A New Prenylated Flavanone from Derris trifoliata Lour

A new flavanone, 4′,5,7-trihydroxy-6,8-di-(2-hydroxy-3-methylbut-3-enyl)- flavanone, was isolated from the aerial parts of Derris trifoliate, together with eleven known compounds: rotenone, tephrosin, 12a-hydroxyrotenone, deguelin, 6a,12a-dehydro-rotenone, dehydrodeguelin, 7a-O-methyldeguelol, 7a-O-methylelliptonol, 5,7,3',4'-tetra-hydroxy-6,8-diprenylisoflavone, daidzein and 4'-hydroxy-7-methoxyflavanone. 7a-O-Methylelliptonol was isolated for the first time from the genus Derris. Their structures were characterized on the basis of spectral data. Eight of the isolated compounds were found to be significantly toxic to brine shrimp (LC50 range 0.06–9.95 μg/mL). The new compound showed weak toxicity (LC50 = 211.31 μg/mL).


Introduction
Derris trifoliata Lour., a mangrove associate occurring in the tropical regions of Asia and East Africa [1], belongs to the Leguminosae, subfamily Papilionoideae.D. trifoliata is a poisonous plant, used locally for catching fish, and nowadays also used extensively for the control of insect pests [2].Different parts of the plant were used in traditional medicine for treatment of wounds, calculus, rheumatism and dysmenorrhea and asthma [3].Extracts and metabolites from this plant have been found to possess significant larvicidal, pesticidal, cytotoxic, anti-fungal, anti-inflammatory, antimicrobial, nitric oxide inhibitory, and cancer chemopreventive activities [4][5][6][7].Previous phytochemical investigations on D. trifoliata show that flavonoids, including rotenoids, are the most characteristic compounds of this plant [8].
The brine shrimp lethality assay (BSLA) is considered a useful tool for assessment of toxicity, and has been widely used in chemical ecology studies on chemical defense in plants.The extract from aerial part of D. trifoliata was found to be toxic to brine shrimp (LC 50 = 10.00 μg/mL) [9].However, to the best of our knowledge, there are no reports on brine shrimp toxicity of compounds from D. trifoliata.As a part of our search for chemical defence compounds of mangrove plants in South China, a chemical investigation of the brine shrimp toxic extract from D. trifoliata has been carried out.In this study, we report the structural elucidation of a new prenylated flavanone, together with 11 known compounds from the aerial part of D. trifoliata, as well as their toxicity against brine shrimp.).Comparison of the 1D NMR spectral data of compound 1 with those of 6-(2-hydroxy-3-methyl-3-butenyl)-8prenyleriodictyol suggested that 1 was a flavnanone with two isoprenyls on ring A [10].In the HMBC spectrum of 1, the correlations from H-4 to C-2, 3, from H-5 to C-3, 4, and 2, and from H-1 to C-2, 3 confirmed the identity of the 2-hydroxy-3-methylbut-3-enyl moiety.The partial structure of another 2-hydroxy-3-methylbut-3-enyl in 1 was confirmed by HMBC correlation peaks of H-4″ with C-2″, 3″;
Results of the brine shrimp lethality assay on three crude extracts and nine isolated compounds are shown in Table 1.Eight flavonoids exhibited significant brine shrimp toxicity (LC 50 < 30 μg/mL) [20].The LC 50 value obtained in present study for 2 was similar to those reported in literature: 0.049~1.8μg/mL for rotenone [21,22].The LC 50 values for 3 and 4 reported here were somewhat lower than literature data, 2.2 μg/mL for tephrosin, and 1.9 μg/mL for 12a-hydroxyrotenone [10].The brine shrimp toxicity of compounds 1, 5-8, and 12 had never been reported in literature.The rotenonoids with sp 3 hybridizaiton at C-6a and C-12a, namely compounds 2-5, showed extreme toxicity with LC 50 values less than 1 μg/mL, and this is consistent with the previously reported cytotoxic effects of compounds from D. trifoliata [14].

General
Optical rotations were measured on a Jasco 1020 polarimeter.NMR spectra were obtained on a Bruker AVANCE 500 spectrometer (operating at 500 MHz for 1 H-NMR, 125 MHz for 13 C-NMR).HREIMS spectra were recorded on a Finnigan MAT TSQ 700 mass spectrometer.UV spectra were obtained in a Beckman DU-640 UV spectrophotometer.Semipreparative HPLC was carried out using a Phenomenex Luna 5 μL C18 100A ODS column (250 × 20 mm) on a system comprised of a Waters 600E Multisolvent Delivery System and a Waters 996 Photodiode Array Detector.

Plant Material
The aerial parts of D. trifoliata were collected in September 2008 from the mangrove area of Sanya, Hainan Province, Southern China.The material was identified by Prof. Si Zhang, Key Laboratory of Marine Bio-resourses Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences.A voucher specimen (No.GKLMMM22) is deposited at the herbarium of South China Sea Institute of Oceanology.

Brine Shrimp Assay
Toxicity against brine shrimp was evaluated using the method reported by Wanyoike [20].Artificial seawater was prepared by dissolving sea salt (38 g) in distilled water (1 L).The 60-mm culture dishe were used for hatching.To seawater (50 mL), brine shrimp eggs (10 mg) were added.The culture dish was placed in the light incubator at 25 °C for 48 h.The larvae were attracted to one side of the vessel with a light source and collected with pipette.Nauplii were separated from eggs by aliquoting them three times in small beakers containing seawater.The brine shrimp are then put in wells of a 96 flat-bottom well plate (Nunc Microwell plate).Each column (12 per plate) contains a two-fold dilution series representing 8 doses.Potassium dichromate was used as a positive control, as well as the solvent DMSO was used as a negative control.Each sample was repeated six times (columns) in each experiment.After 24 hours of exposure to the samples, the number of died and total (determined after killing by freezing) brine shrimp for each well were determined.The data were analyzed by probit regression (SPSS) to calculate the LC 50 .

Table 1 .
The mean LD 50 values ± S.D for crude extracts and compounds screened against brine shrimp larvae (Artemia salina).