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.

Occurrence and Control of Brown Patch in Energy Oil Plants Euphorbia lathyris L.

[ Objective] The purpose was to study the occurrence law and control effect of brown patch of E. lathyris. [ Method ] The pathogen of brown patch in E. lathyris was isolated and identified in Xingyi of Guizhou Province in 2008 -2009, and disease symptom observation and field fungicide efficacy test were conducted. [ Result ] The pathogen of brown patch in E. lathyris was fungus in Pseudoc ercospora sp., its suitable growth temperature was 20 -35 ℃, its occurrence peak was from June to September, and its field incidence were 30% -60%. Field efficacy test showed that 5 kinds of fungicides such as Thiophanate-methyl, Mancozeb, etc. had certain control effect on the disease, the relative control effect was 54. 3% -82.5%, in which mancozeb had better control effect, while Thiediazole copper had worse control effect. [Conclusion] The study would provide theoretical reference for further research on the cultivation of E. lathyris with hiah vield and aualitv and control of brown hatch.

The diterpenoid esters from Euphorbia lathyris L. induce intestinal inflammation via LXRα/ABCA1-regulated lipid rafts and TLR4-mediated pathway

Background:Diterpenoid esters are considered to be the main toxic components and bioactive constituents of Euphorbia lathyris L.(EL).Euphorbia factors L1(EF1),L2(EF2),and L3(EF3),the main diterpenoid esters of EL,have been found to cause intestinal diarrhea and induce intestinal inflammation in mice.This research aimed to explore the effects of major diterpenoid esters from EL on intestinal inflammation,as well as to clarify their possible targets and molecular mechanisms in vivo and vitro.Methods:Caco-2 cells and BALB/c mice were intervened with EFL1,EFL2,and EFL3,respectively.The expressions of TLR4,NLRP3,NF-κB p65,LXRα,ABCA1,TNF-αand IL-1βwere measured by Real-time PCR and ELISA.Cholesterol efflux levels were examined using cholesterol efflux kit.Flow cytometry was applied to detect lipid rafts abundance.Confocal microscopy was applied to investigate co-localization of lipid rafts and TLR4.Results:Our results revealed that EFL1,EFL2,and EFL3 inhibited LXRα,ABCA1 expression,and cholesterol efflux,promoted colocalization of TLR4 and lipid rafts,and up-regulated TLR4,NLRP3,NF-κB p65,TNF-αand IL-1βexpressions.Conclusion:These findings reveal that the mechanisms by which EFL1,EFL2,and EFL3 induce intestinal inflammation may be associated with LXRα/ABCA1-regulated lipid rafts and TLR4-mediated pathways.

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^＊.

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.

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.

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.