TY  - THES
T1  - Isolation, characterization and potential agro-pharmaceutical applications of phorbol esters from Jatropha curcas oil
A1  - Devappa,Rakshit K.
Y1  - 2012/02/27
N2  - Biodiesel is generally prepared from renewable biological sources such as vegetable oils by transesterification.  Jatropha curcas seed oil is a promising feedstock for biodiesel production. During biodiesel production from Jatropha oil, many co-products such as glycerol, fatty acid dis-tillate and seed cake, among others, are obtained. The efficient use of these co-products would enhance the economic viability of the Jatropha based biofuel industry. However, the possible presence of phorbol esters (PEs) in these co-products restricts their efficient utilization. During biodiesel production, Jatropha oil is subjected to many treatments (stripping, degumming and esterification) wherein PEs present in the oil undergo partial or complete destruction depending on the treatment conditions. One of aims of this study was to develop and integrate methodolo-gies for using the PEs as a value added product instead of simply allowing them to be destroyed during biodiesel production. Potential uses of the phorbol ester enriched fraction (PEEF), ob-tained from Jatropha oil in agro-pharmaceutical applications were also investigated. The reason for choosing this group of compounds (PEs) was that they are highly bioactive both in vitro and in vivo, but they are currently considered to be merely toxic, unwanted biomaterial in the Jatro-pha biodiesel production chain.  The recent increase in the cultivation of Jatropha cultivation means that there are potentially huge quantities of PEs that could be used for many purposes.
This study revealed that a large proportion (85.7%) of PEs was localized in the endosperm portion of the Jatropha seed. Interestingly, the kernel coat contained PEs in high concentration. The endosperm portion of the kernel also contained antinutritional factors such as phytate (96.5%) and trypsin inhibitor (95.3%). The presence of high levels of antinutritional/toxic com-ponents in the kernel was presumed to be one of the factors that protect Jatropha seeds against predatory organisms during post harvest storage. 
Based on the presence or absence of PEs, a qualitative method was developed to differentiate between toxic/nontoxic Jatropha genotypes. In this method the methanol extract of seeds is passed through a solid phase extraction (SPE) column and the absorption (280 nm) of the result-ing eluate is measured. After screening Jatropha seeds collected from different parts of the world for toxic and non-toxic genotypes using the pre-established HPLC method for PEs, a cut off value of the absorbance was set up to differentiate toxic and nontoxic genotypes. Raw kernels whose SPE eluates had an absorbance ≥0.056 were considered as toxic and ≤0.032 as nontoxic. Corresponding absorbances for the SPE eluates of defatted kernel meal were ≥0.059 (toxic) and 	≤0.043 (nontoxic). However, confirmation of the presence of PEs especially in Jatropha products for food applications should be carried out using the pre-established and validated HPLC method. The developed qualitative method could find its applications for screening the toxicity of products and co-products obtained from the Jatropha biodiesel industry. 
Conditions were optimized for the extraction of PEs as a phorbol ester enriched fraction (PEEF) from Jatropha oil using methanol as a solvent and a magnetic stirrer/Ultra-turrax as ex-traction tools. The extent of PE reduction in Jatropha oil was >99.4% using methanol as the sol-vent. The PEEF obtained (48.4 mg PEs/g) was 14 fold higher in PEs than in the original oil and this fraction was highly bioactive as determined by the most sensitive snail bioassay (LC100, 1 ppm) (see below). As the removal of PEs from oil took 60 min, which might be considered a long time in an industrial process, further conditions were optimized to extract maximum PEs in the shortest possible time with minimum solvent. The tools used for PE extraction (Ultra-turrax and magnetic stirrer) were effective with a treatment time of 2 and 5 min, resulting in 80 and 78% extraction of PEs, respectively. The biodiesel prepared from both the residual oils met European (EN 14214:2008) and American biodiesel standard (ASTM D6751-09) specifications. It was evident from the study that PEs could be easily extracted by either of the two methods with a high yield and the residual oil could be processed to produce high quality biodiesel. Also the residual oil with a lower PE content is expected neither to harm the environment nor the workers who had to handle it.
The extracted PEEF was evaluated for its agricultural potential as a bio control agent. The PEEF had a high biological activity in aquatic bioassays using snails (Physa fontinalis), brine shrimp (Artemeia salina) and daphnia (Daphnia magna), when compared with microorganisms. The EC50 (48 h) of the PEEF was 0.33, 26.48 and 0.95 ppm PEs for snail, brine shrimp and daphnia respectively. High MIC (minimum inhibitory concentration) values (≥215 ppm) and EC50 values (≥58 ppm) were obtained for both the bacterial and fungal species. Among the bio-assays tested, the snail bioassay was the most sensitive, producing LC100 at 1 μg of PEs/ml. The snail bioassay could be used to monitor the presence of PEs in various Jatropha derived products, contaminated soil and other matrices in the ecosystem that might be involved in the production or use of Jatropha and its products. The study also demonstrated that the PEs exhibit molus-cicidal, antifungal and antibacterial activities.
The shelf life of the PEEF was investigated. The PEEF was more susceptible to degradation when stored at room temperature (50% degradation after 132 days) than when stored at 4 °C or -80 °C (8% and 4% degradation respectively). Similarly, the PEEF lost biological activity (the snail bioassay) more rapidly at room temperature becoming ineffective after 260 days; while at 4 °C and -80 °C, only 27.5% and 32.5% activity was lost after 870 days. The degradation of PEs was due to auto-oxidation. Changes in fatty acid composition, increase in peroxide value and decrease in free radical scavenging activity of the PEEF reflected the auto-oxidation. Inclusion of antioxidants as additives (butylated hydroxyanisole (BHA), Baynox and α-tocopherol) pro-tected the PEs against degradation. The study demonstrated that the PEEF was susceptible to oxidation and addition of antioxidant stabilised the PEs during storage.
In soil, PEs present in both the PEEF (2.6 mg/g soil mixture) (silica was used to adsorb PEs) and Jatropha seed cake (0.37 mg/g soil mixture) were completely degraded as the temperature and moisture content of the soil increased. PEs from silica-bound PEEF were completely de-graded after 19, 12, 12 days (at 13% moisture) and after 17, 9, 9 days (at 23% moisture) at room temperature (22	−23°C), 32 °C and 42 °C respectively. Similarly, at these temperatures, PEs from seed cake were degraded after 21, 17 and 17 days (at 13% moisture) and after 23, 17, and 15 days (at 23% moisture). The toxicity of PE-amended soil extracts when tested using the snail bioassay decreased with the decrease in PE concentration. The study demonstrated that PEs pre-sent in the PEEF or Jatropha seed cake are completely biodegradable in soil and the degraded products are innocuous.
In preliminary studies, the PEEF exhibited potent insecticidal activity against Spodoptera frugiperda, which is a common pest in corn fields damaging maize crop across the tropi-cal/subtropical countries such as Mexico and Brazil. The PEEF produced contact toxicity with an LC50 of 0.83 mg/ml (w/v). The PEEF at higher concentration (0.25 mg/ml, w/v) also reduced food consumption, relative growth rate and food conversion efficiency (FCE) by 33%, 42% and 38% respectively. The study demonstrated that the PEEF has a potential to be used as a bio-control agent. Further in-depth field experiments on the effects of the PEEF on S. frugiperda will pave the way for its use under field conditions.
The pharmaceutical potential of Jatropha PEs was also investigated. The PEs from Jatropha oil were purified. At least six purified PEs (designated as factors C1 to C6) were present in Jatropha oil. The identities of the purified PEs (factors C1 and C2) were confirmed by NMR. Whereas, factor C3 and factors (C4 + C5) were both obtained as mixtures. However, comparison of peak areas for phorbol 12-myristate 13-acetate (PMA) and Jatropha factor C1 in the HPLC method showed a difference in sensitivity of absorption at 280 nm of 41.3 fold. All the individual purified Jatropha PEs (factors C1, C2, C3mixture and (C4+C5)) and PEs-rich extract (factors C1 to (C4 + C5)) were biologically active when tested in the snail and brine shrimp bioassays. In ad-dition, all the Jatropha PEs produced platelet aggregation in vitro with an effective order of (based on ED50 (&mu;M)): Jatropha factor C2 < factor C3mixture < factor C1 < factor (C4+C5). The PEs-rich extract (contains factor C1 to C6) was toxic to mice upon intra gastric administration, with an LD50 of 27.34 mg/kg body mass as PMA equivalent or 0.66 mg/kg body mass as factor C1 equivalent. The prominent histopathological symptoms were observed in lung and kidney. 
The Jatropha purified PEs-rich extract, purified PEs (factor C1, factor C2, factor C3mixture and factors (C4+C5)) and toxic Jatropha oil produced severe cellular alterations/disintegration of the epithelium and also increased the inflammatory response (interleukin-1&alpha; and prostaglandin E2 release) when applied topically to reconstituted human epithelium (RHE) and human corneal epithelium (HCE). In RHE, the nontoxic oil (equivalent to the volume used for toxic oil) pro-duced a lower cellular and inflammatory response than the toxic oil and the response increased with an increase in concentration of the PEs. In HCE, nontoxic oil (equivalent to the volume used for toxic oil) produced marked cellular alterations. The study demonstrated that the pres-ence of PEs in Jatropha oil increased the toxicity, both towards RHE and HCE. In addition, all the purified Jatropha PEs gave positive responses in the tumour promotion assay and negative responses in the tumour initiation assay in vitro (the assay was based on foci formation in Bhas 42 cells). In the tumour promotion assay, the order of transformed foci/well formation was: PEs-rich extract > factor (C4+C5) > factor C3mixture > factor C1 > factor C2. The tumour promotion ac-tivity was mediated by the hyper activation of protein kinase C (PKC). The aforementioned studies demonstrated that Jatropha PEs are toxic when administered orally or when applied topically to the skin or eye tissues. The data obtained should help in establishing safety measures for people working with Jatropha PEs.
The potential of Jatropha PEs as a feedstock intermediate for the synthesis of Prostratin, a promising adjuvant in anti HIV therapy, was evaluated. The studies demonstrated that the Jatro-pha PEs could be synthesized sequentially by converting them first to crotophorbolone and then to prostratin. As analyzed by Nano-LC-ESI-MS/MSR, the prostratin synthesized from Jatropha PEs had similar mass and peak retention time to the reference prostratin (Sigma, St. Louis), The study showed that prostratin could be synthesized from Jatropha PEs. However, further optimi-zation studies are required to ascertain the synthesis reactions and yield of prostratin synthesized from Jatropha PEs.
Some of the preliminary requirements for any successful bio-control agent are that it should have a high bioactivity on the target organism, a long shelf-life and a high biodegradability in soil. In addition, the bioactive phytochemical should be available in large quantities, it should be easily extractable and continuously available. The PEEF potentially satisfies these aforesaid re-quirements. The abundance and novelty of PEs present in Jatropha species could form a new ?stock? for the agro-pharmaceutical industries. Considering the projected oil yield of 26 million tons/annum by 2015 (GEXSI, 2008), huge amount of raw materials will be available for both biodiesel and pharmaceutical industries. PEs in the form of the PEEF could be used either as in-sect controlling agents in agricultural applications or as a ?stock? biomaterial for synthesizing prostratin in pharmaceutical applications. 
KW  - Jatropha
KW  - Phorbolester 
KW  - Toxizität
KW  - Biotest 
KW  - Charakterisierung
CY  - Hohenheim
PB  - Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim
AD  - Garbenstr. 15, 70593 Stuttgart
UR  - http://opus.uni-hohenheim.de/volltexte/2012/684
ER  -