Two New Epoxysteroids from Helianthus tuberosus

Two new epoxy steroids, 5α,8α-epidioxy-22β,23β-epoxyergosta-6-en-3β-ol (1) and 5α,8α-epidioxy-22α,23α-epoxyergosta-6-en-3β-ol (2), and ten known steroids including (24R)-5α,8α-epidioxyergosta-6-en-3β-ol (3), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (4), (22E,24R)-5α,8α-epidioxyergosta-6,9(11),22-trien-3β-ol (5), β-sitosterol (6), sitost-5-en-3β-ol acetate (7), 7α-hydroxysitosterol (8), schleicheol 2 (9), (24R)-24-ethyl-5α-cholestane-3β,5α,6β-triol (10), 7α-hydroxystigmasterol (11), and stigmasterol (12) were isolated from Helianthus tuberosus grown in Laizhou salinized land of coastal zone of Bohai Sea, China. The structures of these compounds were unambiguously established by 1D, 2D NMR and mass spectroscopic techniques. The new compounds 1 and 2 exhibited weak antibacterial activity and no antifungal activity.

The antimicrobial activity of new epoxy steroids 1 and 2 were evaluated using a standard agar diffusion test at 30 μg/disk. Compound 1 showed weak inhibitory activity against Escherichia coli and Staphylococcus aureus (inhibition diameter 7 mm), and 2 exhibited weak inhibitory activity against E. coli (inhibition diameter 7 mm). However, 1 and 2 were found no antifungal activity against plant pathogens Colletotrichum lagenarium and Fusarium oxysporium. Additionally, 1 and 2 exhibited inhibitory rates of 58.7% and 22.5%, respectively, in the toxicity assay against brine shrimp (Artemia salina) at 100 μg/mL.

General
NMR spectra were recorded at 500 and 125 MHz for 1 H and 13 C, respectively, on a Bruker Avance III 500 NMR spectrometer in CDCl 3 using TMS as internal standard. Low and high resolution mass spectra were determined on an Autospec Premier P776 mass spectrometer. IR spectra were obtained on a JASCO FT/IR-4100 Fourier Transform InfraRed spectrometer. HPLC separation was carried out on an Elite HPLC system (P270 pump, UV230+ detector, Dalian Elite Analytical Instruments Co., Ltd, Dalian, China) using an Eclipse XDB-C18 (5μm, 9.4 × 250 mm) column. Column chromatography was performed with silica gel (100-200 and 200-300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China) and Sephadex LH-20 (Pharmacia). Precoated silica gel plates (GF-254, Qingdao Haiyang Chemical Co., Qingdao, China) were used for preparative TLC purification. All solvents were of analytical grade.

Plant Material
Helianthus tuberosus Linn was grown by Qin-Tai Zhao in Laizhou salinized land of coastal zone of Bohai Sea, China, which was collected in December, 2008. A voucher specimen (SP0812) has been deposited at the Bio-Resource Laboratory of Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences.

Bioassays
Antibacterial and antifungal activities were assayed using chloramphenicol as positive control with inhibition diameters of 28 and 30 mm for E. coli and S. aureus, respectively, as described previously [17]. Toxicity against brine shrimp (Artemia salina) was also tested as described previously [18].

Conclusions
Enriched steroids with ergosterol (1-5), sitosterol (6-10), and stigmasterol (11,12) skeletons were isolated and identified from H. tuberosus planted in coastal salinized land, and ergosterol derivatives were reported from this species for the first time. The isolation of two new epoxy sterols (1, 2) was a new addition to the molecular diversity of H. tuberosus, which exhibited weak antibacterial activity and toxicity against brine shrimp.