Analysis and determination of diterpenoids in unprocessed and processed Euphorbia lathyris seeds by HPLC-ESI-MS

Euphorbia lathyris （Caper spurge） is a toxic and potent Chinese materia medica （T/PCMM）. This study sought a method for identifying five diterpenoids （Euphorbia factors LI-L3, L7a, and Ls） with the spectra of UV and mass, quantifying three diterpenoids L1, L2, and L8 in crude extracts of unprocessed and processed E. lathyris seeds by liquid chromatography/ electrospray ionization mass spectrometry （LC-ESI-MS）. The analysis was achieved on an Agilent Eclipse XDB-C18 column （4.6 mm× 150mm i.d., 5 μm） with an isocratic elution with a mobile phase consisting of water and acetonitrile at a flow rate of 0.25 mL/min at column temperature of 30 ℃ and UV detection was set at 272 nm. An ESI source was used with a positive ionization mode. The calibration curve was linear in the ranges of 9.9-79 μg/mL for Euphorbia factor Lb 3.8-30.5μg/mL for Euphorbia factor L2, and 1.0-20.6 μg/mL for Euphorbia factor LB. The average recoveries （n=6） of three diterpenoids were 98.39%, 91.10% and 96.94%, respectively, with RSD of 2.5%, 2.4% and 2.1%, respectively. The contents of the three diterpenoids in processed E. lathyris seeds were 3.435, 1.367 and 0.286 mg/g, respectively, which decreased more sharply than those in unprocessed E. lathyris seeds which were 4.915, 1.944 and 0.425 mg/g, respectively. The method is simple, accurate, reliable and reproducible, and it can be applied to control the quality of unprocessed and processed E. lathyris seeds.

Configuration Confirmation of Euphorbia Factor L_1 by Single-crystal X-ray Diffraction

A 6,（17）-epoxylathyrol diterpenoid （（2S＊3S＊4R＊5R＊6S＊9S＊11S＊15R＊）-5,15-diacetoxy-3- phenylacetoxy-14-oxolathyra-6（17）,（12E）-diene-6（17）-epoxide） was isolated from the seeds of Euhporbia lathyris L. Its configuration was puzzled because of the incomplete X-ray results reported before. In this work, the atom connectivity and configuration were confirmed by single-crystal X-ray diffraction together with ESI-MS, ^1H-, and ^13C-NMR spectroscopy. The compound crystallizes in monoclinic, space group P21 with a = 11.386（1）, b = 8.2839（7）, c = 17.192（2） A, β = 108.305（2）°, Z = 2, V = 1539.5（2）A^3, C32H40O8, Mr = 552.64, Dc = 1.192 g/m^3, /7（000） = 592,μ（MoKα） = 0.085 mm^-l, T = 293（2） K, the final R = 0.0398 and wR = 0.0950 for 2057 observed reflections with Ⅰ 〉 2α（Ⅰ）. The molecule shows a tricyclic terpenoid skeleton, consisting of fused five-, eleven- and three-membered rings. The configuration at C（5） is R^＊ and that at C（6） S^＊.

Plant triterpenoid saponins function as susceptibility factors to promote the pathogenicity of Botrytis cinerea

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen that causes diseases in hundreds of plant species,including high-value crops.Its polyxenous nature and pathogenic success are due to its ability to perceive host signals in its favor.In this study,we found that laticifer cells of Euphorbia lathyris are a source of susceptibility factors required by B.cinerea to cause disease.Consequently,poor-in-latex(pil)mutants,which lack laticifer cells,show full resistance to this pathogen,whereas lot-of-latex mutants,which produce more laticifer cells,are hypersusceptible.These S factors are triterpenoid saponins,which are widely distributed natural products of vast structural diversity.The downregulation of laticifer-specific oxydosqualene cyclase genes,which encode the first committed step enzymes for triterpene and,therefore,saponin biosynthesis,conferred disease resistance to B.cinerea.Likewise,the Medicago truncatula Iha-1 mutant,compromised in triterpenoid saponin biosynthesis,showed enhanced resistance.Interestingly,the application of different purified triterpenoid saponins pharmacologically complemented the diseaseresistant phenotype ofpil and hla-1 mutants and enhanced disease susceptibility in different plant species.We found that triterpenoid saponins function as plant cues that signal transcriptional reprogramming in B.cinerea,leading to a change in its growth habit and infection strategy,culminating in the abundant formation of infection cushions,the multicellular appressoria apparatus dedicated to plant penetration and biomass destruction in B.cinerea.Taken together,these results provide an explanation for how plant triterpenoid saponins function as disease susceptibility factors to promote B.cinerea pathogenicity.

Euphorbia factor L2 induces apoptosis in A549 cells through the mitochondrial pathway

Euphorbia factor L2, a lathyrane diterpenoid isolated from caper euphorbia seed(the seeds of Euphorbia lathyris L.), has been traditionally applied to treat cancer. This article focuses on the cytotoxic activity of Euphorbia factor L2 against lung carcinoma A549 cells and the mechanism by which apoptosis is induced. We analyzed the cytotoxicity and related mechanism of Euphorbia factor L2 with an MTT assay, an annexin V-FITC/PI test, a colorimetric assay, and immunoblotting. Euphorbia factor L2 showed potent cytotoxicity to A549 cells. Euphorbia factor L2 led to an increase in reactive oxygen species(ROS) generation,a loss of mitochondrial electrochemical potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase, suggesting that Euphorbia factor L2 induced apoptosis through a mitochondrial pathway. The cytotoxic activity of Euphorbia factor L2 in A549 cells and the related mechanisms of apoptotic induction provide support for the further investigation of caper euphorbia seeds.

Opposing roles of plant laticifer cells in the resistance to insect herbivores and fungal pathogens

More than 12,000 plant species(ca.10%of flowering plants)exude latex when their tissues are injured.Latex is produced and stored in specialized cells named“laticifers”.Laticifers form a tubing system composed of rows of elongated cells that branch and create an internal network encompassing the entire plant.Laticifers constitute a recent evolutionary achievement in ecophysiological adaptation to specific natural environments;however,their fitness benefit to the plant still remains to be proven.The identification of Euphorbia lathyris mutants(pil mutants)deficient in laticifer cells or latex metabolism,and therefore compromised in latex production,allowed us to test the importance of laticifers in pest resistance.We provided genetic evidence indicating that laticifers represent a cellular adaptation for an essential defense strategy to fend off arthropod herbivores with different feeding habits,such as Spodoptera exigua and Tetranychus urticae.In marked contrast,we also discovered that a lack of laticifer cells causes complete resistance to the fungal pathogen Botrytis cinerea.Thereafter,a latex-derived factor required for conidia germination on the leaf surface was identified.This factor promoted disease susceptibility enhancement even in the non-latex-bearing plant Arabidopsis.We speculate on the role of laticifers in the coevolutionary arms race between plants and their enemies.