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.

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.

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.

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.

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.

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.

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.

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.

Localisation of antinutrients and qualitative identification of toxic components in Jatropha curcas seed.

BACKGROUND: Jatropha curcas seed oil is a promising feedstock for biodiesel production. The seeds contain major toxic (phorbol esters, PEs) and antinutritional (phytate and trypsin inhibitor) factors. In the present study the localisation of antinutrients and a rapid qualitative method for detecting the presence of PEs were investigated. RESULTS: Kernels were separated into cotyledon, hypocotyl, kernel coat and endosperm. The majority of phytate (96.5%), trypsin inhibitor (95.3%) and PEs (85.7%) were localised in the endosperm. Based on PEs, a qualitative method was developed to differentiate between toxic and non-toxic Jatropha genotypes. In this method, PEs were easily detected by passing methanol extracts of kernels (Jatropha toxic and non-toxic genotypes) through a solid phase extraction (SPE) column and measuring the absorption of the resulting eluates at 280 nm. For raw kernels, SPE eluates with absorbance ≥ 0.056 were considered as toxic and those with absorbance ≤0.032 as non-toxic. For defatted kernel meals, SPE eluates with absorbance ≥ 0.059 were considered as toxic and those with absorbance ≤0.043 as non-toxic. CONCLUSION: The majority of antinutrients/toxic compounds are localised in the endosperm of the kernel. The qualitative method developed for rapid identification of toxic PEs could be useful in screening the toxicity of Jatropha-based products in the biodiesel industry. Further confirmation of PEs should be established by high-performance liquid chromatography.

Experimental assessment of toxic phytochemicals in Jatropha curcas: oil, cake, bio-diesel and glycerol.

BACKGROUND: Jatropha curcas seed is a rich source of oil; however, it can not be utilised for nutritional purposes due to presence of toxic and anti-nutritive compounds. The main objective of the present study was to quantify the toxic phytochemicals present in Indian J. curcas (oil, cake, bio-diesel and glycerol). RESULTS: The amount of phorbol esters is greater in solvent extracted oil (2.8 g kg⁻¹) than in expeller oil (2.1 g kg⁻¹). Liquid chromatography-mass spectroscopy analysis of the purified compound from an active extract of oil confirmed the presence of phorbol esters. Similarly, the phorbol esters content is greater in solvent extracted cake (1.1 g kg⁻¹) than in cake after being expelled (0.8 g kg⁻¹). The phytate and trypsin inhibitory activity of the cake was found to be 98 g kg⁻¹ and 8347 TIU g⁻¹ of cake, respectively. Identification of curcin was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the concentration of curcin was 0.95 g L⁻¹ of crude concentrate obtained from cake. CONCLUSION: Higher amounts of phorbol esters are present in oil than cake but bio-diesel and glycerol are free of phorbol esters. The other anti-nutritional components such as trypsin inhibitors, phytates and curcin are present in cake, so the cake should be detoxified before being used for animal feed.

Bioactive compounds and biological activities of Jatropha curcas L. kernel meal extract.

Defatted Jatropha curcas L. (J. curcas) seed kernels contained a high percentage of crude protein (61.8%) and relatively little acid detergent fiber (4.8%) and neutral detergent fiber (9.7%). Spectrophotometric analysis of the methanolic extract showed the presence of phenolics, flavonoids and saponins with values of 3.9, 0.4 and 19.0 mg/g DM, respectively. High performance liquid chromatography (HPLC) analyses showed the presence of gallic acid and pyrogallol (phenolics), rutin and myricetin (flavonoids) and daidzein (isoflavonoid). The amount of phorbol esters in the methanolic extract estimated by HPLC was 3.0 ± 0.1 mg/g DM. Other metabolites detected by GC-MS include: 2-(hydroxymethyl)-2 nitro-1,3-propanediol, ß-sitosterol, 2-furancarboxaldehyde, 5-(hydroxymethy) and acetic acid in the methanolic extract; 2-furancarboxaldehyde, 5-(hydroxymethy), acetic acid and furfural (2-furancarboxaldehyde) in the hot water extract. Methanolic and hot water extracts of kernel meal showed antimicrobial activity against both Gram positive and Gram negative pathogenic bacteria (inhibition range: 0-1.63 cm) at the concentrations of 1 and 1.5 mg/disc. Methanolic extract exhibited antioxidant activities that are higher than hot water extract and comparable to ß-carotene. The extracts tended to scavenge the free radicals in the reduction of ferric ion (Fe(3+)) to ferrous ion (Fe(2+)). Cytotoxicity assay results indicated the potential of methanolic extract as a source of anticancer therapeutic agents toward breast cancer cells.

[Hydrolysis technology optimization of phorbol esters by orthogonal experiment].

OBJECTIVE: To establish the best hydrolytic conditions from phorbol esters. METHOD: The orthogonal experiment was used to optimize 4 factors, which were reaction time, ratio of solid-to-liquid, hydrolytic times, and temperature. Diamonsil C18 column (4. 6 mm x 250 mm, 5 microm) was used and the mobile phase was consisted of acetonitrile and water for HPLC detection. The detection wavelength was set at 234 nm, the flow rate was 1 mL x min(-1), and the column temperature was 25 degrees C. RESULT: The optimum conditions were 10 h of reaction time, 1:6 of solid-to-liquid (BaOH/MeOH) ratio, 25 degrees C of temperature, and one time of hydrolysis. There was a good linear relationship of phorbol in the range of 4.28-107 mg x L(-1) (r = 0.999 9), and the average recovery was 97.89%, with RSD 0.78%. CONCLUSION: The method is steady, reliable and reproducible, and it provides a mean for future study.

Toxic compound, anti-nutritional factors and functional properties of protein isolated from detoxified Jatropha curcas seed cake.

Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications.

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.

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.

Toxicity studies of detoxified Jatropha meal (Jatropha curcas) in rats.

Jatropha curcas, a tropical plant introduced in many Asian and African countries is presently used as a source of biodiesel. The cake after oil extraction is rich in protein and is a potential source of livestock feed. In view of the high toxic nature of whole as well as dehulled seed meal due to the presence of toxic phorbol esters and lectin, the meal was subjected to alkali and heat treatments to deactivate the phorbol ester as well as lectin content. After treatment, the phorbol ester content was reduced up to 89% in whole and dehulled seed meal. Toxicity studies were conducted on male growing rats by feeding treated as well as untreated meal through dietary source. All rats irrespective of treatment had reduced appetite and diet intake was low accompanied by diarrhoea. The rats also exhibited reduced motor activity. The rats fed with treated meals exhibited delayed mortality compared to untreated meal fed rats (p0.02). There were significant changes both in terms of food intake and gain in body weight. Gross examination of vital organs indicated atrophy compared to control casein fed rats. However, histopathological examination of various vital organs did not reveal any treatment related microscopic changes suggesting that the mortality of rats occurred due to lack of food intake, diarrhoea and emaciation. Further studies are in progress for complete detoxification of J. curcas meal for use in livestock feed.

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.

Radioimmunoassay for phorbol esters using rabbit antisera against phorbol succinate.

The phorbol nucleus was succinylated and then conjugated to bovine albumin using dicyclohexylcarbodiimide. Rabbits given injections of the conjugate developed antibodies which rose in titer progressively with repeated immunization. By the ninth bleeding, the binding of one antiserum, diluted 1:15,000, was saturated with about 10 nM [3H]phorbol-12,13-dibutyrate [( 3H]-PDBU) and had an average association constant, Ka, of 2.6 X 10(8) M-1. The serological specificity of the antisera was characterized by examining the inhibition of the [3H]PDBU-anti-phorbol succinate immune system by 18 phorbol-related compounds. The specificities of antibodies from two rabbits tested in detail were qualitatively similar. The rank order of inhibitory activity for certain phorbol-related compounds was PDBU [concentration of inhibitor required to give 50% inhibition of PDBU binding (IC50) = 7.6 nM] = phorbol-13-acetate [IC50 = 8.2 nM] greater than phorbol-12,13-dibenzoate greater than 4-beta-phorbol [IC50 = 124 nM] greater than or equal to phorbol-12,13-diacetate greater than or equal to phorbol-12-myristate-13-acetate [IC50 = 184 nM] greater than phorbol-13,20-diacetate greater than phorbol-12-acetate [IC50 = 2300 nM]. The following compounds showed no detectable serological activity: mezerein, 4-0-methylphorbol-12-myristate-13-acetate, ingenol, 4-alpha-phorbol, teleocidin B, and dihydroteleocidin B. These and other results indicated that the 4-beta-phorbol nucleus was required for serological activity, that esterification of the C-13 position with benzoate, acetate, or butyrate enhanced the immunoreactivity of 4-beta-phorbol, and that among the phorbol-related compounds examined there was no direct relationship between serological activity and biological potency as tumor promoters. Using the [3H]PDBU-anti-phorbol succinate immune system, we measured the concentrations of immunoreactive phorbol-related material in crude mixtures such as croton oil and performed pharmacokinetic studies in rats given PDBU s.c.

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.