A New Eudesmane Sesquiterpene Glucoside from Liriope muscari Fibrous Roots

The screening of several Chinese medicinal herbs for nematocidal properties showed that the ethanol extract of Liriope muscari fibrous roots possessed significant nematocidal activity against the pine wood nematode (Bursaphelenchus xylophilus). From the ethanol extract, a new constituent (1,4-epoxy-cis-eudesm-6-O-β-D-glucopyranoside) and three known glycosides [1β,6α-dihydroxy-cis-eudesm-3-ene-6-O-β-D-glucopyranoside (liriopeoside A), 1β,6β-dihydroxy-cis-eudesm-3-ene-6-O-β-D-glucopyranoside, and 1α,6β-dihydroxy-5,10-bis-epi-eudesm-4(15)-ene-6-O-β-D-glucopyranoside] were isolated by bioassay-guided fractionation. The structures were elucidated by 1D and 2D NMR and MS techniques. 1,4-Epoxy-cis-eudesm-6-O-β-D-glucopyranoside possessed moderate nemato-cidal activity against B. xylophilus with a LC50 value of 339.76 μg/mL, while liriopeoside A (LC50 = 82.84 μg/mL) and 1β,6β-dihydroxy-cis-eudesm-3-ene-6-O-β-D-glucopyranoside (LC50 = 153.39 μg/mL) also exhibited nematocidal activity against B. xylophilus. The crude extract of L. muscari fibrous roots exhibited nematocidal activity against the pine wood nematode with a LC50 value of 182.56 μg/mL.

The coupling constant (J = 7.7 Hz) of the anomeric proton at δ H = 4.19 indicated the D-glucose moiety was in the β-form. The glycosidic position was established by HMBC, with a long-range correlation observed between H-1′ (δ H = 4.19, d, J = 7.7 Hz) and C-6 (δ C = 76.2).

General
Melting points were measured on a Buchi 535 apparatus and were uncorrected. High-resolution mass spectra were determined on a Bruker micrOTOF-Q spectrometer, equipped with Apollo II electrospray ionization source with ion funnel, operated in the positive ion mode. 1 H-and 13 C-NMR spectra were recorded on Bruker Avance DRX 500 instrument using DMSO-d 6 as a solvent with TMS as internal standard.

Plant Material
Fresh fibrous roots of L. muscari were collected from Quanzhou City (24.54 N latitude and 118.37 E longitude), Fujian Province, China in May 2010. The species was identified by Dr. Liu. Q.R. (College of Life Sciences, Beijing Normal University), and the voucher specimens (BNU-CMH-Dushuahan-2010-05-24-012 were deposited at the Herbarium (BNU) of College of Life Sciences, Beijing Normal University. The roots were air-dried and ground to a powder using a grinding mill (Retsch Muhle, Germany).

Extraction and Isolation of Active Ingredients
The powder (5 kg) was extracted with 80% ethanol (10 L) at room temperature over a period of 2 weeks. The extracts were concentrated to afford a syrup (1.8 Kg), which was dissolved in water (1,800 mL) and chromatographed on a AB-8 resin (Nankai University, Tianjin, China) column (120 mm in diameter and 1.0 m in height) eluting with a gradient of EtOH-H 2 O (0:100, 10: 90, 50: 50, 90: 10) to give four fractions. The 90% ethanol eluent was concentrated under vacuum to obtain the crude glycosides (51 g) which were dissolved in water (500 mL), and then fractionated with n-BuOH (500 mL × 5) to yield the n-BuOH-soluble extract (45 g) after evaporation of the solvent.

Nematocidal Assay
The pine wood nematode (B. xylophilus) was isolated from chips of infested pine wood collected in Taizhou city (28.41 N latitude and 121.27 E longitude), Zhejiang Province, China and extracted by Baermann funnel techniques [13]. The pine wood nematode isolate was rinsed three times with sterile distilled water and reared on a lawn of Botrytis cinerea cultured on potato dextrose agar medium in the dark at 25 C. Petri-dishes (9 cm in diameter) with fully grown fungus were inoculated with B. xylophilus and left until fungal mycelia were completely consumed (3-5 days). The cultured nematodes (mixed stage) were separated from fungal cultures by centrifuging at 8,500 rpm at 20 C, rinsed with sterile distilled water and collected. An aqueous suspension of the nematodes (ca. 5,000 nematodes per mL) was prepared by appropriate dilution for use as a working stock.
Range-finding studies were run to determine the appropriate testing concentrations. A serial dilution of the ethanol extract (pure compounds, five concentrations) was prepared. The crude extract/compounds were first dissolved in ethanol and the final concentration of the ethanol solution was 2%. The in vitro tests were performed in wells of 12-well plates. Solution containing test compounds/extract (20 μL) and dilution with about 100 nematodes (20 μL) were added to each well and the final volume of each vial was 1 mL. The plates were incubated in an incubator at 25 C. Dead and active nematodes were recorded in an interval of 24 h for 72 h using a microscope (×20). The nematodes were considered dead if they gave no response to physical stimuli such as mechanical stirring and pricking with the point of a needle. Six replicates were performed for each treatment. A 2% alcohol in H 2 O solution was used as a negative control and carbofuran as a positive control. Results from all replicates for the pure compounds and ethanol extract were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC 50 values and their 95% confidence intervals [14].

Conclusions
Based on mass screening of medicinal herbs, the ethanol extract of L. muscari fibrous roots was found to possess toxicity against the pine wood nematode (B. xylophilus). A novel and three known eudesmane sesquiterpene glycosides were isolated and identified from the ethanol extract of L. muscari by bioactivity-guided fractionation. The four isolated constituents and the crude extract exhibited nematocidal activity against the pine-wood nematode (B. xylophilus).