Chemistry of Phorbol Ester Toxicity in Jatropha curcas Seed – a Review

The toxicity of Jatropha curcas seeds emanates mainly from phorbol esters. It is known that the seeds are a valuable source of oil, which can be used to produce biodiesel. The cake generated as a by-product, after oil pressing is a protein-rich potential stock for animal feed. Researchers, the world over, have studied these phorbol esters and investigated ways of improving the applications of the seed meal. This review paper outlines the previous research done on the Jatropha curcas plant, the toxicity of phorbol esters and the efforts made to detoxify the seed. It also highlights the knowledge gaps concerning the chemistry of phorbol ester toxicity and the probable areas of future research.

There are many other uses of J. curcas. Traditionally, the plant has been used as a hedge and living fence, not edible by livestock. Growing Jatropha reclaims eroded and waste land. The seed oil can be used as a fuel for lighting and is a raw material for making high quality soap. Residue from seed pressing is good organic fertilizer [17]. All parts of J. curcas (seeds, leaves and bark) have been used in traditional medicine and for veterinary purposes for a long time [1,18]. This is typical of most members of the Euphorbiaceae family, whose medicinal properties may be due to stress factors such as drought and extreme temperatures that induce synthesis of survival and defence chemicals [19]. A 2% etheral solution of J. curcas seed oil was observed to have insecticidal properties against Callosobruchus maculatus -a seed beetle in cowpeas [20]. Just 0.05% methanolic solution of the seed oil caused a 70% larval mortality for Cnaphalocrocis medinalis -rice leaf folder and Helicoverpa armigera -cotton boll worm [21]. Ethanolic root extract of J. curcas was found to have antibiotic effect on Escherichia coli, Neisseria gonorrhoea and Staphylococcus aureus, with a minimum inhibitory quantity of 0.5ml [22]. The toxicity of J. curcas to mollusks has been investigated because of its relevance in schistosomiasis control [23]. Methanolic extracts of J. curcas seed oil showed toxicity against tested organisms, with LC 100 values (concentrations killing 100% of the organism) of 25ppm for cercariae and the snail Biomphalaria glabrata and 1ppm for the snails Bulinus truncatus and B. natalensis [24]. Phorbol esters were seen to be the active compounds in activity against snails [25]. At least 1% ethanolic solution of crude extract caused complete inhibition of mycelial growth of Fusarium oxysporum, Pythium aphanidermatum, Lasiodiplodia theobromae, Curvularia lunata, Fusarium semitectum, Colletotrichum capsici and Colletotrichum gloeosporioides [26].

PHORBOL ESTERS
The term 'phorbol esters' is used today to describe a naturally occurring group of compounds mainly distributed in plant species of the Euphorbiaceae family. Phorbol esters are esters of phorbol, a tetracyclic diterpenoid with a tigliane skeletal structure [27]. A B

C D
Phorbol itself was isolated as a non-toxic crystalline solid from the plant, Croton tiglium L. [28], but its ester, TPA (12-O-tetradecanoylphorbol-13-acetate), present in croton oil, is the most toxic and most studied phorbol ester, known to be a co-carcinogen [29,30]. However, phorbol esters from J. curcas are derivatives of 12-deoxy-16-hydroxyphorbol [31]. Phorbol esters with the same diterpenoid moiety have also been extracted from the latex of Euphorbia cooperi [32,33].
Adolf et al. [34] were the first to identify phorbol esters in J. curcas. Although they concluded that the irritant principles of the plant represented 12-deoxy-16-hydroxyphorbol-13-acylates with highly unsaturated acid moieties, they could not elucidate their exact chemical structures. Hirota et al. [35] identified the macrocyclic, dicarboxylic acid, diester structure of one of the phorbol esters and showed that it is a tumor promoter with weaker activity than TPA. Later, Haas et al. [31] isolated and did a thorough structural determination on all phorbol esters found in J. curcas.
Six phorbol esters have been characterized from J. curcas seed oil and designated as Jatropha factors C 1 , C 2 , C 3 , epimers C 4 & C 5 and C 6 , with the molecular formula C 44 H 54 O 8 . All of them are intra-molecular diesters of the same diterpenoid. Jatropha factor C 6 was found to be the least stable. It has been suggested that the diester group biosynthetically arises from two originally separated monoacids [31]. The full structure of jatropha factor C 3 is shown below; the other esters are given without the 12-deoxy-16-hydroxyphorbol portion. Jatropha factor C 6 The content of phorbol esters in J. curcas can vary according to the region in which the plant grows. Ahmed and Salimon [36] have shown a variation in the phorbol ester content of the J. curcas seed oil from Malaysia (0.23%), Indonesia (1.58%) and India (0.58%). Saetae and Suntornsuk [37] demonstrated a variation in phorbol ester content in J. curcas seeds from different provinces of Thailand. However, Makkar et al. [7] had previously found similar levels of phorbol esters in seed kernels from Cape Verde (0.27%), Nicaragua (0.22%) and Nigeria (0.23%).
Although the phorbol esters are distributed in different parts of the J. curcas plant, they are concentrated in the seed kernel, of which 70% of them report to the oil and the remainder being found in the deoiled seed cake [8]. Hipal et al. [38] analyzed J. curcas seeds collected from various regions of Gujarat, India; the phorbol ester content in the oil had an average of 4.56%, whereas that of the seed cake was 1.21%. Studies carried out by Devappa et al. [39] showed that 86% of kernel phorbol esters are localized in the endosperm.

PHORBOL ESTER TOXICITY
Phorbol esters are the most potent tumor promoters known. They exhibit a remarkable ability to amplify the effect of a carcinogen but are themselves not carcinogenic [40]. Their promoter dose can be as low as 5 nanomoles [41]. The seeds from J.curcas have been reported to be orally toxic to humans, rodents and ruminants and phorbol esters have been identified as the main toxic agent [42]. Pure phorbol esters can kill when administered in quantities of micrograms [1]. Symptoms of intoxication in humans are: burning and pain in mouth and throat, vomiting, delirium, muscle shock, decrease in visual capacity and high pulse [6]. In Nigeria and Burkina Faso, Jatropha seeds and seed oil are added to Strophanthus arrow poison, whereas in Gabon the seeds are grated with palm oil to kill rats [6]. The Shambaa of Usambara region of Tanzania used Jatropha seeds as an ordeal poison [43]. According to Adam and Magzoub [44], toxicity to goats was recorded, with doses of seed at 10g/kg per day having fatal consequences in two days. Post-mortem examination of the goats revealed hemorrhage in the rumen, reticulum, kidney, spleen and heart [44]. Ahmed and Adam [45] studied toxicity of J. curcas seeds on calves; they found that feeding the animals at doses of 0.25g/kg caused deaths within 19 hours. Some of the symptoms of toxicity were abdominal pain, salivation, diarrhea, sunken eyes and dehydration [45]. Common symptoms of topical application of isolated phorbol esters to rabbits, mice and rats are erythema, oedema, necrosis, diarrhea, scaling and thickening of skins [46]. Ingestion of phorbol esters (LD 50 for mice being 27mg/kg body mass) can cause lung and kidney damage, resulting in fatality [47].
A summary of the effects of phorbol esters on different animals is given in Table 1 below. Ingestion Lower metabolic rate, increase in fecal mucus production, rejection of feed [42] The toxicity of phorbol esters is mainly due to their action on biological cell membranes [23]. The phorbol esters are amphiphilic molecules and have a tendency to bind to phospholipid membrane receptors. The receptors are usually the primary targets of the phorbol esters [23]. The most investigated activity of the phorbol esters is their binding and activation of protein kinase C (PKC), an enzyme that plays a critical role in signal transduction pathways and also regulates cell growth and differentiation [49,50,51]. It has been proposed that the phorbol esters convert PKC into a constitutive active form that is irreversibly inserted into the membrane [49]. During normal signal transduction, the enzyme is activated by diacylglycerol, which is then rapidly hydrolyzed. Upon activation, PKC enzymes are translocated to the plasma membrane to conduct various other signal transduction pathways [52]. The phorbol esters act as analogues for diacylglycerol and are stronger PKC activators that are hardly metabolized by the cell [51]. They hyperactivate PKC and trigger cell proliferation, thus amplifying the efficacy of carcinogens [23].
Because phorbol esters activate PKC, they affect insulin binding by stimulating phosphorylation of insulin receptors on the protein [53].
The relationship between the chemical structure and toxicity of phorbol esters has received considerable attention from scientists, without conclusive results. For instance, the lack of toxicity of phorbol, as contrasted to its esters, both in vivo and in vitro had strongly indicated that adequate hydrophobicity was one of the requirements for activity [54]. Studies on effects of phorbol myristate acetate (PMA), also known as PTA, in mouse skin showed that its metabolite, phorbolol myristate acetate (PHMA) was nearly as effective a tumor promoter as PMA [55] -here, the carbonyl group in PMA is converted to a hydroxyl group to give PHMA.
It has been observed that shorter chain derivatives of phorbol are irritant but not promoting, these being two biological effects that could be considered as independent [59]. The presence of a C 20 hydroxyl group has been seen to be important for the irritant and tumor promoting activities of phorbol esters. Introduction of an acetyl group in the 20-position gives rise to lower irritancy [60]. Therefore, it may be postulated that the spatial position of this function, as determined by their particular carbon skeleton and relative to specifically binding cellular structures, may play a delicate role in the hydrophilic interaction of the esters with membranes and receptors [41]. One way to weaken or abolish tumor promoting efficacy of a phorbol ester without impairing its mitogenic and irritant activities is to introduce double bonds into the long chain fatty acid residue at position 12 of the phorbol moiety [61].
Although many biological activities of phorbol esters are mediated through their direct interaction and activation of PKC, there are some phorbol esters that are active without being tumor promoters, such as ostodin and prostratin [62,63]. Studies by El-Mekkawy et al. [63,64] reported that 12-O-acetylphorbol-13-decanoate potently inhibits the cytopathic effects of human immunodeficiency virus (HIV) type 1 without appreciable activation of PKC.
Phorbol, the parent diterpenoid of phorbol esters, contains five hydroxyl groups with different reactivities towards acylation (Hecker, 1987). The three most widely used, biologically active phorbol esters, TPA (4β-12-O-tetradecanoylphorbol-13-acetate), PDBu (4β-phorbol-2,13dibutyrate) and PMA (phorbol 12-myristate-13-acetate), differ only by their substitutions at positions 12 and 13 of the 6-membered ring. Their epimeric α-phorbol esters, which are often used as inactive controls, differ on the OH group at position 4, thus affecting the spatial arrangement of the cyclopentenone ring. [65] Information about structure of PKC has been limited by its large size, which makes nuclear magnetic resonance (NMR) structural analysis quite difficult. It should be emphasized that the competitive binding to PKC, exhibited by diacylglycerol and the different phorbol esters, does not require that they possess similar recognition subunits. They could be interacting differently with functionality in the receptor domain. [40]

DETOXIFICATION
The J. curcas seed cake is a by-product of oil extraction from the seed. Although the seed cake has been found to be protein-rich, its utilization as animal feed is hindered by the presence of toxic phorbol esters and the antinutritional factors that include lectin (curcin), saponins, phytic acid, and trypsin inhibitor [6,12,66]. According to Makkar et al. [4], lectin and trypsin inhibitor can be deactivated by heat treatment, whereas phytic acid can be diminished by addition of phytase. However, phorbol esters must be removed from the seed cake to ensure complete detoxification. Several biological, chemical and physical detoxification methods have been suggested.
Makkar et al. [12] showed that roasting of seeds inactivates trypsin inhibitor and reduces lectin activity by 50%, on average. However, saponins, phytic acid and phorbol esters are not affected by roasting. In Mexico, Quintana Roo state, non-toxic J. curcas seeds are consumed by humans, after roasting. The consumption of raw seeds is known to produce cramps and uneasy feeling in the stomach [12]. It is not possible to destroy phorbol esters by heat treatment because they are heat stable and can withstand roasting temperature as high as 160ºC for 30 minutes [67].
Complete degradation of phorbol esters by stripping or deodorization of J. curcas oil at 260 o C, with 3 mbar pressure and 1% steam injection has been reported by Makkar et al. [68].
El Diwani et al. [69] claimed a 75% removal of phorbol esters in J. curcas seed cake by treatment with an equal portion of 0.2M NaHCO 3 , followed by addition of 50mg/l ozone for 3 minutes.
Devappa and Swamylingappa [70] reported total elimination of phorbol esters during preparation of protein isolates from J. curcas kernel meal. They suggested that the effectiveness of phorbol-ester removal could be due to the processing steps such as steam injection, washing and removal of oil in the whey. Solvent extraction is among the most successful means of detoxifying J. curcas seed cake. Chivandi et al. [71] reported that a hexane/ethanol solvent system reduced phorbol ester content in the seed meal by 70%. Sequential extraction involving non-polar solvent followed by polar solvent has been recommended by Gaur et al. [72]. Approximately 80% of phorbol esters present in Jatropha oil were extracted using methanol, in a single extraction of 1:2 (w/v) oil-methanol ratio [73]. Aqueous ethanol (1:5 w/v) was reported by Makkar and Becker [16] to have removed 93% phorbol esters in Jatropha seed cake, after four extractions. Saetae and Suntornsuck [26] claimed that 90% ethanol was just as effective as 99.5% methanol for complete phorbol ester extraction from J. curcas seed cake. They preferred ethanol since it is safer for humans and other animals than methanol. Ethanol was also found to be efficient in removing lectin and reducing saponin, phytic acid and trypsin inhibitor in the seed cake [74]. Research done by Martínez-Herrera et al. [75] indicated that extraction with ethanol, followed by treatment with 0.07% NaHCO 3 decreased phorbol ester content by 98% in Jatropha seeds; there was also a considerable decrease in lectin activity. Organic solvents (alkaline methanol) were used by Makkar and Becker to reduce phorbol esters in J. curcas kernel meal to undetectable levels (< 5µg/g); their procedure was patented in 2010 [76].
Makkar and Becker [77] reported that rumen microbes cannot degrade phorbol esters and ruminants, like cattle, are as prone to phorbol ester toxicity as monogastric animals. Hydrolytic enzymes such as phorbol-diester hydrolase in mouse plasma [78] and liver carboxyesterase [79] have been reported to degrade phorbol esters.
Devappa et al. [80] have demonstrated that Jatropha phorbol esters are biodegradable in the soil, within nine days, at 42ºC and 230g/kg moisture. The degraded products are innocuous. Degradation rates of phorbol esters in the soil are influenced by temperature and moisture levels. The occurrence of storage fungi in J. curcas seeds points to the use of these microorganisms in detoxifying the seed meal. Different species of Aspergillus, Penicillium, Mucor and Rhizopus were isolated from stored Jatropha seeds [81]. Solid-state fermentation of J. curcas seed cake, using various microorganisms is a technique that has received attention from researchers. Here, the method constitutes a fermentation process performed on a non-soluble material that acts both as physical support and source of nutrients in the absence of free flowing liquid [82]. The same technique had been employed to eliminate or reduce antinutritional factors in other plants, such as saponin from whole soybean tempe by Rhizopus oligosporus, caffeine and tannins from coffee husk by Aspergillus sp. and gossypol from cotton seed by Candida tropicalis [83].
Belewu and Akande [84] studied the effect of Aspergillus niger and Penicillium sp. treated J. curcas kernel cake, as stock feed for West African dwarf goat. They concluded that fungitreated seed meal can partially replace soybean cake for growing goat by as much as 50%. Other work by Belewu and Sam [85] showed that solid-state fermentation of J. curcas kernel cake with Aspergillus niger, for 7 days, achieved a 77% reduction in phorbol esters. Rosa et al. [86] reported that Aspergillus niger is not only a detoxifier but a nutritional enrichment additive. Joshi et al. [83] have claimed that phorbol esters can be completely degraded in 9 days by Pseudomonas aeruginosa PseA, under optimized solid-state fermentation of deoiled J. curcas seed cake. Studies by de Barros et al. [87] showed that incubation of J. curcas seed cake with the white-rot fungi, Bjerkandera adusta and Phlebia rufa, for 30 days, decreased phorbol esters by 91% and 97%, respectively. Phengnuam and Suntornsuck [88] performed submerged fermentation experiments of J. curcas seed cake with Bacillus licheniformis (GRAS), in distilled water, for 5 days and observed a reduction in phorbol esters by 62%.

FUTURE RESEARCH
The role of chemists in finding the most appropriate detoxification method for the J. curcas seed cannot be overlooked. Research on structure-toxicity relationships and detoxification mechanisms of phorbol esters is important. Investigations on possible chemical detoxification methods are also useful.

Structure-Toxicity Relationship
The active sites that are responsible for phorbol ester toxicity are still unknown. Conflicting suggestions have been made about the polarity [41,54], conformation [40,65] and critical groups [41,60,61] being contributory for toxicity in these compounds. However, the universally agreed configuration and functional groups of phorbol esters that make them toxic have not yet been determined, considering that the Jatropha molecules are larger than those previously studied. More work is required to characterize the appropriate structure or particular groups that give rise to toxic properties of phorbol esters.

Detoxification Mechanisms
Biological methods of detoxification, where microorganisms are used, imply that reduction of phorbol esters occurs by degradation. The same applies to the physical detoxification methods, such as the deodorization process described by Makkar et al. [68] and chemical methods that involve digestion with reagents like those proposed by El Diwani et al. [69]. Despite their various degrees of success, such detoxification processes should be used to determine the mechanisms of degradation of phorbol esters. Solvent extraction techniques do not involve phorbol ester degradation.

Chemical Detoxification Methods
Detoxification of the J. curcas seed by chemical digestion can be quite fast and economically efficient. The major challenge, however, could be the generation of secondary toxins. Another option would be to identify and isolate an enzyme that quickly degrades the phorbol esters; this protein can then be produced in bulk.

CONCLUSION
The toxicity of J. curcas seeds emanates mainly from phorbol esters. Their structure-toxicity relationships have not been well studied. Biological, physical and some chemical detoxification methods of Jatropha seed have been proposed, but further research on detoxification mechanisms and chemical detoxifying methods is necessary.