Untargeted metabolomic analysis reveals a potential role of saponins in the partial resistance of pea (Pisum sativum) against a root rot pathogen, Aphanomyces euteiches.

In soil-borne diseases, the plant-pathogen interaction begins as soon as the seed germinates and develops into a seedling. Aphanomyces euteiches, an oomycete, stays dormant in soil and gets activated by sensing the host through chemical signals present in the root exudates. The composition of plant exudates may, thus, play an important role during the early phase of infection. To better understand the role of root exudates in plant resistance, we investigated the interaction between partially resistant lines (PI660736 and PI557500) and susceptible pea cultivars (CDC Meadow and AAC Chrome) against Aphanomyces euteiches during the pre-invasion phase. The root exudates of two sets of cultivars clearly distinguished from each other in inducing oospore germination. PI557500 root exudate not only had diminished induction but also inhibited the oospore germination. The contrast between the root exudates of resistance and susceptible cultivars was reflected in their metabolic profiles. Data from fractionation and oospore germination inhibitory experiments identified a group of saponins that accumulated differentially in susceptible and resistant cultivars. We detected 56 saponins and quantified 44 of them in pea root and 30 from root exudate; the majority of them, especially Soyasaponin I and dehydrosoyasaponin I with potent in vitro inhibitory activities, were present in significantly higher amounts in both roots and root exudates of PI660736 and PI557500 as compared to Meadow and Chrome. Our results provide evidence for saponins as deterrents against Aphanomyces euteiches, which might have contributed to the resistance against root rot in the studied pea cultivars.

Comprehensive Analysis of Biomass from Chlorella sorokiniana Cultivated with Industrial Flue Gas as the Carbon Source.

Chlorella sorokiniana, isolated from a pond adjacent to a cement plant, was cultured using flue gas collected directly from kiln emissions using 20 L and 25000 L photobioreactors. Lipids, proteins, and polysaccharides were analyzed to understand their overall composition for potential applications. The lipid content ranged from 17.97% to 21.54% of the dry biomass, with carotenoid concentrations between 8.4 and 9.2 mg/g. Lutein accounted for 55% of the total carotenoids. LC/MS analysis led to the identification of 71 intact triacylglycerols, 8 lysophosphatidylcholines, 10 phosphatidylcholines, 9 monogalactosyldiacylglycerols, 12 digalactosyldiacylglycerols, and 1 sulfoquinovosyl diacylglycerol. Palmitic acid, oleic acid, linoleic acid, and α-linolenic acid were the main fatty acids. Polyunsaturated fatty acid covers ≥ 56% of total fatty acids. Protein isolates and polysaccharides were also extracted. Protein purity was determined to be ≥75% by amino acid analysis, with all essential amino acids present. Monomer analysis of polysaccharides suggested that they are composed of mainly D-(+)-mannose, D-(+)-galactose, and D-(+)-glucose. The results demonstrate that there is no adverse effect on the metabolite profile of C. sorokiniana biomass cultured using flue gas as the primary carbon source, revealing the possibility of utilizing such algal biomass in industrial applications such as animal feed, sources of cosmeceuticals, and as biofuel.

Characterization of Neutral Lipids of the Oleaginous Alga Micractinum inermum.

An oleaginous microalga Micractinum inermum isolated from Mariana Lake, AB, Canada was cultured in a 1000 L photobioreactor with an f/2 medium to study its lipid content and neutral lipid profile. Algal biomass was collected at the stationary phase contained a significant amount of lipids (44.2%), as determined by Folch's method. The lipid was fractionated into neutral lipid, glycolipid and phospholipid fractions. The neutral lipid constitutes almost 77.3% of the total lipid species and is mainly composed of triacylglycerols (TAGs) determined by a proton NMR study. UHPLC-HRMS analysis allows us for the first time to identify 81 TAGs in the neutral lipid fraction of M. inermum. The fatty acid acyl side chains were identified based on fragment ions observed in MSMS analysis. TAGs with fatty acid acyl chains 18:1/18:1/18:1, 18:1/18:1/16:0, 18:2/18:1/16:0, and 18:2/18:2/18:0 were the major ones among the identified TAGs. Fatty acid analysis further supports the fact that oleic acid was the major fatty acid present in the neutral lipid fraction of M. inermum constituting 41.7%, followed by linoleic acid at 21.5%, and palmitic acid at 21.2%. The saturated and monounsaturated fatty acids were 67.8% or higher in the lipid fraction. Long-chain fatty acids were only present in a minor quantity. The results clearly demonstrate that M. inermum is an excellent source for TAGs.

Analysis of Polar Lipids in Hemp (Cannabis sativa L.) By-Products by Ultra-High Performance Liquid Chromatography and High-Resolution Mass Spectrometry.

Polar lipids were extracted from residual biomass of hemp (Cannabis sativa L.) by-products with EtOH and partitioned into aqueous and chloroform fractions. The chloroform fractions were studied for their lipid composition using solid-phase extraction (SPE) followed by UHPLC/HRMS and NMR analyses. The 1H NMR and gravimetric yield of SPE indicated triacylglycerols covered ≥ 51.3% of the chloroform fraction of hemp seed hulls and hemp cake. UHPLC/HRMS analyses of remaining polar lipids led to the identification of nine diacylglycerols (DAGs), six lysophosphatidylcholines (LPCs), five lysophosphatidylethanolamines (LPEs), eight phosphatidylethanolamines (PEs), and thirteen phosphatidylcholines (PCs) for the first time from hemp seed hulls. The regiospecificity of fatty acyl substitutes in glycerol backbone of individual phospholipids were assigned by analyzing the diagnostic fragment ions and their intensities. The heat-map analysis suggested that DAG 18:2/18:2, 1-LPC 18:2, 1-LPE 18:2, PE 18:2/18:2, and PC 18:2/18:2 were the predominant molecules within their classes, supported by the fact that linoleic acid was the major fatty acid covering > 41.1% of the total fatty acids determined by GC-FID analysis. The 31P NMR analysis confirmed the identification of phospholipids and suggested PC covers ≥ 37.9% of the total phospholipid present in hemp by-products. HPLC purification led to the isolation of 1,2-dilinoleoylphosphatidylcholine and 1-palmitoyl-2-linoleoylphosphatidylcholine. These two major PCs further confirmed the UHPLC/HRMS finding.

Biochemical Characterization and In Vitro Digestibility of Protein Isolates from Hemp (Cannabis sativa L.) By-Products for Salmonid Feed Applications.

Hemp (Cannabis sativa L.) processing by-products (hemp cake and hemp seed hulls) were studied for their protein content, extraction of protein isolates (PIs), and their in vitro protein digestibility (IVPD). Crude protein contents of hemp cake and hemp seed hulls were 30.4% and 8.6%, respectively, calculated based on generalized N-to-P conversion factor (N × 5.37). Extraction efficiency of PIs from defatted biomass ranged from 56.0 to 67.7% with alkaline extraction (0.1 M NaOH) followed by isoelectric precipitation (1.0 M HCl). Nitrogen analysis suggested that the total protein contents of PIs extracted using three different alkaline conditions (0.5 M, 0.1 M, and pH 10.0 with NaOH) were >69.7%. The hemp by-product PIs contained all essential amino acids (EAAs) required for fish with leucine, valine, and phenylalanine belonging to the five dominant amino acids. Overall, glutamate was the dominant non-EAA followed by aspartate. Coomassie staining of an SDS-PAGE gel revealed strong presence of the storage protein edestin. High IVPD of >88% was observed for PIs extracted from hemp seeds and by-products when evaluated using a two-phase in vitro gastric/pancreatic protein digestibility assay. PIs extracted from by-products were further tested for their antioxidant activities. The tested PIs showed dose-dependent DPPH radical scavenging activity and possessed strong ORAC values > 650 µM TE/g.

Triacylglycerols and Other Lipids Profiling of Hemp By-Products.

Hemp seed by-products, namely hemp cake (hemp meal) and hemp hulls were studied for their lipid content and composition. Total lipid content of hemp cake and hemp hulls was 13.1% and 17.5%, respectively. Oil extraction yields using hexane, on the other hand, were much lower in hemp cake (7.4%) and hemp hulls (12.1%). Oil derived from both hemp seeds and by-products were primarily composed of neutral lipids (>97.1%), mainly triacylglycerols (TAGs), determined by SPE and confirmed by NMR study. Linoleic acid was the major fatty acid present in oils derived from hemp by-products, covering almost 55%, followed by α-linolenic acid, covering around 18% of the total fatty acids. For the first time, 47 intact TAGs were identified in the hemp oils using UPLC-HRMS. Among them, TAGs with fatty acid acyl chain 18:3/18:2/18:2 and 18:3/18:2/18:1 were the major ones, followed by TAGs with fatty acid acyl chain of 18:3/18:3/18:2, 18:2/18:2/16:0, 18:2/18:2/18:1, 18:3/18:2.18:0, 18:2/18:2/18:0, 18:2/18:1/18:1 and 18:3/18:2:16:0. Besides TAGs, low levels of terpenes, carotenoids and cannabidiolic acid were also detected in the oils. Moreover, the oils extracted from hemp by-products possessed a dose-dependent DPPH radical scavenging property and their potencies were in a similar range compared to other vegetable oils.

9,10-Dihydro-5-hydroxy-2,3,6-trimethoxyphenanthrene-1,4-dione: a new dihydrophenanthrene from commercial cannabis and its effect on zebrafish larval behaviour.

A new dihydrophenanthrene derivative namely 9,10-dihydro-5-hydroxy-2, 3,6-trimethoxyphenanthrene-1,4-dione (1) was isolated from commercial cannabis product together with 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), combretastatin B-2 (4) and isocannbispiradienone (5). Structure elucidation of the isolated compounds were done based on the interpretation of the mass spectrometry (MS) and nuclear magnetic resonance (NMR) data. New dihydrophenanthrene derivative (1) was tested for its effect on zebrafish larval behaviour. Preliminary results suggested that the new dihydrophenanthrene derivative (1) exhibits similar effect on zebrafish larval behaviour as cannabidiol (CBD), a biologically active component of Cannabis.

A Cultivated Form of a Red Seaweed (Chondrus crispus), Suppresses ß-Amyloid-Induced Paralysis in Caenorhabditis elegans.

We report here the protective effects of a methanol extract from a cultivated strain of the red seaweed, Chondrus crispus, against ß-amyloid-induced toxicity, in a transgenic Caenorhabditis elegans, expressing human Aß1-42 gene. The methanol extract of C. crispus (CCE), delayed ß-amyloid-induced paralysis, whereas the water extract (CCW) was not effective. The CCE treatment did not affect the transcript abundance of amy1; however, Western blot analysis revealed a significant decrease of Aß species, as compared to untreated worms. The transcript abundance of stress response genes; sod3, hsp16.2 and skn1 increased in CCE-treated worms. Bioassay guided fractionation of the CCE yielded a fraction enriched in monogalactosyl diacylglycerols (MGDG) that significantly delayed the onset of ß-amyloid-induced paralysis. Taken together, these results suggested that the cultivated strain of C. crispus, whilst providing dietary nutritional value, may also have significant protective effects against ß-amyloid-induced toxicity in C. elegans, partly through reduced ß-amyloid species, up-regulation of stress induced genes and reduced accumulation of reactive oxygen species (ROS).

Neuroprotective effects of the cultivated Chondrus crispus in a C. elegans model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in the elderly people, currently with no cure. Its mechanisms are not well understood, thus studies targeting cause-directed therapy or prevention are needed. This study uses the transgenic Caenorhabditis elegans PD model. We demonstrated that dietary supplementation of the worms with an extract from the cultivated red seaweed Chondrus crispus decreased the accumulation of α-synulein and protected the worms from the neuronal toxin-, 6-OHDA, induced dopaminergic neurodegeneration. These effects were associated with a corrected slowness of movement. We also showed that the enhancement of oxidative stress tolerance and an up-regulation of the stress response genes, sod-3 and skn-1, may have served as the molecular mechanism for the C. crispus-extract-mediated protection against PD pathology. Altogether, apart from its potential as a functional food, the tested red seaweed, C. crispus, might find promising pharmaceutical applications for the development of potential novel anti-neurodegenerative drugs for humans.

Polar lipids from the marine macroalga Palmaria palmata inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells.

The EtOAc soluble fraction of a MeOH/CHCl3 extract of Palmaria palmata showed strong nitric oxide (NO) inhibitory activity against lipopolysaccharide (LPS)-induced NO production in murine RAW264.7 cells. NO inhibition-guided isolation led to identification of three new polar lipids including a sulfoquinovosyl diacylglycerol (SQDG) (2S)-1-O-eicosapentaenoyl-2-O-myristoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-glycerol (1) and two phosphatidylglycerols, 1-O-eicosapentaenoyl-2-O-trans-3-hexadecenoyl-3-phospho-(1'-glycerol)-glycerol (3) and 1-O-eicosapentaenoyl-2-O-palmitoyl-3-phospho-(1'-glycerol)-glycerol (4) from the EtOAc fraction. Seven known lipids were also isolated including a SQDG (2), a phospholipid (5) and five galactolipids (6-10). Structures of the isolated lipids were elucidated by spectral analyses. The isolated SQDGs, phosphatidylglycerols and phospholipid possessed strong and dose-dependent NO inhibitory activity compared to N(G)-methyl-L-arginine acetate salt (L-NMMA), a well-known NO inhibitor used as a positive control. Further study suggested that these polar lipids suppressed NO production through down-regulation of inducible nitric oxide synthase (iNOS).

Components of the cultivated red seaweed Chondrus crispus enhance the immune response of Caenorhabditis elegans to Pseudomonas aeruginosa through the pmk-1, daf-2/daf-16, and skn-1 pathways.

Marine macroalgae are rich in bioactive compounds that can, when consumed, impart beneficial effects on animal and human health. The red seaweed Chondrus crispus has been reported to have a wide range of health-promoting activities, such as antitumor and antiviral activities. Using a Caenorhabditis elegans infection model, we show that C. crispus water extract (CCWE) enhances host immunity and suppresses the expression of quorum sensing (QS) and the virulence factors of Pseudomonas aeruginosa (strain PA14). Supplementation of nematode growth medium with CCWE induced the expression of C. elegans innate immune genes, such as irg-1, irg-2, F49F1.6, hsf-1, K05D8.5, F56D6.2, C29F3.7, F28D1.3, F38A1.5 ZK6.7, lys-1, spp-1, and abf-1, by more than 2-fold, while T20G5.7 was not affected. Additionally, CCWE suppressed the expression of PA14 QS genes and virulence factors, although it did not affect the growth of the bacteria. These effects correlated with a 28% reduction in the PA14-inflicted killing of C. elegans. Kappa-carrageenan (K-CGN), a major component of CCWE, was shown to play an important role in the enhancement of host immunity. Using C. elegans mutants, we identified that pmk-1, daf-2/daf-16, and skn-1 are essential in the K-CGN-induced host immune response. In view of the conservation of innate immune pathways between C. elegans and humans, the results of this study suggest that water-soluble components of C. crispus may also play a health-promoting role in higher animals and humans.

Nitric oxide inhibitory activity of monogalactosylmonoacylglycerols from a freshwater microalgae Chlorella sorokiniana.

Chemical investigation of the freshwater microalgae Chlorella sorokiniana led to the isolation of a new monogalactosylmonoacylglycerol, namely, (2S)-1-O-(7Z,10Z-hexadecadienoyl)-3-O-ß-D-galactopyranosylglycerol (1) together with a known glycolipid (2S)-1-O-(7Z,10Z,13Z-hexadecatrienoyl)-3-O-ß-D-galactopyranosylglycerol (2). Both monogalactosylmonoacylglycerols showed dose-dependent nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced NO production in RAW264.7 macrophage cells suggesting their possible use as anti-inflammatory agents.

Monogalactosyldiacylglycerols, potent nitric oxide inhibitors from the marine microalga Tetraselmis chui.

Methanolic extracts of some marine and freshwater microalgae were tested for their nitric oxide (NO) inhibitory activity on lipopolysaccharide-induced NO production in RAW264.7 macrophage cells. Among the tested extracts, Tetraselmis chui extract showed the strongest NO inhibitory activity, thus selected for further study. NO inhibitory activity guided isolation led to identification of two monogalactosyldiacylglycerols (MGDGs) (2S)-1-O-(6Z,9Z,12Z,15Z-octadecatetranoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-O-ß-D-galactopyranosylglycerol (1) and (2S)-1-O-(9Z,12Z,15Z-octadecatrinoyl)-2-O-(4Z,7Z,10Z,13Z-hexadecatetranoyl)-3-O-ß-D-galactopyranosylglycerol (2) from the MeOH extract of T. chui. The stereo-chemistry of 1 was elucidated by classical degradation method. MGDGs 1 and 2 showed strong NO inhibitory activity compared to N(G)-methyl-L-arginine acetate salt, a well known NO inhibitor used as a positive control. Isolated MGDGs suppressed NO production through down-regulation of inducible NO synthase protein. A structure activity relationship study suggested that the polyunsaturated fatty acids of the MGDGs are responsible for NO inhibition. Moreover, increasing unsaturation on the fatty acid side chains enhanced the NO inhibitory potency of the MGDGs.

TLN-05220, TLN-05223, new Echinosporamicin-type antibiotics, and proposed revision of the structure of bravomicins(*).

The deposited strain of the hazimicin producer, Micromonospora echinospora ssp. challisensis NRRL 12255 has considerable biosynthetic capabilities as revealed by genome scanning. Among these is a locus containing both type I and type II PKS genes. The presumed products of this locus, TLN-05220 (1) and TLN-05223 (2), bear a core backbone composed of six fused rings starting with a 2-pyridone moiety. The structures were confirmed by conventional spectral analyses including MS, and 1D and 2D NMR experiments. Comparison of both the 1H and 13C NMR data of the newly isolated compound with those of echinosporamicin and bravomicin A led us to propose a revision of the structure of the latter to include a 2-pyridone instead of the pyran originally postulated. Both compounds (1 and 2) possessed strong antibacterial activity against a series of gram-positive pathogens including several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. The TLN compounds are the first of this group with reported anticancer activity.

Biosynthesis of diazepinomicin/ECO-4601, a Micromonospora secondary metabolite with a novel ring system.

The novel microbial metabolite diazepinomicin/ECO-4601 (1) has a unique tricyclic dibenzodiazepinone core, which was unprecedented among microbial metabolites. Labeled feeding experiments indicated that the carbocyclic ring and the ring nitrogen of tryptophan could be incorporated via degradation to the 3-hydroxyanthranilic acid, forming ring A and the nonamide nitrogen of 1. Genomic analysis of the biosynthetic locus indicated that the farnesyl side chain was mevalonate derived, the 3-hydroxyanthranilic acid moiety could be formed directly from chorismate, and the third ring was constructed via 3-amino-5-hydroxybenzoic acid. Successful incorporation of 4,6-D2-3-hydroxyanthranilic acid into ring A of 1 via feeding experiments supports the genetic analysis and the allocation of the locus to this biosynthesis. These studies highlight the enzymatic complexity needed to produce this structural type, which is rare in nature.

Genomic analyses lead to novel secondary metabolites. Part 3. ECO-0501, a novel antibacterial of a new class.

Genomic analyses of Amycolatopsis orientalis ATCC 43491 strain, deposited as a vancomycin producer, revealed the presence of genetic loci for the production of at least 10 secondary metabolites other than vancomycin. One of these gene clusters, which contained a type I polyketide synthase, was predicted to direct the synthesis of novel class of compound, a glycosidic polyketide ECO-0501 (1). Screening of culture extracts for a compound with the predicted physicochemical properties of the product from this locus, led to the isolation of the 13-O-glucuronide of 13-hydroxy-2,12,14,16,22-pentamethyl-28-(N-methyl-guanidino)-octacosa-2,4,6,8,10,14,20,24-octaenoic acid (2-hydroxy-5-oxo-cyclopent-1-enyl)-amide (ECO-0501, 1). The structure, confirmed by spectral analyses including MS, and ID and 2D NMR experiments, were in accord with that predicted by genomic analyses. ECO-0501 possessed strong antibacterial activity against a series of Gram-positive pathogens including several strains of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). ECO-0501 was chemically modified by esterification (1a-1c), N-acetylation (1d) and hydrogenation (1e) in order to explore structure activity relationships (SAR).

Isolation and identification of three new 5-alkenyl-3,3(2H)-furanones from two streptomyces species using a genomic screening approach.

Analyses of biosynthetic gene clusters derived from Streptomyces aculeolatus NRRL 18422 and Streptomyces sp. Eco86 indicated that both microorganisms have similar type I polyketide synthase (PKS) gene clusters with relatively few genes encoding post-PKS elaborative enzymes. However both gene clusters included a sequence coding for a relatively uncommon oxidative enzyme related to Baeyer-Villiger, flavin-type monooxygenases. Screening of culture extracts for compounds with the predicted physicochemical properties of the end products from these loci, led to the isolation of three 5-alkenyl-3,3(2H)-furanones, one (E-837, 1) from the former and two (E-492, 2, E-975, 3) from the latter strain. The structures, confirmed by spectral analyses including MS, and ID and 2D NMR experiments, were in accord with those predicted by genomic analyses. Baeyer-Villiger type oxidation is postulated to be involved in the formation of the furanone moieties in these molecules. All three new compounds were tested for their electron transport inhibitory activities. They had IC50 values of 1-4 microg/ml against Ascaris suum NADH-fumarate reductase and 1-12 microg/ml against bovine heart NADH oxidase.

Cassane- and norcassane-type diterpenes from Caesalpinia crista of Indonesia and their antimalarial activity against the growth of Plasmodium falciparum.

The CH2Cl2 extract of the seed kernels of Caesalpinia crista, which exhibited promising antimalarial activity against Plasmodium berghei-infected mice in vivo, was examined and resulted in the isolation of seven new furanocassane-type diterpenes [caesalpinins C-G (1-5) and norcaesalpinins D and E (6, 7)] together with norcaesalpinins A-C (8-10) and 11 known compounds (norcaesalpinins A-C, 2-acetoxy-3-deacetoxycaesaldekarin e, caesalmin B, caesaldekarin e, caesalpin F, 14(17)-dehydrocaesalpin F, 2-acetoxycaesaldekarin e, 7-acetoxybonducellpin C, and caesalmin G). Their structures were determined on the basis of spectroscopic analysis. The isolated diterpenes showed significant dose-dependent inhibitory effects on Plasmodium falciparum FCR-3/A2 growth in vitro. Their IC50 values ranged from 90 nM to 6.5 microM, and norcaesalpinin E (7) showed the most potent inhibitory activity (IC50, 90 nM).

Cassane- and norcassane-type diterpenes of Caesalpinia crista from Myanmar.

From the CH(2)Cl(2) extract of seed kernels of Caesalpinia crista from Myanmar, five new cassane-type diterpenes, caesalpinins MA-ME (1-5), and three new norcassane-type diterpenes, norcaesalpinins MA-MC (6-8), have been isolated, together with 12 known cassane-type diterpenes, 14(17)-dehydrocaesalmin F, caesaldekarin e, caesalmin B, caesalmin C, caesalmin E, 2-acetoxy-3-deacetoxycaesaldekarin e, 2-acetoxycaesaldekarin e, caesalpinin C, 7-acetoxybonducellpin C, caesalpinin E, norcaesalpinin B, and 6-acetoxy-3-deacetoxycaesaldekarin e. The structures of the isolated compounds were elucidated by analysis of their spectroscopic data.

Secoisolariciresinol and isotaxiresinol inhibit tumor necrosis factor-alpha-dependent hepatic apoptosis in mice.

The effects of secoisolariciresinol (1) and isotaxiresinol (2), two major lignans isolated from the wood of Taxus yunnanensis, on tumor necrosis factor-alpha (TNF-alpha)-dependent hepatic apoptosis induced by D-galactosamine (d-GalN)/lipopolysaccharide (LPS) were investigated in mice. Co-administration of d-GalN (700 mg/kg) and LPS (10 microg/kg) resulted in a typical hepatic apoptosis characterized by DNA fragmentation and the formation of apoptotic bodies. Serum glutamic pyruvic transaminase (sGPT) and glutamic oxaloacetic transaminase (sGOT) levels were also raised at 8 h after d-GalN/LPS intoxication due to a severe necrosis of hepatocytes. Pre-administration of 1 or 2 (50, 10 mg/kg, i.p.) 12 and 1 h before d-GalN/LPS significantly reduced DNA fragmentation and prevented chromatin condensation, apoptotic body formation and hepatitis. Pro-inflammatory cytokines such as TNF-alpha and interferon-gamma (IFN-gamma) secreted from LPS-activated macrophages are important mediators of hepatocyte apoptosis in this model. Pre-treatment with 1 or 2 significantly inhibited the elevation of serum TNF-alpha and IFN-gamma levels. In a separate experiment, both lignans had a significant dose-dependent protective effect on d-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes and TNF-alpha-mediated cell death in murine L929 fibrosarcoma cells. These results indicated that 1 and 2 prevent d-GalN/LPS-induced hepatic injury by inhibiting hepatocyte apoptosis through the blocking of TNF-alpha and IFN-gamma production by activated macrophages and direct inhibition of the apoptosis induced by TNF-alpha.