The worldwide search for new fuel sources has grown during the last decades due to two main factors: the global concern about environmental issues and the high price of petroleum. Biodiesel is a type of biofuel that is already used in many countries, and its usage will most likely increase over the next few years. Biodiesel can be produced using different technologies and raw materials, such as vegetable oils, animal fats and microalgae oil. However, despite the wide range of oil sources for biodiesel production, vegetable oils are primarily used for this purpose. The choice of oilseed to be planted for biodiesel production depends on many factors, including the regional climate and soil conditions. The biodiesel industries in the US primarily use soybean oil, whereas in Europe, rapeseed is primarily used for biodiesel production. In tropical countries, biodiesel is produced from plants that grow in these tropical areas, such as palm, physic nut and castor bean.
In addition to biodiesel production using vegetable oils, the by-products generated at different steps during the production process have garnered increasing attention. Some of these by-products are generated in large amounts, making it both economically necessary and interesting to find a use for them. Currently, the residual cake, also known as the seed cake or press cake, has been shown to be a noteworthy by-product. The seed cake consists of the organic waste obtained during the oil extraction process by the pressing of seeds. Large amounts of residual cakes are generated during the oil extraction process. For example, for each ton of castor bean pressed for oil, a half-ton of cake is produced . The residual cake can be used as fertiliser because of the macro- and microelements composition. Moreover, the protein content makes it useful as a component of animal feed.
Several countries from South and Central America and Asia are attempting to use new oilseed sources for biodiesel production. Two of the oilseeds that are expected to be used for this purpose are the castor bean (
Castor bean seeds have long been known for their toxicity. They are the source of the most potent phytotoxin known, the protein ricin. Moreover, the toxic alkaloid, ricinin, is also found in the castor bean; however, this compound is different from ricin in that it is not as toxic and can easily be removed from the castor cake.
The toxin, ricin, has been known since ancient times because of its use in criminal practices. According to Olsnes , in 1887, Dixon had hypothesised that the
Ricin is a type 2 ribosome-inactivating protein (RIP) that is found exclusively in the endosperm of castor bean seeds. As a type 2 RIP, ricin is a dimeric protein comprised of an A chain (32 kDa) and a B chain (34 kDa) linked by a disulfide bond . The ricin A chain (RTA) is responsible for the enzymatic activity of the protein. This N-glycosidase enzymatic activity removes a specific adenine, depurination, (A4324) residue from a region of rRNA known as the α-sarcin/ricin loop (SRL) (Figure 1).
The absence of this adenine residue inhibits binding of the elongation factor, thereby stopping protein synthesis . The B chain (RTB) is a lectin that binds to glycoproteins and glycolipids on the cell surface and cytosol and mediates the internalisation and intracellular translocation of the toxin [10,11].
The ricin toxin is very efficient and a single molecule may inactivate 2,000 ribosomes per minute . Because ricin can be used as a bioterrorism agent , many assays to detect ricin have been described. Some of these assays are highly accurate and can detect very low concentrations of the toxin. However, there is no standard methodology to use as a quality control for castor cake detoxification processes. Many methodologies to eliminate ricin toxicity from castor cakes have been described, and there are several promising processes when economic aspects are considered . Therefore, to use castor cakes as animal feedstock, efficient methods to detect ricin toxicity after the detoxification process are needed to ensure quality control and safety before the material can be commercialised.
2.1.1. Detection of ricin
Because ricin can be used as a bioterrorism agent, the search for fast and sensitive detection methods began soon after the first studies describing the mechanism of action of ricin. The earliest proposed detection method was the enzyme-linked immunosorbent assay (ELISA) . In this assay, rabbit anti-ricin antibodies (reduced IgG and Fab’ fragments) conjugated withβ-D-galactosidase was used. Using the rabbit anti-ricin Fab'-β-D-galactosidase complex, it was possible to detect as little as 4 ng/mL of ricin with the sandwich ELISA technique. However, less sensitivity was observed when this method was utilised for determining the amount of ricin added to rabbit body fluids. In this case, the lowest concentration of ricin that could be assayed was 40 ng/mL. During the next two years, new methods based on radioimmunoassays were proposed [16, 17]. These radioimmunoassays were very sensitive and could detect 50-100 pg RTA and 500 pg RTB; however, the sensitivity was reduced to intact ricin. The matrix used for these assays consisted of 0.1% sodium azide and 0.1% bovine serum albumin (BSA) in 0.05 M sodium phosphate buffer. Limitations of these assays include the difficulties in handling radioisotopes and the long incubation period. Therefore, despite the high sensitivity of these assays, the drawbacks associated with radioimmunoassays make them less preferable than ELISA. Poli et al.  developed an enhanced colorimetric and chemiluminescent ELISA to detect ricin in biological fluids. This assay utilised an affinity-purified goat polyclonal antibody (pAb) to adsorb ricin from the solution. The same pAb was then used to form a sandwich, and avidin-linked alkaline phosphatase was used for colour development. Enhancement of the colourimetric assay was obtained because of the increased biotinylated antibody content and a reduction in the dilution ratio of the avidin-linked alkaline phosphatase. This assay could detect 100 pg/mL ricin in phosphate-buffered saline (PBS), human urine and human serum. This sandwich assay could also be used with a chemiluminescence detection reagent; however, the quantitation was limited to a range of 0.1–1 ng/mL and was subject to greater variability compared to the colourimetric assay. An ELISA using monoclonal antibodies (mAb) was performed to detect ricin in biological fluids . This method was also based on the sandwich format using an anti-ricin B chain mAb to adsorb ricin from the solution and an anti-ricin A chain mAb conjugated to peroxidase as the second antibody that is then used to form a sandwich. The peroxidase allows for colour development and measurement of optical density at 450 nm. The sensitivity of this assay is 5 ng/mL and is lower than the sensitivity reported for the amplified and chemiluminescent immunoassays . The ELISA is still used to detect ricin, and a commercial ELISA kit specific for ricin detection can be obtained . However, ELISA has several disadvantages that prevent it from being the best method to detect of ricin. ELISAs consume too much time because of the washing steps involved and they also have limited throughput. ELISAs may also underestimate the actual ricin content in situations where antigen concentrations are high (hook effect) and specialised personnel are also required to perform the ELISAs.
To reduce the time necessary to assay for ricin, a method based on a fiber-optic sensor was developed [21, 22] and optimised . A sandwich immunoassay scheme was used in which an anti-ricin IgG was immobilised onto the surface of an optical fiber. The limits of detection for ricin, as detected by laser-induced fluorescence, in a buffer solution and river water were 100 pg/mL and 1 ng/mL, respectively. The complete assay can be performed in 20 minutes.
The first immunochromatography assay to detect ricin was performed using antibody anti A-Chain mAb with two distinct specificities. An anti-RTB mAb (1G7) was immobilised to a defined detection zone on a porous nitrocellulose membrane, whereas an anti-RTA mAb (5E11) was conjugated to colloidal gold particles that worked as the detection agent . The ricin-containing mixture was added to the membrane and allowed to react with the mAb 5E11-coated particles. This mixture moved across the porous membrane by capillary action until it reach the extremity containing the anti-RTB mAbs, which bound to the particles of ricin that were attached to the gold-labelled anti-RTA mAbs. The detection limit of this assay was 50 ng/mL ricin in phosphate-buffered saline (PBS). This sensitivity could be enhanced further to 100 pg/mL with the use of a silver enhancer. The advantages of these gold particles were their superior mobility, decreased aggregation and commercial availability. An immunochromatography assay was also used to shown differences in ricin content among different castor bean cultivars . All the ricin isoforms were detected in the range of 1 to 2.5 ng/ mL in buffer.
In addition to using a better antibody for improved sensitivity, there was also a development regarding the technology of the solid phase surface of the immunoassay. The conventional microplate was exchanged for magnetic micro beads. Immunomagnetic (IM) assays to detect ricin were first used by Gatto-Menking et al. . They used immunomagnetic electrochemiluminescence (IM-ECL) to detect ricin and other toxic agents, such as botulinus A, cholera β subunit, ricin and staphylococcal enterotoxoid B. Antibody-conjugated magnetic micro beads were used to capture the target toxins and ruthenium trisbipyridal chelate-labelled antibodies were used as the reporter. High sensitivity levels were obtained for all the tested toxins. All IM-ELC assays could be performed in a maximum combined incubation and assay time of approximately 40 minutes, and the sensitivity to ricin was 5 pg/mL. Some years later, an enhanced ECL assay had a detection limit of 0.5 pg/mL for ricin in PBS . The same study demonstrated the detection of ricin by fluorogenic-chemiluninescence (FCL), and the sensitivity was 1 ng/mL. Advantages of these micro beads were due to their large surface area (Figure 2) that leads to enhanced sensitivity, to free moving microspheres coated with antibody that accelerates the reaction rates and reduces the assay time, and to easy detection using a simple magnetic field. Both the FCL and ECL had similar formats, except that the FCL used alkaline phosphatase as the label and detected the ricin through the measurement of fluorescence, whereas the ECL used ruthenium-trisbipyridal as the label and detected the ricin through photoemission. For a magnetoelastic surface sensor instead of microspheres, the detection technology was a sandwich immunoassay on the sensor surface. Biocatalytic precipitation was then used to cause a change in mass, which resulted in a change in the resonance frequency that allowed for quantitation of ricin at a detection limit of 5 ng/mL in aqueous media, such as water, blood or serum . This magnetoelastic sensor had a sensitivity that was comparable to the ELISA; however, this assay had a much lower cost, was disposable and had a relatively quick analysis time.
The search for an assay to detect several toxins simultaneously led to the use of array systems. Three different toxins, ricin, SEB and
Sano et al.  developed a method to detect antigens that combined the specificity of immunological analysis with the exponential amplification of PCR. This immuno-polymerase chain reaction (IPCR) was an interesting method to monitor the presence of ricin in samples . A schematic representation of this method is shown in Figure 3.
Ricin was dissolved at different concentrations in PBS, and detection was performed revealing a detection limit of 10 fg/mL. The assay was then performed with ricin dissolved in human serum revealing a detection limit of 0.5 fg/mL. The method has also been used for post-intoxication evaluation of the biological half-life of ricin. IPCR analysis of sera from mice fed ricin showed that the toxin was rapidly sequestered from the sera (30 minutes) with a half-life (t1/2α) of 4 minutes . The time required to complete the entire IPCR process is 9 hours. Compared with conventional immunological methods, IPCR requires a greater amount of time because of the PCR itself and the post-PCR analysis. Moreover, the use of more expensive reagents and the increased reagent consumption make this technique less attractive than conventional immunological methods. However, these limitations are counterbalanced by greater sensitivity (8 million times greater than conventional ELISA), enabling a broader range of applications.
In recent years, highly sophisticated mass-spectrometry (MS)-based methods for the detection and quantification of ricin have been developed. It was shown that ricin could be unequivocally identified by liquid chromatography-electrospray (LC-ES) MS/MS experiments with reduced, cysteine-derivatised, trypsin-digested material . It was also shown that MALDI-MS could be used to detect intact ricin and to screen samples for ricin peptides. The amount of crude sample required was a few milligrams containing less than 5% ricin. According to the authors, the selection of a few marker peptides from the A and B chains can be used as a method to improve the sensitivity and efficiency of this method. A method combining immunocapture and analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for ricin detection was also described . Ricin samples were applied to magnetic spheres coated with a monoclonal anti-B-chain antibody. After acidic elution, tryptic peptides of the A and B chains were obtained by accelerated digestion with trypsin in the presence of acetonitrile. Three of the 20 peptides obtained were used for ricin detection by MALDI-TOF MS. This assay had a limit of detection estimated at 50 ng/mL, and the result could be obtained in approximately 5 hours. These results are not as exciting compared to other more sensitive and faster methodologies; however, an interesting feature is that MS detection provides increased specificity because of the simultaneous monitoring of several characteristic ricin-specific peptides. Furthermore, the possible miniaturisation of MALDI-TOF technology suggests that the assay could be adapted for use with a portable mass spectrometer. A recent study described the combination of a multiplex-immunoaffinity purification approach followed by MALDI-based detection for the simultaneous identification of different toxins, including ricin . Selected antibodies against each toxic agent allowed for the specific and simultaneous capture of these toxins. The toxins were subsequently identified by MALDI-TOF MS following a tryptic digest, and after an assay time of 8 hours, the ricin could be detected at a minimum of 200 ng/mL. The time requirement and detection limit were not satisfactory for this assay; however, ricin could be detected in complex matrices, such as milk and juice.
Aptamers are artiﬁcial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and reampliﬁcation. Because of their high thermostability when compared with antibodies, aptamers have potential applications in analytical devices, including biosensors, and as therapeutic agents . Assays for protein identification and quantitation were developed and applied to ricin detection [39, 40]. A multiplex aptamer microarray was generated by printing an anti-ricin RNA aptamer onto either streptavidin (SA)- or neutravidin (NA)-coated glass slides. The limit of detection in a sandwich assay format after optimisation studies was 15 ng/mL in PBS. This assay was also used to detect other proteins and showed satisfactory results. Capillary electrophoresis (CE) has been shown to be a viable alternative to traditional immunoassays when coupled with laser-induced fluorescence detection. Haes et al.  demonstrated that capillary electrophoresis could be used to detect ricin by monitoring its interaction with a fluorescently tagged aptamer under non-equilibrium conditions. The quantitative response revealed a detection limit as low as 14 ng/mL. This study also revealed that the presence of nucleases in the sample leads to a slight decrease in the ability of the aptamer to detect ricin; however, it is still possible to detect the toxin at very low concentrations. This assay can be performed in less than 10 minutes, consumes minimum quantities of material, and generates a low amount of waste.
Liquid-crystal (LC) based sensors that can be used as rapid and effective detection technologies have attracted a significant amount of attention in recent years , and their utility regarding ricin detection has previously been demonstrated . This method relied on the use of LCs 5CB to amplify and report the presence of ricin captured by an affinity ligand. One merit of this approach is that the ricin can be imaged on chemically functionalised surfaces and transduced into an optical signal. The optical signal caused by the orientational transition of the LCs could easily be identified with polarised light microscopy. However, despite the success of the LC-based sensor, which did not use complex instrumentations and did not involve any labelling steps, the limit of detection of 10μg/mL was not as good compared to other methods. Similar to other assays, this interesting technology must be improved to become among the most sensitive methods for ricin detection.
Despite the many methods to detect the presence of ricin, the detection of the toxin in castor cakes subjected to detoxification is not performed in a standard manner. Anandan et al.  used different physical and chemical treatments to detoxify castor cakes, and the ricin content was determined based on electrophoretic analysis. They reported that ricin bands did not appear in SDS-PAGE samples of autoclaved (15 psi, 60 minutes) and lime treated (40 g/kg) castor cakes. Solid-state fermentation by
The greatest problem that affects not only electrophoresis, but also all the ricin detection methods described in this chapter, is the inability to detect the biological activity of the toxin. Each proposed assay can detect the presence of ricin at minimal concentrations and many of these are able to do so in a very sensitive and specific way; however, they cannot determine whether the toxin is biologically active. To validate the castor cake detoxification processes, it is important to be able to detect the biological activity of ricin. This is because some of the described toxin inactivation processes can be related to modifications in the active site of the enzyme, and although ricin may be present in processed cake, it may be not active and the product would be safe to use in animal feed.
2.1.2. Detection of ricin biological activity
The first method of detecting ricin activity was based on measuring the inhibition of protein synthesis in a rabbit reticulocyte cell-free system mediated by toxic tryptic peptides from ricin . The method was justified because of the long period of time required to observe intoxication symptoms in animals. It was reported that similar to the native protein, toxic ricin peptides could inhibit protein synthesis in a cell-free system. This information reinforces the necessity for assaying ricin biological activity after subjecting the castor cake to detoxification processes.
The ability of the RIPs in inhibit protein synthesis can be monitored with
The inhibition of protein synthesis was also the target of a method to detect ricin in a “well-in-well” device . The miniaturised system presented a mechanism to supply nutrients continuously and remove by-products, leading to higher protein expression yields and larger detection signals. This method showed a detection limit of 0.3 ng/mL ricin. The nested-well device was also used for measuring the toxicity of ricin after physical or chemical treatment. The good results obtained with inactivated ricin make this method a good choice for use in castor cake detoxification processes.
The N-glycosidase activity removes an adenine residue from the α-sarcin/ricin loop of rRNA. The removed adenine can be used as a positive indicator of biologically active ricin. The most common method for quantifying free adenine in a variety of applications is the detection of fluorescent-derivatised adenine by HPLC . To detect ricin activity based on rRNAdepurination, a high-throughput, enzyme-based colorimetric adenine quantification assay was developed . The key step of this assay is the conversion of adenine to AMP and concurrent release of pyrophosphate from PRPP. Pyrophosphate is then cleaved to phosphate by inorganic pyrophosphatase. To enhance the signal, the AMP formed is converted by 5’-nucleotidase to adenosine and inorganic phosphate, finally resulting in three phosphates for each adenine. Inorganic phosphate was quantified by a modified procedure with a commercially available kit. All four enzyme reactions of the assay, including colour development, occur simultaneously in approximately 15 minutes inside the same reaction tube, and the rate of adenine released by the commercially obtained RTA was determined to be 43 pmol adenine/pmol RTA per hour.
Recently, several methods using electrochemiluminescence (ECL) to detect ricin activity were also developed [52, 53]. First, a deadenylation assay using paramagnetic beads could detect ricin in crude extracts [52, 54]. Synthetic biotinylated RNA substrates were cleaved by the combined actions of the ricin holotoxin and a chemical agent, N,N’-dimethylethylenediamine. The annealing of the product with a ruthenylatedoligodeoxynucleotide resulted in the capture of ruthenium chelate onto magnetic beads, enabling the electrochemiluminescence (ECL)-based detection of RNA N-glycosidase activities of toxins. Compared to ECL immunoassays , the ECL activity assay presented lower sensitivity, reaching a detection limit of 100 pg/mL. The disadvantage of the ECL immunoassay compared to the ECL activity assay is that the antibodies recognise surface features of the proteins (epitopes) that may be unrelated to any enzymatic activity or other mechanism of toxicity. Therefore, it may be possible for inactive protein toxins to cause positive signals in these immunoassays resulting in an over-estimation of the threat. The plate-based assay unlike the bead-based assay, included wash steps that enabled the removal of food particles, thereby maximising the matrix effects and improving the limits of detection. The limits of detection for ricin in apple juice, vegetable juice, and citrate buffer using the bead-based assay were 0.4, 1, and 0.1 μg/mL, respectively. By contrast, the limits of detection for ricin using the plate-based assay were 0.04, 0.1, and 0.04 μg/mL in apple juice, vegetable juice, and citrate buffer, respectively. These data suggest that the plate-based assay is the best method for detecting ricin activity by ECL.
The ricin detection methods based on adenine liberation and direct infusion electron spray ionisation mass spectrometry have been shown to provide rapid, selective, and sensitive detection of various peptides and small nucleic acids, and these methods should provide a sensitive method for the real-time analysis of RIP enzymatic activity by monitoring adenine release. Therefore, high-performance liquid chromatography (HPLC) and selected ion monitoring mass spectrometry (MS) were used to develop a quantitative assay for adenine release from a synthetic RNA substrate by the ricin A chain . The sensitivity of this MS assay made it possible to measure RIP activity at approximately 0.6- to 600 ng/mL. A more specific assay to detect ricin by MS was developed by Becher et al.  in which they used an anti-B chain mAb immobilised on magnetic beads to capture the toxin. Ricin toxicity was measured through quantification of the free adenine by HPLC-MS. The immunoaffinity step combined with enzymatic activity detection led to a specific assay for the entire functional ricin protein with a lower limit of detection of 100 pg/mL.
When mass spectrometry was used to detect ricin activity, a combination of three techniques, all performed on the same sample, provided a sensitive and selective analysis of ricin isolated from a food or clinical sample and measured the activity of the toxin . First, ricin was isolated from abundant proteins in a food or clinical sample, such as milk, apple juice, serum or saliva through immunoaffinity capture on antibody-coated beads. Second, the activity of ricin was examined through interaction of the toxin with a DNA substrate that simulated the
The mass spectrometry based methods for detecting ricin activity through monitoring adenine liberation have some disadvantages that make them not suitable for use in the validation of the detoxification processes of the castor cakes. These disadvantages include complications regarding the handling of mass spectrometers and the interpretation of results that requires highly specialised personnel. Another problem is that adenine liberation may not be the most efficient method to detect biologically active ricin because depurination activity is not a unique mechanism involved in ricin toxicity. It was previously shown that non-cytotoxic RTA mutants could depurinate ribosomes in yeast cells without the occurrence of cell death and apoptosis signals .
Toxicology assays to detect ricin based on the activity against animals could be the best way to evaluate the efficiency of castor cake detoxification processes because of the desire to use this by-product as animal feedstock. However, despite the ethical questions surrounding the use of
The possibility of using cell culture models to evaluate ricin toxicity by colorimetric assays, such as the LDH assay, seem to be a good idea for use as a biological test to determine the efficiency of the castor bean cake detoxification process. It was reported that solid-state fermentation (SSF) reduced the ricin levels in castor bean alkaline waste from Petrobras (the national petroleum company of Brazil) during the biodiesel production process [45, 62]. This was determined by molecular exclusion chromatography and electrophoresis. To verify the biological activity of ricin after SSF at different time intervals, an
When the cell counting and LDH assays were compared to determine the cytotoxicity of ricin against Vero cells, it was reported that both methods are efficient and detected ricin at a minimum concentration of 10 ng/mL . After adjusting the method to detect the purified protein, they used the Vero cell cytotoxicity assay to evaluate the following two castor cake detoxification processes: SSF using
Two main toxic components are present in the physic nut plant, the ribosome-inactivating protein, curcin, and phorbol esters. Among these toxins, the phorbol esters are the most dangerous toxic components in
Curcin (28.2 kDa) is a type 1 RIP that is found in
3.2. Phorbol esters
Phorbol esters (PE) are polycyclic compounds in which two hydroxyl groups in neighbouring carbons are esterified to fatty acids, and these substances are present in many different plants, including
The PEs and their different derivatives are known for their tumour induction activity. They activate protein kinase C (PKC), which plays a critical role in signal transduction pathways and regulates cell proliferation . By contrast, it was reported that some types of PEs could induce apoptosis .
Several detoxification processes used to eliminate PEs from Jatropha cake have been previously described , and some of the existing detection methods were used to confirm the effectiveness of these processes.
3.2.1. Detection of phorbol esters
Many of the phorbol ester detection methods are related to using Jatropha cake as animal feedstock, which are different from ricin containing cakes that can be used as bioterrorism agents. Therefore, there are few techniques for PE detection compared to ricin detection methods.
The determination of irritant activity caused by phorbol esters was first demonstrated by Adolf et al. . The irritant activity of PE isolated from different Jatropha species was assayed in rat ears, and the irritant dose 50 for
The most commonly used method to detect and quantify PE from
Similar to ricin detection methods, the biological activity of phorbol esters must to be assayed to guarantee the efficiency of the Jatropha cake detoxification processes. Because Jatropha cake is used as feedstock, quality control of detoxification processes is often performed using live animals, such as rats [77, 79]), sheep , pigs  and fish [97, 98]. With a few exceptions, this type of biological activity control is usually preceded by RP-HPLC detection and quantification of PEs. Therefore, it remains necessary to continue using RP-HPLC and sacrificing animals to detect the presence and biological activity of PEs because toxicity evaluation using live animals is not the best method for use on a large scale. Other biological tests have previously been described for assaying PE toxicity, and some of these assays are very sensitive and simple to perform on a large scale.
Earlier reports regarding
The efficacy of phorbol esters against insects has been shown recently. Termites (
Some crustaceans are widely used as toxicity indicators in bioassay systems. Phorbol ester toxicity has previously been assayed to
Similar to the molluscicidal, insecticidal and antiparasitic activity, PE toxicity against microorganisms was also reported. It was demonstrated that phorbol esters from
Because PEs are activators of protein kinase C (PKC), a biochemical assay to detect PEs based on this property was described . In this method, PKC is incubated with Mg-ATP and a synthetic peptide which is labelled with a fluorescent dye. When a PKC activator is present, the active enzyme phosphorylates the peptide. When the reaction mixture is separated by electrophoresis, the phosphorylated peptide becomes negatively charged and migrates to the positive pole. The fluorescently labelled peptide can then be quantified by densitometric analysis. This assay was used by Wink et al.  to determine the activity of PEs sequestered by
Although many methods have been described to detect
Currently, several processes to detoxify castor bean and Jatropha cakes have been developed however, it is essential to choose a method that is universally accepted to validate such processes of detoxification. The literature indicates that the method to be used to evaluate the toxicity of castor cake is different from what should be used for jatropha cake.
Among the different methods that can be used to assess the presence of ricin some are more suitable to control attacks bioterrorist. They are sensitive methods that detect the presence of ricin, but need not evaluate the biological activity.
In this review, methods based on Vero cell viability are best suited to validate the processes of castor cake detoxification. Vero cells, epithelial cell line isolated from African green monkey are indicated since these cells maintain cell organelles characteristics and stable structure when in contact with the cake detoxified. Evaluation procedures for Jatropha are still under development. The detection of phorbol esters by reverse phase chromatography, associated with toxicity tests on snails are recommended.