the hydroxyl substituent was at C-8 not C-8'. In addition,the signals of C-7 correlated with H-2,H-6,which indicated that C-7 was substituted by an aromatic ring and a lactone ring. The signal of C-7' correlated with H-2',H-6',which indicated that C-7' was substituted by a aromatic ring and a lactone ring. The CD spectrum exhibited a positive cotton effect at 228 nm and 276 nm ^{[13, 14, 15]}. From these data,the structure of compound 2 (Fig. 3) was identified as (8R,8'S)-4,40,8-trihydroxyl-3,3'- dimethoxyl-90 lignanolide (Fig. 4).

Fig. 3

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Fig. 3.The structure of compound 2.

Fig. 4

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Fig. 4.Key 1H-1H COSY and HMBC correlations of compound 2.

Compound 2 was obtained as light yellow acicular crystals. Its molecular formula was determined as C₁₈H₁₈O₈ by HREIMS (m/z 362.0984; cacld. 362.0996). The IR spectrum revealed the presence of hydroxyl (3325 cm^-1),carbonyl (1678 cm^-1),aromatic (1612, 1560 and 1512 cm^-1) groups. Its UV spectrum showed a maximum absorption band at 330 nm. The ¹H NMR spectrum of compound 2 (Table 1) showed two 3,4,5-trisubstituted benzenes at δ 7.12(s, 4H),four aromatic methoxy groups at δ 3.79 (s,12H). The ¹³C NMR and DEPT,HSQC spectrum showed the presence of two C=O at δ 193.3,four methoxy carbons at δ 55.8,as well as carbon signals of two aromatic rings (δ 107.6-148.3). In the HMBC spectrum,two C=O groups showed correlations with H-2,H-6 and H-2',H-6', respectively. From these data,the structure of compound 2 (Fig. 5) was identified as 4,4'-dihydroxyl-3,3',5,5'-dimethoxyldiphenyldiketone (Figs. 6 and 7).

Fig. 5

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Fig. 5.The structure of compound 3.

Fig. 6

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Fig. 6.Key 1H-1H COSY and HMBC correlations of compound 3.

Fig. 7

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Fig. 7.Effect of compounds on PC-12 cells induced by hypoxia/reoxygenation. *: Compared with the control group,p < 0.05; #: Compared with H/R,p < 0.05; H/R: hypoxia/reoxygenation

Additionally,we investigated the protective effect of compounds on the injury of PC-12 cells induced by hypoxia/ reoxygenation ^{[16, 17]}. These three compounds displayed potent protective effect on PC-12 cells induced by hypoxia/reoxygenation at 10_-5 mol/L,10_-6 mol/L and 10_-7 mol/L (Fig. 3) with good dose dependency. We will continue to conduct pharmacological activity evaluations on the three compounds and explore the underlying mechanism of action. 4. Conclusion

In this study on the chemical constituents of the genus Selaginella involven Spring,we reported the isolation and structural elucidation of two new compounds named as 3β,12,16-trihydroxy- 6,8,11,13-abietatrien (1),(8R,80S)-4,4',8-trihydroxyl-3,3'- dimethoxyl-9'-lignanolide (2) and a new natural product 4,4'- dihydroxyl-3,3',5,5'-dimethoxyldiphenyldiketone (3) along with their potent protective effect against the injury of PC-12 cells induced by hypoxia/reoxygenation for the first time.

This research was supported by National Natural Science Foundation (No. 31370370),the Doctoral Program Foundation of Institutions of Higher Education of China (No. 20100162110057), Chinese Medicine Research Program of Hunan Province (Nos. 2009059 and 2010004).