Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Comparison of the removal of uraemic toxins with medium cut-off and high-flux dialysers: a randomized clinical trial.

BACKGROUND: Accumulation of middle-weight uraemic toxins in haemodialysis (HD) patients results in increased morbidity and mortality. Whether medium cut-off HD (MCO-HD) improves removal of middle-weight uraemic toxins remains to be demonstrated. METHODS: This cross-over prospective study included 40 patients randomly assigned to receive either 3 months of MCO-HD followed by 3 months of high-flux HD (HF-HD), or vice versa. The primary endpoint was myoglobin reduction ratio (RR) after 3 months of MCO-HD. Secondary endpoints were the effect of MCO-HD on other middle-weight toxins and protein-bound toxins, and on parameters of nutrition, inflammation, anaemia and oxidative stress. RESULTS: Compared with HF-HD, MCO-HD provided higher mean RR of myoglobin (36 ± 8 versus 57 ± 13%, P < 0.0001), beta2-microglobulin (68 ± 6 versus 73 ± 15%, P = 0.04), prolactin (32 ± 13 versus 59 ± 11%, P < 0.0001), fibroblast growth factor 23 (20 ± 21 versus 41 ± 22%, P = 0.0002), homocysteine (43 ± 7 versus 46 ± 9%, P = 0.03) and higher median RR of kappa [54 (48-58) versus 70 (63-74)%, P < 0.0001] and lambda free light chain (FLC) [15 (9-22) versus 44 (38-49)%, P < 0.0001]. Mean ± SD pre-dialysis levels of beta2-microglobulin (28.4 ± 5.6 versus 26.9 ± 5.1 mg/L, P = 0.01) and oxidized low-density lipoprote (6.9 ± 4.4 versus 5.5 ± 2.5 pg/mL, P = 0.04), and median (interquartile range) kappa FLC [145 (104-203) versus 129 (109-190) mg/L, P < 0.03] and lambda FLC [106 (77-132) versus 89 (62-125) mg/L, P = 0.002] were significantly lower. Mean albumin levels decreased significantly (38.2 ± 4.1 versus 36.9 ± 4.3 g/L, P = 0.004), without an effect on nutritional status as suggested by unchanged normalized protein catabolic rate and transthyretin level. CONCLUSIONS: Compared with HF-HD, MCO-HD provides higher myoglobin and other middle molecules RR and is associated with moderate hypoalbuminemia. The potential benefits of this strategy on long-term clinical outcomes deserve further evaluation.

New Advanced Materials and Sorbent-Based Microextraction Techniques as Strategies in Sample Preparation to Improve the Determination of Natural Toxins in Food Samples.

Natural toxins are chemical substances that are not toxic to the organisms that produce them, but which can be a potential risk to human health when ingested through food. Thus, it is of high interest to develop advanced analytical methodologies to control the occurrence of these compounds in food products. However, the analysis of food samples is a challenging task because of the high complexity of these matrices, which hinders the extraction and detection of the analytes. Therefore, sample preparation is a crucial step in food analysis to achieve adequate isolation and/or preconcentration of analytes and provide suitable clean-up of matrix interferences prior to instrumental analysis. Current trends in sample preparation involve moving towards "greener" approaches by scaling down analytical operations, miniaturizing the instruments and integrating new advanced materials as sorbents. The combination of these new materials with sorbent-based microextraction technologies enables the development of high-throughput sample preparation methods, which improve conventional extraction and clean-up procedures. This review gives an overview of the most relevant analytical strategies employed for sorbent-based microextraction of natural toxins of exogenous origin from food, as well as the improvements achieved in food sample preparation by the integration of new advanced materials as sorbents in these microextraction techniques, giving some relevant examples from the last ten years. Challenges and expected future trends are also discussed.

Antimalarials and Phytotoxins from Botryosphaeria dothidea Identified from a Seed of Diseased Torreya taxifolia.

The metabolic pathways in the apicoplast organelle of Plasmodium parasites are similar to those in plastids in plant cells and are suitable targets for malaria drug discovery. Some phytotoxins released by plant pathogenic fungi have been known to target metabolic pathways of the plastid; thus, they may also serve as potential antimalarial drug leads. An EtOAc extract of the broth of the endophyte Botryosphaeria dothidea isolated from a seed collected from a Torreya taxifolia plant with disease symptoms, showed in vitro antimalarial and phytotoxic activities. Bioactivity-guided fractionation of the extract afforded a mixture of two known isomeric phytotoxins, FRT-A and flavipucine (or their enantiomers, sapinopyridione and (-)-flavipucine), and two new unstable γ-lactam alkaloids dothilactaenes A and B. The isomeric mixture of phytotoxins displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.

Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin.

Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria.

Improved dialytic removal of protein-bound uremic toxins by intravenous lipid emulsion in chronic kidney disease rats.

BACKGROUND: Protein-bound uremic toxins (PBUTs) have received extensive attention, as their accumulation leads to pleiotropic toxic biological effects, while the removal of these solutes by conventional dialysis therapies is severely hampered. This study aimed to examine whether increased removal of PBUTs could be achieved with intravenous lipid emulsion (ILE). METHODS: PBUTs such as 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), p-cresyl sulfate (PCS) and indoxyl sulfate (IS) were spiked with human serum albumin (HSA) solution and the inhibitory effects of free fatty acid (FFA) on the binding of CMPF, PCS and IS to HSA were examined separately in vitro by ultrafiltration. In vitro dialysis of albumin solution was then performed to investigate the effects of fatty acid (FAs) mixtures infusion on the fractional removal of PBUTs. Finally, the inhibitory effect of FFA on the binding of PBUTs to albumin was examined in uremic rats, and blood purification therapy was conducted to calculate the reduction ratio (RR) and the total solute removal (TSR) of solutes. RESULTS: The percentage protein binding of CMPF, PCS and IS decreased significantly with increasing FFAs concentrations, and the inhibitory effect was more remarkable with the addition of oleic acid or linoleic acid than that of eicosapentaenoic acid and docosahexaenoic acid. In vitro infusion of FAs increased the fractional removal of CMPF to 14.40 ± 2.38%. PCS, IS and indole-3-acetic acid removal increased from 8.00 ± 2.43%, 11.68 ± 1.54% and 15.38 ± 3.97%, respectively, at baseline to 28.21 ± 5.99%, 35.42 ± 5.27% and 40.18 ± 5.05%, respectively, when FAs were present. In vivo, rat serum concentrations of free PBUTs were significantly higher in the ILE group than in the control group, and administration of ILE resulted in higher RRs and more TSR for PBUTs after 3 h of hemodialysis (HD) therapy compared with the control group. CONCLUSIONS: Administration of ILE effectively increased the dialytic removal of PBUTs. This method could be applied to current HD therapy.

Effect of prebiotic (fructooligosaccharide) on uremic toxins of chronic kidney disease patients: a randomized controlled trial.

BACKGROUND: Microbial-derived uremic toxins, p-cresyl sulfate (PCS), indoxyl sulfate (IS) and indole 3-acetic acid (IAA), have been associated with the burden of chronic kidney disease (CKD). Prebiotics have emerged as an alternative to modulate the gut environment and to attenuate toxin production. This trial aims to investigate the effect of a prebiotic fructooligosaccharide (FOS) on uremic toxins of non-dialysis-dependent CKD (NDD-CKD) patients. METHODS: A double-blind, placebo-controlled, randomized trial was conducted for 3 months. In all, 50 nondiabetic NDD-CKD patients [estimated glomerular filtration rate (eGFR) <45 mL/min/1.73 m2], aged 18-80 years, were allocated to prebiotic (FOS, 12 g/day) or placebo (maltodextrin, 12 g/day) groups. Primary outcomes were changes in serum (total and free) and urinary (total) PCS. Secondary outcomes included changes in IS, IAA, serum markers of intestinal permeability (zonulin), gut-trophic factors (epidermal growth factor and glucagon-like peptide-2), eGFR, inflammation (high sensitive c-reactive protein and interleukin-6), homeostatic model assessment-insulin resistance, lipid profile and gastrointestinal symptoms. RESULTS: From 50 participants (54% men, 57.3 ± 14.6 years and eGFR 21.4 ± 7.6 mL/min/1.73 m2), 46 completed the follow-up. No changes in dietary intake or gastrointestinal symptoms were observed. There was a trend in the difference of serum total ΔPCS (treatment effect adjusted for baseline levels: -12.4 mg/L; 95% confidence interval (-5.6 to 0.9 mg/L; P = 0.07) and serum-free Δ%PCS [intervention -8.6 (-41.5 to 13.9%) versus placebo 3.5 (-28.8 to 85.5%); P = 0.07] between the groups. The trend in the difference of serum total ΔPCS was independent of eGFR and dietary protein:fiber ratio intake. No difference was found in urinary PCS. Aside from the decreased high-density lipoprotein cholesterol in the intervention, no differences were observed in the change of IS, IAA or other secondary outcome between the groups. CONCLUSIONS: Our result suggests the potential of FOS in reducing serum total and free PCS in nondiabetic NDD-CKD patients.

Isolation and characterization of the insecticidal, two-domain toxin LaIT3 from the Liocheles australasiae scorpion venom.

A novel insecticidal peptide (LaIT3) was isolated from the Liocheles australasiae venom. The primary structure of LaIT3 was determined by a combination of Edman degradation and MS/MS de novo sequencing analysis. Discrimination between Leu and Ile in MS/MS analysis was achieved based on the difference in side chain fragmentation assisted by chemical derivatization. LaIT3 was determined to be an 84-residue peptide with three intrachain disulfide bonds. The sequence similarity search revealed that LaIT3 belongs to the scorpine-like peptides consisting of two structural domains: an N-terminal α-helical domain and a C-terminal cystine-stabilized domain. As observed for most of the scorpine-like peptides, LaIT3 showed significant antibacterial activity against Escherichia coli, which is likely to be caused by its membrane-disrupting property.

Increasing the removal of protein-bound uremic toxins by liposome-supported hemodialysis.

Protein-bound uremic toxins (PBUTs) accumulate at high plasma levels and cause various deleterious effects in end-stage renal disease patients because their removal by conventional hemodialysis is severely limited by their low free-fraction levels in plasma. Here, we assessed the extent to which solute removal can be increased by adding liposomes to the dialysate. The uptake of liposomes by direct incubation in vitro showed an obvious dose-response relationship for p-cresyl sulfate (PCS) and indoxyl sulfate (IS) but not for hippuric acid (HA). The percent removal of both PCS and IS but not of HA was gradually increased with the increased concentration of liposomes in a rapid equilibrium dialysis setup. In vitro closed circulation showed that adding liposomes to the dialysate markedly increased the dialysances of PBUTs without greatly altering that of urea and creatinine. In vivo experiments in uremic rats demonstrated that adding liposomes to the dialysate resulted in higher reduction ratios (RRs) and more total solute removal (TSR) for several PBUTs compared to the conventional dialysate, which was approximately similar to the addition of bovine serum albumin to the dialysate. These findings highlight that as an adjunct to conventional hemodialysis, addition of liposomes to the dialysate could significantly improve the removal of protein-bound uremic solutes without greatly altering the removal of small, water-soluble solutes.

Uremic Toxins and their Relation to Dialysis Efficacy.

Toxin retention is felt to be a major contributor to the development of uremia in patients with advanced chronic kidney disease and end-stage renal disease (ESRD). Uremic retention compounds are classically divided into 3 categories: small solutes, middle molecules, and protein-bound toxins. Compounds comprising the first category, for which the upper molecular weight limit is generally considered to be 500 Da, possess a high degree of water solubility and minimal or absent protein binding. The second category of middle molecules has largely evolved now to be synonymous with peptides and proteins that accumulate in uremia. Although not precisely defined, low-molecular weight proteins as a class have a molecular weight spectrum ranging from approximately 500 to 60,000 daltons. The final category of uremic retention compounds is protein-bound uremic toxins (PBUTs). As opposed to the above small, highly water-soluble toxins, which are largely by-products of protein metabolism, PBUTs have diverse origins and possess chemical characteristics that preclude the possibility of circulation in an unbound form despite being of low molecular weight. This review is the first in a series of papers designed to provide the current state of the art for extracorporeal treatment of ESRD. Subsequent papers in this series will address membranes, mass transfer mechanisms, and future directions. For small solutes and middle molecules, particular emphasis is placed on the important clinical trials that comprise the evidence base regarding the influence of dialytic solute removal on outcome. Because such trials do not exist for PBUTs, the discussion here is instead focused on solute characteristics and renal elimination mechanisms.

Adsorption of Protein-Bound Uremic Toxins Using Activated Carbon through Direct Hemoperfusion in vitro.

BACKGROUND/AIMS: Accumulation of protein-bound uremic toxins (PBUTs) is associated with mortality due to various systemic disorders in patients with chronic kidney disease (CKD), especially in those undergoing dialysis treatment. The clinical outcomes of such patients could be improved by removing sufficient amounts of PBUTs; however, conventional dialysis lacks this ability. We examined the efficacy of activated carbon in adsorbing circulating PBUTs through direct hemoperfusion (DHP) in vitro. METHODS: An in vitro blood circulating system was constructed with 8.5 mL blood circulating around a column containing activated carbon (50, 100, or 200 mg). Bovine blood containing a kind of PBUT (at the same concentration as that found in the blood of dialysis patients) and blood from hemodialysis patients (n = 8) were used. After circulation for the designated amount of time, sera were collected and the levels of PBUTs, including indoxyl sulfate (IS), p-cresyl sulfate, indole acetic acid (IAA), phenyl sulfate, and hippuric acid, were analyzed with mass spectrometry. RESULTS: Activated carbon decreased the PBUT level in bovine blood in a dose-dependent manner (e.g., reduction rate of IS: 67.9 ± 3.8, 83.3 ± 1.9, and 94.5 ± 1.1% after 60-min circulation in columns containing 50, 100, and 200 mg activated carbon respectively). IS, PCS, and IAA were dramatically adsorbed by activated carbon from the blood of patients undergoing hemodialysis (pre vs. post 240-min reaction: IS 2.835 ± 0.876 vs. 0.455 ± 0.108 mg/dL [p < 0.01], PCS 3.208 ± 2.876 vs. 0.768 ± 0.632 mg/dL [p < 0.01], IAA 0.082 ± 0.045 vs. 0.016 ± 0.005 mg/dL [p < 0.01]). CONCLUSION: Activated carbon effectively adsorbed blood PBUTs in vitro. DHP with activated carbon could be a promising strategy for removing circulating PBUTs from the blood of patients with CKD.

Predilution On-Line Hemodiafiltration Improves Albumin Redox in Maintenance Hemodialysis Patients.

BACKGROUND/AIM: In this study, we compared the dialysis efficiency, oxidative stress, and nutritional conditions between predilution on-line hemodiafiltration (pre-OL-HDF) and conventional hemodialysis (HD) using a super-flux dialyzer (CHD). METHOD: This was a crossover study of 38 maintenance HD patients. All patients were treated with CHD for the first 4 months (1st CHD period), then were switched to pre-OL-HDF for 4 months (pre-OL-HDF period), and were returned to CHD for the next 4 months (2nd CHD period). RESULTS: We found no significant difference in the removal ratio of small uremic substances or the indices of inflammation or nutritional states between the pre-OL-HDF and CHD periods. However, we found higher removal of ß2 micro-globulin in the pre-OL-HDF period, and the human mercapto-albumin (Alb)/human serum Alb ratio was significantly higher in the pre-OL-HDF period. CONCLUSION: Treatment with pre-OL-HDF enabled enhanced removal of middle molecule uremic toxins and better Alb redox than did CHD.

Is There Enough Evidence to Prove That Hemodiafiltration Is Superior?

The evidence concerning online hemodiafiltration (HDF) includes the following: (1) the increase of removal of uremic toxin, (2) the prevention of dialysis rerated hypotension, (3) the recovery of survival, and (4) the recovery of dialysis related uncertain symptoms. Although clinical evidence has been noted, the hypothesis of the efficacy of HDF are still indefinite and require further precise research.

Rapid Detection of Abrin Toxin and Its Isoforms in Complex Matrices by Immuno-Extraction and Quantitative High Resolution Targeted Mass Spectrometry.

Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 µg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.

New paradoxical three-finger toxin from the cobra Naja kaouthia venom: Isolation and characterization.

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of "orphan" toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.

Functional Nanoparticles Activate a Decellularized Liver Scaffold for Blood Detoxification.

Extracorporeal devices have great promise for cleansing the body of virulence factors that are caused by venomous injuries, bacterial infections, and biological weaponry. The clinically used extracorporeal devices, such as artificial liver-support systems that are mainly based on dialysis or electrostatic interaction, are limited to remove a target toxin. Here, a liver-mimetic device is shown that consists of decellularized liver scaffold (DLS) populated with polydiacetylene (PDA) nanoparticles. DLS has the gross shape and 3D architecture of a liver, and the PDA nanoparticles selectively capture and neutralize the pore-forming toxins (PFTs). This device can efficiently and target-orientedly remove PFTs in human blood ex vivo without changing blood components or activating complement factors, showing potential application in antidotal therapy. This work provides a proof-of-principle for blood detoxification by a nanoparticle-activated DLS, and can lead to the development of future medical devices for antidotal therapy.

Immuno-PCR: Achievements and Perspectives.

The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization" of antigen-antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Magnetic separation-based blood purification: a promising new approach for the removal of disease-causing compounds?

Recent studies report promising results regarding extracorporeal magnetic separation-based blood purification for the rapid and selective removal of disease-causing compounds from whole blood. High molecular weight compounds, bacteria and cells can be eliminated from blood within minutes, hence offering novel treatment strategies for the management of intoxications and blood stream infections. However, risks associated with incomplete particle separation and the biological consequences of particles entering circulation remain largely unclear. This article discusses the promising future of magnetic separation-based purification while keeping important safety considerations in mind.

Suaveolic acid: a potent phytotoxic substance of Hyptis suaveolens.

Hyptis suaveolens (Lamiaceae) is an exotic invasive plant in many countries. Earlier studies reported that the aqueous, methanol, and aqueous methanol extract of H. suaveolens and its residues have phytotoxic properties. However, to date, the phytotoxic substances of this plant have not been reported. Therefore, the objectives of this study were isolation and identification of phytotoxic substances of H. suaveolens. Aqueous methanol extract of this plant was purified by several chromatographic runs through bioassay guided fractionation using garden cress (Lepidium sativum) as a test plant. Final purification of a phytotoxic substance was achieved by reverse phase HPLC and characterized as 14α-hydroxy-13ß-abiet-8-en-18-oic acid (suaveolic acid) by high-resolution ESI-MS, (1)H-,(13)C-NMR, CD, and specific rotation. Suaveolic acid inhibited the shoot growth of garden cress, lettuce (Lactuca sativa), Italian ryegrass (Lolium multiflorum), and barnyard grass (Echinochloa crus-galli) at concentrations greater than 30 µM. Root growth of all but lettuce was also inhibited at concentrations greater than 30 µM. The inhibitory activities were concentration dependent. Concentrations required for 50% growth inhibition of suaveolic acid for those test plant species were ranged from 76 to 1155 µM. Therefore, suaveolic acid is phytotoxic and may be responsible for the phytotoxicity of H. suaveolens plant extracts.