Untargeted Metabolomic Analysis of Leaves and Roots of Jatropha curcas Genotypes with Contrasting Levels of Phorbol Esters.

AIMS: Phorbol esters (PE) are toxic diterpenoids accumulated in physic nut (Jatropha curcas L.) seed tissues. Their biosynthetic pathway remains unknown, and the participation of roots in this process may be possible. Thus, we set out to study the deposition pattern of PE and other terpenoids in roots and leaves of genotypes with detected (DPE) and not detected (NPE) phorbol esters based on previous studies. OUTLINE OF DATA RESOURCES: We analyzed physic nut leaf and root organic extracts using LC-HRMS. By an untargeted metabolomics approach, it was possible to annotate 496 and 146 metabolites in the positive and negative electrospray ionization modes, respectively. KEY RESULTS: PE were detected only in samples of the DPE genotype. Remarkably, PE were found in both leaves and roots, making this study the first report of PE in J. curcas roots. Furthermore, untargeted metabolomic analysis revealed that diterpenoids and apocarotenoids are preferentially accumulated in the DPE genotype in comparison with NPE, which may be linked to the divergence between the genotypes concerning PE biosynthesis, since sesquiterpenoids showed greater abundance in the NPE. UTILITY OF THE RESOURCE: The LC-HRMS files, publicly available in the MassIVE database (identifier MSV000092920), are valuable as they expand our understanding of PE biosynthesis, which can assist in the development of molecular strategies to reduce PE levels in toxic genotypes, making possible the food use of the seedcake, as well as its potential to contain high-quality spectral information about several other metabolites that may possess biological activity.

Growth, metabolism and digestibility of Nile tilapia fed diets with solvent and extrusion-treated Jatropha curcas cake.

Physic nut Jatropha curcas cake/meal obtained after oil extraction has a high protein content, however, the presence of antinutrients (trypsin inhibitor, lectin and phytate) and toxic compounds (phorbol esters) limit their use as an alternative feedstuff. Thus, the detoxification process in cake/meal is necessary to allow their inclusion in fish diets. The present study aimed to evaluate the effects of solvent and extrusion-treated jatropha cake (SETJC) in Nile tilapia (Oreochromis niloticus) diets on growth, body composition, nutrient utilization, metabolic and hematological responses, and digestibility of experimental diets. Five experimental diets were formulated to be isonitrogenous (28.50% digestible protein) and isoenergetic (13.39 MJ/kg digestible energy) with graded levels of SETJC (0, 3, 6, 9, and 12%). The experimental design was completely randomized with five treatments and four replicates. The detoxification treatments reduced the phorbol esters (PE) of jatropha cake by 96% (0.58 mg/g of PE before and 0.023 mg/g of PE after treatments). Increased levels of SETJC depressed growth, feed efficiency, and protein digestibility. A similar trend was observed for hematological and biochemistry parameters. Aspartate and alanine aminotransferase, as well as phosphorus and magnesium concentrations in the fillets, increased at the highest levels of SETJC. Thus, the data of the present study suggests that the residual content, different structural forms of phorbol ester and its biological activity, as well as some antinutritional factors, can influence negatively the growth, metabolism and digestibility of experimental diets for Nile tilapia.

Detoxification of jatropha kernel meal to utilize it as aqua-feed.

BACKGROUND: Jatropha is an oilseed crop with high kernel oil (55-58%) and protein (26-29%) contents, which makes it a good source of biodiesel and animal/aqua-feed. However, the presence of anti-nutritional toxins, such as phorbol esters, lectins, trypsin inhibitor, phytate, and saponins, restricts its use as feed. This paper describes chemical, ultraviolet (UV) radiation, and biological treatments for detoxification of jatropha kernel meal. Raw, defatted, and one-time and two-times mechanically expressed oil samples were analyzed for toxins. Chemical treatment involved heating with 90% methanol and 4% sodium hydroxide. UV treatment was carried out at UV light intensity of 53.4 mW cm-2 for 30 min. For biological treatment, cell-free extract from Pseudomonas aeruginosa (strain PAO1) was mixed with kernel meal for detoxification. RESULTS: Among treatments, chemical treatment was most effective in reducing all toxins, with phorbol esters in the range 0.034-0.052 mg g-1 , lectin 0.082-10.766 mg g-1 , trypsin inhibitor 10.499-11.350 mg g-1 , phytate 2.475-5.769 mg g-1 , and saponins 0.044-0.098 mg g-1 . Biological treatment reduced all toxins except phytate, whereas UV treatment could not reduce any of toxins and, hence, cannot be used for aqua-feed preparation. Pellets prepared from chemically detoxified kernel meal with the least oil content (defatted) resulted in the highest strength (70.93 N). CONCLUSION: Chemically treated jatropha kernel meal can be used for aqua-feed pellet preparation because of its low toxin content. The highest compressive strength was obtained for pellets with the least oil content (defatted). Biological treatment time must have been extended for many hours instead of 24 h. Jatropha kernel meal treated chemically can be recommended for aqua-feed manufacturing. © 2021 Society of Chemical Industry.

Lab-made solid phase microextraction phases for off line extraction and direct mass spectrometry analysis: Evaluating the extraction parameters.

The analyses of drugs and metabolites in complex matrices have been widely studied in recent years. However, due to high levels endogenous compounds and matrix complexity, these analyses require a sample pre-treatment step. To this aim, two lab-made extractive phases were integrated to probe electrospray ionization mass spectrometry (PESI-MS) technique for direct analysis of illicit drugs in biological fluids and phorbol esters in Jatropha curcas extract. The polypyrrole (PPy) phase was electropolymerized onto a platinum wire surface by cyclic voltammetry. The molecularly imprinted polymer (MIP) was synthesized and adhered onto a stainless-steel needle with epoxy resin. The PPy-PESI-MS method showed to be linear in a concentration range from 1 to 500 µg L-1, with accuracy values between -2.1 and 14%, and precision values between 0.8 and 10.8%. The MIP-PESI-MS method showed to be linear in a concentration range from 0.9 to 30 mg L-1, with accuracy values between -1.6 and -15.3%, and precision values between 4.1 and 13.5%.

Traditional detoxification of Jatropha curcas L. seeds.

ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha curcas L. is a plant with high cultural significance for quilombola communities of Oriximiná (Pará State, Brazil). Although the plant is highly toxic, its seeds are used in these communities to treat tuberculosis and related diseases and symptoms. AIM OF THE STUDY: This study was designed to provide a scientific rationale for the traditional detoxification method and use of J. curcas seeds in quilombola communities of Oriximiná. MATERIALS AND METHODS: J. curcas seeds were manually separated into testa, tegmen, endosperm, and embryo, and then methanolic extracts of each sample were prepared. The traditional preparation of J. curcas seeds consists of a water extract of endosperms that is known as "milk of pinhão-branco". The content of phorbol esters (PEs) in the extracts was analyzed by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD). The cytotoxic activity was evaluated in human monocytic cell line THP-1 by Resazurin Reduction Assay, and antimycobacterial activity was assessed by determining Minimal Inhibitory Concentration (MIC) values against H37Rv and BCG strains using the Resazurin Microtiter Assay (REMA). RESULTS: The content analysis revealed that the distribution of PEs within the seeds is not homogeneous. High contents were found in tegmens (4.22 ±â€¯0.25-15.52 ±â€¯0.06 mg/g) and endosperms (1.61 ±â€¯0.07-5.00 ±â€¯0.42 mg/g), while concentrations found in testas and embryos were all below 0.5 mg/g. The traditional preparation derived from the endosperm of J. curcas contained significantly less PEs than the endosperms (0.01 ±â€¯0.005 mg/g). Against THP-1 cells, all the parts of the seed showed cytotoxic activity, while the traditional preparation was considered non-cytotoxic. Nevertheless, only the tegmen and endosperm of J. curcas were considered active against M. tuberculosis and M. bovis (MIC = 200 µg/mL). CONCLUSION: The results of this study indicated that the traditional processing performed by the quilombola people from Oriximiná is effective in reducing the toxicity of J. curcas seeds. Although inactive against mycobacteria, the extensive use of the traditional preparation and its low toxicity encourage further studies to investigate other biological activities.

Paper spray portable mass spectrometry for screening of phorbol ester contamination in glycerol-based medical products.

The Jatropha curcas plant (Jatropha) has been proposed as a source of biodiesel fuel, as it yields crude glycerol as an abundant by-product. Its by-products could serve as a starting material in making glycerol for FDA-regulated products. Jatropha is not regarded as a source of edible vegetable oil since it contains phorbol esters (PEs). PEs, even at very low exposure concentrations, demonstrate various toxicities in humans and animals, but may not be detected by routine impurity analyses. Here, we demonstrate the development of a rapid and simplified method for the detection and quantification of Jatropha-derived PE toxins using ambient ionization mass spectrometry. To do this, we successfully coupled a paper spray ambient ionization source with an ion trap portable mass spectrometer. The paper spray source was assembled using chromatography papers, and analyte ions were generated by applying a high voltage to a wetted paper triangle loaded with PE standards. For method development, we used commercially available PE standards on an ion trap portable mass spectrometer. Standard solutions were prepared using ethanol with PE concentrations ranging from 1.0 to 0.0001 mg mL-1. Spike and recovery experiments were performed using USP grade and commercially available glycerol. To discern chemical differences between samples, we applied multivariate data analysis. Based on the results obtained, paper spray coupled with a portable mass spectrometric method can be successfully adopted for the analysis of toxic contaminants present in glycerol-based consumer products with LOD and LOQ of 0.175 µg mL-1 and 0.3 µg mL-1 respectively. This direct, simple design, and low-cost sampling and ionization method enables fast screening with high sensitivity in non-laboratory settings.

Rapid and selective screening for toxic phorbol esters in Jatropha curcas seed oil using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

Jatropha curcas L. is an inedible plant whose seed oil is an interesting source for biodiesel production. Seed cake, the main byproduct remaining (about 70% w/w) after the oil extraction process, has a high nutritional value but the presence in Jatropha curcas seed of phorbol esters (PEs), a family of toxic compounds with a tigliane skeleton, prevents application of seed cake and other byproducts (e.g. glycerin) in animal feed without an efficient detoxification. Considering the high toxicity of PEs, it is important to have a sensitive analytical method to evaluate the presence of these compounds in Jatropha curcas derivatives. In this paper we present the study of the ESI-MS/MS fragmentation pattern of the [M+Na]+ ion at m/z 733.5 of the six known PEs, namely Jatropha factors (JFs) C1-C6, which allowed to tentatively identify a series of characteristic and specific fragment ions useful to reveal the presence of JFs in Jatropha curcas seed oil, distinguish them from each other, and identify new PEs (J1-J4). Moreover, the substitution of the usual acetonitrile/water as mobile phase with a mixture of methanol/water (85:15, v/v) allowed to increase the signal of the sodium adduct of about 50-fold during the HPLC-ESI-MS/MS analysis.

Detoxification of Jatropha curcas seed cake by a new soil-borne Enterobacter cloacae strain.

Jatropha curcas seed cake is a by-product generated after oil extraction from J. curcas seeds. Although the protein content is high, the cake contains phorbol esters and antinutritional factors such as phytates, trypsin inhibitors, lectins and tannins. Therefore, it cannot be directly used in food or feed. In this study, the toxic compounds and antinutrients present in J. curcas seed cake were detoxified by fermentation with Enterobacter Z11, a soil-borne isolate. Solid-state fermentation was undertaken under optimized conditions: deoiled cake, 5·0 g; initial moisture content, 50%; temperature, 30°C; and inoculum, 2 × 106 cells per gram of cake. Postfermentation, bacterial growth, pH and the amount of antinutrients were studied. Fermentation reduced the content of phorbol esters, phytates, lectins, tannins and trypsin inhibitors by 51·6, 82·6, 88·9, 37·8 and 90·5%, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain of Enterobacter cloacae Z11 was originally isolated from the soil. To the best of our knowledge, E. cloacae has never been used to remove toxins and antinutritional factors in Jatropha curcas seed cake (JSC). Under the optimized condition, fermentation with the Enterobacter strain decreased the phorbol esters content in JSC by 51·6%, and phytates, tannins, lectins and trypsin inhibitors contents by 83, 38, 89 and 90%, respectively. This study provided a new method with potential to render the seed cake suitable for use in feed. Further study is needed to focus on remaining toxicity and nutritional value post-treatment.

Application of time-of-flight mass spectrometry for screening of crude glycerins for toxic phorbol ester contaminants.

Since 2007, the U.S. Food and Drug Administration (FDA) has received numerous complaints of pet illnesses that may be related to the consumption of jerky pet treats. Many of those treats include glycerin as an ingredient. Glycerin can be made directly from oils such as palm seed oil, but can also be derived from the seed oil of toxic Jatropha plant during biodiesel production. If crude glycerin from biodiesel production from Jatropha curcas is used in the manufacture of animal feed, toxic tigliane diterpene phorbol esters (PEs), namely Jatropha factors (JFs), may be present and could lead to animal illnesses. Considering the numerous uses of glycerin in consumer products there is a need for a rapid method to screen crude glycerin for JF toxins and other PE contaminants. We describe the development of an ultra-high pressure liquid chromatography/quadrupole time of flight (UHPLC/Q-TOF) method for screening crude glycerin for PEs. An exact mass database, developed in-house, of previously identified PEs from Jatropha curcas as well as putative compounds was used to identify possible contaminants.

Fatty Acid Diversity is Not Associated with Neutral Genetic Diversity in Native Populations of the Biodiesel Plant Jatropha curcas L.

Jatropha curcas L. (Euphorbiaceae) is a shrub native to Mexico and Central America, which produces seeds with a high oil content that can be converted to biodiesel. The genetic diversity of this plant has been widely studied, but it is not known whether the diversity of the seed oil chemical composition correlates with neutral genetic diversity. The total seed oil content, the diversity of profiles of fatty acids and phorbol esters were quantified, also, the genetic diversity obtained from simple sequence repeats was analyzed in native populations of J. curcas in Mexico. Using the fatty acids profiles, a discriminant analysis recognized three groups of individuals according to geographical origin. Bayesian assignment analysis revealed two genetic groups, while the genetic structure of the populations could not be explained by isolation-by-distance. Genetic and fatty acid profile data were not correlated based on Mantel test. Also, phorbol ester content and genetic diversity were not associated. Multiple linear regression analysis showed that total oil content was associated with altitude and seasonality of temperature. The content of unsaturated fatty acids was associated with altitude. Therefore, the cultivation planning of J. curcas should take into account chemical variation related to environmental factors.

Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed.

Phorbol esters (PEs) are well known as the main toxic compounds in Jatropha curcas Linnaeus (JCL), the seed oil of which has been considered as a major feedstock for the production of biodiesel. In the present study, we investigated a series of PEs extracted from JCL seed kernels with methanol (MeOH), and identified more than seven components contained in the PEs. The isolation of main five components of a series of PEs was revised using a semi-preparative reversed phase HPLC analysis of ODS-3 column. The five peaks of components were successfully isolated, and peaks of J2, J3, J5, and J7 were assigned to be Jatropha factors C1, C2, C3, and C4/5, but J6 was a mixture of Jatropha factor C6 and its isomer based on the data of UV and LC-MS/MS, and J2 was identified using 1H NMR analysis. By characterization using LC-MS/MS analysis, all components of a series of PEs were elucidated to be the 12-deoxy-16-hydroxyphorbol esters composed of isomeric form of dicarboxylic groups with same m/z value of 380.

Screening for toxic phorbol esters in jerky pet treat products using LC-MS.

Since 2007, the U.S. FDA's Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats. Jerky used in pet treats contains glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Because some biodiesel is produced using oil from Jatropha curcas, a plant that contains toxic compounds including phorbol esters, CVM developed a liquid chromatography-mass spectrometry (LC-MS) screening method to evaluate investigational jerky samples for the presence of these toxins. Results indicated that the samples analyzed with the new method did not contain Jatropha toxins at or above the lowest concentration tested.

Rapid isolation and purification of phorbol esters from Jatropha curcas by high-speed countercurrent chromatography.

In this work, a high-speed countercurrent chromatography (HSCCC) method was established for the preparation of phorbol esters (PEs) from Jatropha curcas. n-Hexane-ethyl acetate-methanol-water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum two-phase solvent system to separate and purify jatropha factor C1 (JC1) with a purity of 85.2%, as determined by HPLC, and to obtain a mixture containing four or five PEs. Subsequently, continuous semipreparative HPLC was applied to further purify JC1 (99.8% as determined by HPLC). In addition, UPLC-PDA and UPLC-MS were established and successfully used to evaluate the isolated JC1 and PE-rich crude extract. The purity of JC1 was only 87.8% by UPLC-UV. A peak (a compound highly similar to JC1) was indentified as the isomer of JC1 by comparing the characteristic UV absorption and MS spectra. Meanwhile, this strategy was also applied to analyze the PE-rich crude extract from J. curcas. It is interesting that there may be more than 15 PEs according to the same quasi-molecular ion peaks, highly similar sequence-specific fragment ions, and similar UV absorption spectrum.

Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test.

Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.

Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98.

This study focused on the solid-state fermentation of Jatropha seed cake (JSC), a byproduct generated after biodiesel production. Presence of anti-nutritional compounds and toxins restricts its application in livestock feed. The disposal of the JSC is a major environmental problem in the future, due to the generation of huge quantity of JSC after biodiesel extraction. Hence the JSC was assessed for its suitability as substrate for production and optimization of lipase and protease from Aspergillus versicolor CJS-98 by solid-state fermentation (SSF). The present study was also focused on the biodetoxification of anti-nutrients and toxins in JSC. The SSF parameters were optimized for maximum production of lipase and protease. Under the optimized conditions, the JSC supplemented with maltose and peptone (2%), adjusted to pH 7.0, moisture content 40%, inoculated with 1 × 10(7) spores per 5 g cake and incubated at 25°C, produced maximum lipase, 1288 U/g and protease, 3366 U/g at 96 h. The anti-nutrients like phytic acid (6.08%), tannins (0.37%), trypsin inhibitors (697.5 TIU/g), cyanogenic glucosides (692.5 µg/100 g), and lectins (0.309 mg/ml), were reduced to 1.70%, 0.23%, 12.5 TIU/g, 560.6 µg/100 g and 0.034 mg/ml respectively. The main toxic compound phorbol esters content in the JSC was reduced from 0.083% to 0.015% after SSF. Our results indicate that viability of SSF to utilize the huge amount of seed cake generated after extraction of biodiesel, for production of industrial enzymes and biodetoxification of anti-nutrients, toxins.

Potential treatments to reduce phorbol esters levels in jatropha seed cake for improving the value added product.

Jatropha seed cake contains high amounts of protein and other nutrients, however it has a drawback due to toxic compounds. The aim of this study was to investigate the methods applied to detoxify the main toxin, phorbol esters in jatropha seed cake, to a safe and acceptable level by maintaining the nutritional values. Phorbol esters are tetracyclic diterpenoids-polycyclic compounds that are known as tumor promoters and hence exhibited the toxicity within a broad range of species. Mismanagement of the jatropha waste from jatropha oil industries would lead to contamination of the environment, affecting living organisms and human health through the food chain, so several methods were tested for reducing the toxicity of the seed cake. The results from this investigation showed that heat treatments at either 120°C or 220°C for 1 hour and then mixing with adsorbing bentonite (10%), nanoparticles of zinc oxide (100 µg/g) plus NaHCO3 at 4%, followed by a 4-week incubation period yielded the best final product. The remaining phorbol esters concentration (0.05-0.04 mg/g) from this treatment was less than that reported for the nontoxic jatropha varieties (0.11-0.27 mg/g). Nutritional values of the seed cake after treatment remained at the same levels found in the control group and these values were crude protein (20.47-21.40 + 0.17-0.25%), crude lipid (14.27-14.68 + 0.13-0.14%) and crude fiber (27.33-29.67 + 0.58%). A cytotoxicity test conducted using L929 and normal human dermal fibroblast cell lines confirmed that most of the toxic compounds, especially phorbol esters, were shown as completely eliminated. The results suggested that the detoxification of phorbol esters residues in the jatropha seed cake was possible while it also retained nutritional values. Therefore, the methods to detoxify phorbol esters are necessary to minimize the toxicity of jatropha seed cake. Further, it is essential to reduce the possible environmental impacts that may be generated throughout the jatropha waste-handling process. However additional tests such as digestibility as well as acceptability of the treated jatropha seed cake should be conducted using both in vivo and in vitro studies before recommending the jatropha seed cake as a source of renewable animal feed and other value-added products.

Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation.

Jatropha curcas seed cake is a by-product generated from oil extraction of J. curcas seed. Although it contains a high amount of protein, it has phorbol esters and anti-nutritional factors such as phytate, trypsin inhibitor, lectin and saponin. It cannot be applied directly in the food or animal feed industries. This investigation was aimed at detoxifying the toxic and anti-nutritional compounds in J. curcas seed cake by fermentation with Bacillus spp. Two GRAS (generally recognized as safe) Bacillus strains used in the study were Bacillus subtilis and Bacillus licheniformis with solid-state and submerged fermentations. Solid-state fermentation was done on 10 g of seed cake with a moisture content of 70% for 7 days, while submerged fermentation was carried out on 10 g of seed cake in 100 ml distilled water for 5 days. The fermentations were incubated at the optimum condition of each strain. After fermentation, bacterial growth, pH, toxic and anti-nutritional compounds were determined. Results showed that B. licheniformis with submerged fermentation were the most effective method to degrade toxic and anti-nutritional compounds in the seed cake. After fermentation, phorbol esters, phytate and trypsin inhibitor were reduced by 62%, 42% and 75%, respectively, while lectin could not be eliminated. The reduction of phorbol esters, phytate and trypsin inhibitor was related to esterase, phytase and protease activities, respectively. J. curcas seed cake could be mainly detoxified by bacterial fermentation and the high-protein fermented seed cake could be potentially applied to animal feed.

Chemical characterisation of kernels, kernel meals and oils from Jatropha cordata and Jatropha cardiophylla seeds.

BACKGROUND: Jatropha cordata and Jatropha cardiophylla are native to northwestern Mexico and are adapted to arid and semi-arid conditions (<500 mm of precipitation and temperatures from 8 to 45 °C). The aim of this study was to evaluate the chemical composition of J. cordata and J. cardiophylla kernels and oils as well as antinutrients in the defatted kernel meals of these species. RESULTS: Kernels of J. cordata and J. cardiophylla seeds analysed in this study were rich in crude protein (283 and 289 g kg(-1) respectively) and lipid (517 and 537 g kg(-1) respectively). The main fatty acids in J. cordata and J. cardiophylla oils were linoleic and oleic acids. High levels of trypsin inhibitor and phytates and low levels of saponins were present in the meals. The phorbol ester contents in J. cordata and J. cardiophylla kernel meals were 2.73 and 1.46 mg g(-1) respectively. CONCLUSION: For both J. cordata and J. cardiophylla it could be inferred that (a) the oil and kernel meal were toxic and the kernel meal could be used as livestock feed only after detoxification, (b) the oil could be used for non-alimentary purposes, i.e. biodiesel production, and (c) the seed or oil could be used for isolating various bioactive compounds for pharmaceutical and agricultural applications.

Metabolic profiling assisted quality control of phorbolesters in Jatropha curcas seed by high-performance liquid chromatography using a fused-core column.

In this work, fast and sensitive high-performance liquid chromatography (HPLC) coupled with multivariate analysis was utilized to evaluate the metabolic profiling of Jatropha curcas seed and screen the marker compounds of phorbolesters (PEs), which significantly contributed to the metabolic profiling for quality control of PEs in J. curcas seed. Thirty-two peaks were separated and detected in J. curcas seed on a fused-core C(18) column between 2 and 25 min. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of the chromatographic data demonstrated that 12 batches of J. curcas seed could be well-differentiated and categorized into 5 groups. Especially, one sample obtained from Lijiang Yunan was significantly different from the others. Partial least-squares discriminant analysis (PLS-DA) showed that the metabolic profiling differentiation might be explained by 9 components including J.factors C(1)-C(5) of PEs, peak 21 (similar to J.factor C(1)) and 3 peaks (peaks 1, 9, and 10) significantly different in retention time from the PEs. The observation that the content levels of J.factors C(1) and C(2) plus peak 21 (equivalent to J.factor C(1)) were more relevant to the multivariate chromatographic data than the ones of J.factors C(3)-C(5) was confirmed by the PLS prediction models. The results of the present study indicated not only that J.factors C(1) and C(2) were the more rational markers representing the comprehensive quality of PEs in J. curcas seed but also that peak 21 (similar to J.factor C(1)) was a rational marker, too.

Comparative characteristic of the inflammatory diterpenes in the roots of Euphorbia fischeriana with different preparation method using HPLC-ELSD.

A rapid and simple method was established for the simultaneous determination of ten diterpenes by reversed phase HPLC coupled with evaporative light scattering detection. Chromatographic separation was carried out in gradient mode by using a WondaSil™ C(18) column (250mm×4.6mm, 5µm) with mobile phases of methanol and water at 1mL/min. The drift tube temperature of evaporative light scattering detector was set to 65°C and nitrogen flow-rate was 2.7L/min. The method validated was shown to be specific, precise, accurate and linear. Moreover, it was applied to investigate four samples of E. fischeriana with different extracting methods. Contrast to the dried roots, the fresh roots had much higher content of prostratin which represented much higher inflammatory effects than other diterpenes. The results demonstrated that the dried roots were suitable for the ordinary therapy to avoid intense stimulatory, while the fresh roots could be used in the anticancer treatment. All of the results suggested that comparative analysis of chemical components as biomarker and connecting toxic effects of an herb was helpful for revealing the mechanism of its toxicity, and for guiding safer and better application of the herb in TCM clinic.