Fate of toxic phorbol esters in Jatropha curcas oil by a biodiesel fuel production process

Clean Techn Environ Policy DOI 10.1007/s10098-016-1149-4 ORIGINAL PAPER Fate of toxic phorbol esters in Jatropha curcas oil by a biodiesel fuel production process Duong Huu Huy1,2 • Kiyoshi Imamura3 • Le Tu Thanh2 • Phuong Duc Luu4 • Hoa Thi Truong5 • Hanh Thi Ngoc Le1 • Boi Van Luu4 • Norimichi Takenaka1 Yasuaki Maeda3 • Received: 24 September 2015 / Accepted: March 2016 Ó Springer-Verlag Berlin Heidelberg 2016 Abstract Biodiesel fuel (BDF) is an important alternative fuel because of the carbon neutral nature of biomass and the exhaustion of fossil fuel resources Jatropha curcas oil (JCO) produced from J curcas seeds contains toxic phorbol esters that can cause cancer The behaviors of toxic phorbol esters were investigated during BDF production Liquid chromatography–tandem mass spectrometry and photodiode array analyses revealed that the phorbol esters contained in JCO had a tigliane skeleton The partition coefficients of phorbol esters between methanol (MeOH) and the oil (KMeOH/oil) ranged from 2.4 to 20 As a result, the phorbol esters in the JCO were largely partitioned into the MeOH phase The phorbol esters in the oil were converted stoichiometrically into phorbol and the corresponding fatty acid methyl esters via a transesterification reaction in a potassium hydroxide (KOH)/methanol (MeOH) solution The phorbol produced predominantly & Kiyoshi Imamura k_imamura@riast.osakafu-u.ac.jp Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan Faculty of Environmental Science, University of Science, Vietnam National University - Ho Chi Minh City, 227 Nguyen Van Cu St., Dist 5, Ho Chi Minh City, Vietnam Research Organization for University-Community Collaborations, Osaka Prefecture University, 1-2 Gakuencho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan Faculty of Chemistry, Vietnam National University, Hanoi, 19 Le Thanh Tong St., Hanoi, Vietnam Danang Environmental Technology Center, Institute of Environmental Technology, Vietnam Academy of Science and Technology, Tran Dai Nghia Road, Ngu Hanh Son District, Da Nang, Vietnam partitioned into the glycerin phase A small amount of phorbol residue contained in the BDF could be removed by washing with water These results suggest that it is safe to use BDF produced by the aforementioned transesterification reaction and purification process However, phorbol contamination of glycerin and wastewater from the production process should not be ignored Keywords Phorbol esters Á Phorbol Á Jatropha curcas oil (JCO) Á Transesterification Á Participation Introduction Biodiesel fuel (BDF) is an alternative fuel produced from renewable vegetable oils (Thanh et al 2010b, 2013; Chakraborty et al 2015), animal fats (Halek et al 2013; Thanh et al 2013; Gurusala and Selvan 2015), recycled cooking oil (Thanh et al 2010a; Chuah et al 2015; Delavari et al 2015), and biomass waste (Caetano et al 2014), and it has drawn significant attention because diminishing petroleum reserves and increasing environmental concerns that favor the use of carbon neutral fuels (Glaser 2009) Presently, more than 10 million tonnes of BDF have been produced commercially from vegetable oil, and about three million tonnes have been produced from waste cooking oils in the European Union (EU), which have reduced air pollution and the net emission of greenhouse gases (Freedman et al 1984; Shay 1993; Ma and Hanna 1999; Yuen-May and Ah-Ngan 2000; Parawira 2010) A variety of edible oils, such as rapeseed, soybean, palm, coconut, sunflower, and peanut oils, can be used as raw materials for BDF; however, the use of edible oils for BDF production competes with that for food production in the marketplace This increases the costs of BDF products and 123 H H Duong et al disturbs the stable supply of food products Therefore, it is necessary to identify other raw materials that have high yields and lower prices than edible oils In this context, nonedible oils, such as jatropha, neem, karanja, rubber, and tobacco oils are prominent candidates for BDF production Jatropha curcas, an oil-bearing shrub, can grow at high elevations in dry regions, as well as on wastelands, and is widely distributed in Asian, American and African countries The seed kernels contain up to 60 % oil that is composed of triglycerides, but the seeds and seed oil (JCO) cannot be used as nutrients because they are toxic and cocarcinogenic to humans and animals (Makkar et al 1998; Ahmed and Salimon 2009; Li et al 2010) As a result of the sudden increase in the price of crude and edible oils in 2008, the plantation area of J curcas expanded to a few tens of thousands of hectares (ha) in developing countries, including those in West Africa and India, to increase BDF production (Iiyama 2012) Siang (2009) reported that the expected worldwide land area for J curcas cultivation will be 33 million in 2017 according to an estimate by the International Jatropha Organization, which will result in the production of 160 million tonnes of seeds Phorbol esters have been identified as the major toxic compounds in JCO, and their contents are less than a few percent in the seed kernels (Makkar et al 1997) Phorbol is a naturally occurring tigliane diterpene, and it contains four rings (A, B, C, and D) that are substituted with five hydroxyl (OH) functional groups The epimeric isomer of the beta OH group at the C4 position is biologically active, while that of the alpha OH group is inactive (Silinsky and Searl 2003) The esterification of phorbol at different positions with various kinds of carboxylic acids leads to the formation of a large variety of phorbol ester compounds Many kinds of phorbol esters and deoxyphorbol esters have been identified using liquid chromatography–tandem mass spectrometry (LC/MS/MS) measurements (Vogg et al 1999) Six 12-deoxy-16-hydroxy phorbol esters have been isolated from JCO, and their structures and toxicities have been characterized (Haas et al 2002; Goel et al 2007) (Fig 1) Phorbol esters are well known as cancer-promoting materials that exert a plethora of biological effects, including inflammation, tumor promotion, cell proliferation, and differentiation (Mentlein 1986; Goel et al 2007; Li et al 2010) Devappa et al (2010) reported that the toxicity (EC50, half maximal effective concentration) of phorbol esters extracted from JCO using methanol (MeOH) was 330 lg/L (phorbol 12-myristate-13-acetate (TPA) equivalent) by the snail bioassay and 26.5 mg/L by the Artemia assay Roach et al (2012) reported the EC50s of six compounds, named Jatropha factors C1 to C6, which were isolated and purified from J curcas seeds The EC50s of factor C3 were 280 lg/L by the snail test and 44.6 mg/L 123 by the Artemia test; the EC50s of factor C2 were 270 lg/L and 487 mg/L; the EC50s of factor C1 were 170 lg/L and 17.6 mg/L; and the EC50s of a C4&C5 mixture were 90 lg/ L and 1.8 mg/L, respectively During BDF production, the dry seeds of J curcas are chopped into pieces and pressed and heated to make the oil Crude BDF is produced from the oil by a transesterification reaction in a MeOH solution in the presence of alkaline (KOH and/or NaOH) (Berchmans and Hirata 2008; Thanh et al 2010a, b) The final BDF product is obtained after purification with water washes, followed by distillation under reduced pressure to remove the water Homogeneous transesterification process that is catalyzed by KOH using acetone as a co-solvent had been developed, and the reaction using the co-solvent method terminates within a few minutes to produce crude BDF and glycerin (Maeda et al 2011; Thanh et al 2013; Luu et al 2014) Recently, the heterogeneous catalyst reaction has been developed for both esterification of FFAs and transesterification of triglyceride in a single step (Singh et al 2015) and the use of a helicoidal reactor with ultrasound-assisted for continuous biodiesel production (Delavari et al 2015) As a huge increase in BDF production from JCO is expected in the latter half of this decade, the behaviors of the toxic compounds of phorbol esters in JCO should be examined during BDF production to prevent harmful effects to humans, to minimize the contamination of the environment via the emission of waste materials, and to ensure the safety of BDF as a commercial product The objectives of this study are to investigate the behaviors of toxic phorbol esters during the production of BDF, and to remove phorbol ester contaminants from the BDF products In addition, the distributions of phorbol and phorbol esters into the glycerin and FAME phases by the transesterification of JCO and into the FAME and wastewater phases by the clean-up process of crude BDF are investigated in order to prospect the fate of toxic phorbol and phorbol esters under the process of BDF production from JCO Materials and methods Jatropha curcas oil In this study, JCO produced from J curcas seeds harvested in Son La, Vietnam was used JCO was produced by compressing dry seeds containing 38 wt% of oil The physical and chemical properties were as follows: density, 0.913 g/cm3; acid value, 9.67 mg KOH/g oil; water content, 0.1 wt%; and the components of fatty acid methyl esters (FAME) after transesterification were methyl palmitate (16.2 wt%), methyl stearate (7.4 wt%), methyl oleate (35.5 wt%), and methyl linoleate (37.1 wt%) The Fate of toxic phorbol esters in Jatropha curcas oil by a biodiesel fuel production process CH2 CH2 CH3 CH3 O O H3C O 17 HO 16 OH H3C B H 11 H3C H 18 OH H OH OH O Jatropha factor C1 H3C 19 O 16 15 O OH H H3C O 16 13 14 C 10 OH D H3C 13 A 12 13 OH H3C O O H3C H H OH 20 H3C OH OH O O Jatropha factor C2 12-Deoxy-16-hydroxyphorbol CH3 H2C H2C O O O O H3C O O 16 O 13 OH H3C H3C H OH OH Jatropha factor C4 and C5 CH3 13 H H OH OH H3C O 16 OH H3C H H CH3 O O 13 H3C H3C O O 16 OH H O H3C OH O Jatropha factor C3 H3C OH O Jatropha factor C6 Fig Structures of 12-deoxy-16-hydroxy phorbol and six phorbol esters named Jatropha factor C1 to C6 in Jatropha curcas oil (Haas et al 2002) estimated average molecular weight of the JCO was 840 g/mol Reagents and standards Standards of phorbol and five kinds of phorbol esters (PDA, phorbol 12-, 13-diacetate; PDBu, phorbol 12-, 13-dibutyrate; PDB, phorbol 12-, 13-dibenzoate; TPA, and PDD, phorbol 12-, 13-didecanoate) were purchased from Wako Pure Chemicals (Osaka, Japan) MeOH, ethanol, acetonitrile, and tetrahydrofuran (THF) were a high-performance liquid chromatography (HPLC) analytical grade, and isopropanol, acetone, KOH and phosphoric acid were analytical grade They were purchased from Wako Pure Chemicals (Osaka, Japan) The alkaline solution (KOH/MeOH) for the transesterification reaction was prepared by dissolving 3.6 g of KOH in 100 mL of MeOH Preparation of standard and stock solutions Individual stock standard solutions of phorbol, PDA, PDBu, PDB, TPA, and PDD were prepared at a concentration of 1000 lg/mL Oxygen in the atmosphere of the MeOH solutions was purged for 10 with nitrogen gas Standard concentrations were estimated by measuring the difference in the container weight before and after dissolution of the standards A mixture of six compounds was prepared by mixing the individual standard solutions and diluting them at concentrations ranging from 0.1 to 100 lg/ mL All standard solutions were stored at °C Measurement of phorbol, phorbol esters, and fatty acid methyl esters The HPLC system for quantitative analysis of phorbol and its esters consisted of a series GL 7400 (GL Sciences Inc., 123 H H Duong et al Saitama, Japan) equipped with a UV–Vis detector (GL7450, GL Sciences Inc.) and a photodiode array (PDA) detector (GL-7452A, GL Sciences Inc.) For the analysis using the UV–Vis detector, an Inertsil ODS-4 analytical column (particle size lm, 250 mm mm i.d.) was used Analytical conditions were as follows: the mobile phase was water and acetonitrile, operated in a gradient mode, with an initial water to acetonitrile volume ratio of 60:40, followed by a 50:50 ratio for 10 min, a 25:75 ratio for 30 min, a 0:100 ratio for 15 min, and a 60:40 ratio for 10 Finally, the column was washed with solvent containing 75 % THF and 25 % acetonitrile The separation process was conducted at a column temperature of 30 °C, and the flow rate was 0.4 mL/min The UV-VS detector was operated at wavelength of 280 nm The injection volume was 20 lL For the analysis using the PDA detector, a cartridge guard column E was mounted on an Inertsil ODS-4 column (particle size lm, 100 mm mm i.d.) Analytical conditions were as follows: the initial mobile phase was a mixture of water and acetonitrile (95:5 ratio), followed by a 50:50 ratio for 10 min, a 25:75 ratio for 15 min, and a 0:100 ratio for 15 at a column temperature of 30 °C The injection volume was 50 lL A LC/MS/MS system for qualitative analysis of phorbol esters consisting of a GC 7400 HPLC (GL Sciences Inc., Saitama, Japan) and an Applied Biosystems API 4000 QTrapÒ LC/MS/MS system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electron spray was used The analytical conditions of the HPLC system were the same as those of the PDA analysis described previously The LC/MS/MS system was operated in multiple reactions monitoring (MRM)-positive mode with collisioninduced dissociation The characteristic precursor ion was monitored simultaneously with one of its fragment products, such as m/z 313 to m/z 295 (313/295) and m/z 295 to m/z 267 (295/267) for monitoring the ingenane type of phorbol, while m/z 311 to m/z 293 (311/293) and m/z 293 to m/z 265 (293/265) were used for monitoring the tigliane type (Vogg et al 1999) A group of peaks eluted from 35 to 40 according to the PDA analysis of the HPLC data, and the peaks that eluted from 40 to 45 according to a precursor scan analysis of the LC/MS/MS with m/z 311 to m/z 293 (311/ 293) were assigned as components of the tigliane-type phorbol esters The gas chromatograph (GC) system for FAME analysis was a Hewlett Packard HP 6890 (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) The analytical column was a SPTM-2380 (30 m 0.25 mm i.d., 0.2-lm film thickness) (Supelco, Bellefonte, PA, USA) The column temperature was held at 50 °C for min, and it was programmed to increase to 123 250 °C at a rate of 10 °C/min and held for The injection temperature was 250 °C, and the helium gas flow rate was 1.0 mL/min The gas flow rates for the FID detector were as follows: hydrogen, 40 mL/min; air, 450 mL/min; and the carrier gas supply (helium), 45 mL/ A 1–2 lL sample was injected by split mode with a split ratio of 1:50 Transesterifications Transesterification of JCO was performed as follows: 2.8 mL of MeOH containing 0.1 g KOH was added to 10 g of JCO (molar ratio of MeOH to oil, 6:1; KOH catalyst to oil, wt%), and then the mixture was stirred by a magnetic stirrer for h at room temperature (25 ± °C) After the reaction, the mixture was neutralized with phosphoric acid (5 % v/v) and left to separate into two phases: the upper FAME phase and the lower glycerin phase Twenty lL of each solution was diluted in an appropriate volume of solvent and injected into the HPLC for the determination of phorbol and phorbol esters In the case of the MeOH extract, mL of the MeOH extract containing phorbol esters extracted from JCO was reacted with 0.4 mL of MeOH containing 0.008 g of KOH as a catalyst In the case of the phorbol ester standard solution, 0.2 mL of MeOH containing 0.004 g of KOH was added to mL of the standard solutions They were treated in the same manner as the aforementioned transesterification reactions Results and discussion Phorbol and phorbol esters The PDA chromatogram of the MeOH extract from JCO at wavelengths ranging from 190 to 300 nm is shown in Fig The UV absorption maximum at wavelengths ranging from 260 to 300 nm for a group of peaks that eluted with retention times of 35–40 was coincident with those of phorbol esters The pattern of the peaks consist of six components was very similar to that reported by Makkar et al (1997) According to the results of the MRM LC/MS/MS analysis, a group of peaks eluted with retention times ranging from 35 to 40 was shown to be a tigliane type of phorbol (Vogg et al 1999) The HPLC chromatogram of the MeOH phase extracted from JCO is shown in Fig 3a, and that of authentic samples of phorbol and five phorbol esters are shown in Fig 3b The five phorbol esters eluted in wide range, from PDA (9.5 min) to PDD (66 min) Among them, TPA, which was used as external standard for quantification, had Fate of toxic phorbol esters in Jatropha curcas oil by a biodiesel fuel production process Fig PDA chromatogram of the MeOH extract from JCO at wavelengths ranging from 190 to 300 nm Fig HPLC chromatogram of MeOH extracts a from Jatropha curcas oil and b authentic phorbol ester standards Notes phorbol; PAA, phorbol 12, 13-diacetate; PDBu, phorbol 12, 13-dibutyrate; PDB, phorbol 12, 13-dibenzoate; TPA, phorbol 12-myristate 13-acetate; PDD, phorbol 12, 13-didecanoate Each concentration was ca 8.0 lg/ mL a retention time of 58.7 A group of phorbol esters extracted from JCO eluted at retention times range from 52 to 56 min, as determined by monitoring at the 280 nm wavelength of the UV region; however, the peaks eluted just before the group of phorbol esters could not be assigned as phorbol esters based on the UV spectra and MRM analyses To determine the concentration of phorbol esters in JCO, a 10 g of oil sample was extracted with 10 mL of MeOH, and the extraction was repeated three times After extraction, all extracts were combined After adjusting the volume with solvent, a 20 lL of aliquot was quantitatively analyzed using HPLC The concentration of phorbol esters was estimated from the total area of a group of phorbol 123 H H Duong et al ester components that had retention times ranging from 52 to 56 in the chromatogram The quantification was conducted by the external calibration method using TPA as the standard material The concentrations of phorbol esters contained in the oils produced from J curcas seeds cultivated at three different areas in Vietnam were examined The results are shown in Table Their concentrations ranged from to mg/g for phorbol esters and from 0.2 to 0.8 mg/g for phorbol; the contents of phorbol and phorbol esters would depend on the species of Jatropha, as well as the climatic and geographic conditions in which the species were cultivated Partition coefficients Ten g of JCO was extracted with 10 mL of MeOH The partition coefficients KMeOH/Oil of the phorbol esters between MeOH and JCO were estimated using the method described in reference (Christian 1986) The KMeOH/Oil was calculated using the following formula: KMeOH/Oil = (C1/ C2) - 1, where C1 is the concentration of a component (mg/mL) of the first extraction and C2 is that of the second extraction The results are shown in Table These results indicated that it was necessary to perform more than three times extractions to attain more than 95 % efficiency extraction of the PDD, because of its most hydrophobic property of the phorbol esters tested (Wang et al 2000 and references therein) The most hydrophobic property of PDD could be explained by its retention time on a reversed-phase C18 column because it is the last compound eluted as shown in Fig 3b, and its partition coefficient is the lowest with value of 2.4 (Table 2) Table Concentrations of phorbol and its esters contained in JCO Transesterification Phorbol ester standards A known amount of a TPA standard solution (80 mg/L) (Experiment 1) and a mixture of phorbol and five phorbol esters (PDA, PDBu, PDB, TPA, and PDD) standard solutions (each ca 8.0 mg/L) (Experiment 2) were reacted with MeOH in the presence of KOH as a catalyst After reacting, the mixtures were neutralized by phosphoric acid (5 % v/v), and aliquots of the products were analyzed by HPLC The HPLC chromatogram of the reaction products in Experiment is shown in Fig 4b Five peaks of phorbol esters, as shown in Fig 3b, disappeared, and the intensity of the phorbol peak with a retention time of 4.1 increased The relationship between the concentrations of reactants and products in each experiment is shown in Table In Experiment 1, 13.9 mol of TPA was converted into 14.4 mol of phorbol (the molar ratio of phorbol to TPA was 1.04), and in Experiment 2, 9.0 mol of five phorbol esters and phorbol were converted into 9.3 mol of phorbol (the molar ratio of phorbol to phorbol esters was 1.03) For instance, mol of phorbol and mol of carboxylic acid methyl esters were produced from mol of phorbol 12-myristate-13-acetate by the transesterification process (Eq 1) These molar ratios suggested that the reaction proceeded stoichiometrically, and thus, phorbol was produced quantitatively These results suggest that phorbol esters were transesterified and completely converted into phorbol and the corresponding carboxylic acid methyl esters However, their methyl esters could not be identified because of their lower sensitivities in the HPLC analysis using the UV detector (280 nm) Sample no Sources of location in Vietnam Phorbol esters (mg/g) Phorbol (mg/g) Son La 6.2 0.82 Binh Phuoc 1.9 0.21 Binh Thuan 2.7 0.18 Table Partition coefficients of authentic phorbol esters No Phorbol esters Partition coefficient K (CV %) No Phorbol esters Partition coefficient K (CV %) PDA 19.6 (2.6) TPA 5.0 (2.4) PDBu 15.8 (0.7) PEs 2.4 (15) PDB 6.4 (16) PDD 2.4 (3.8) PDA phorbol 12, 13-diacetate; PDBu phorbol 12, 13-dibutyrate; PDB 12, 13-dibenzoate; TPA phorbol 12-myristate 13-acetate; PDD phorbol 12, 13-didecanoate; PEs a group of phorbol esters extracted from JCO; CV coefficient of variation 123 Fate of toxic phorbol esters in Jatropha curcas oil by a biodiesel fuel production process O O O H3C H3C H H H3C OH H OH CH3 OH H3C CH3 O CH3 + CH3OH KOH H H3C H H OH catalyst O CH3 CH3 O HO O HO OH phorbol 12-myristate 13-acetate OH phorbol + O H3C methyl myristate O + H3C O CH3 CH3 methyl acetate ð1Þ MeOH extract from Jatropha curcas oil The MeOH extract from JCO was reacted with a KOH/ MeOH solution, and the product was analyzed as described in ‘‘Phorbol ester standards’’ section The HPLC chromatogram of the reaction products is shown in Fig 4a Phorbol esters also disappeared, and the intensity of the phorbol peak (4.1 min) increased in the same manner as that of the authentic phorbol esters However, the molar ratio of the phorbol in product to the total amount of phorbol and phorbol esters was 0.62, which was lower than that estimated stoichiometrically This was mainly caused by the different sensitivities (per gram) (Dimitrijevic´ et al 1996) of each phorbol ester to TPA because the sensitivity at 280 nm depends on the absorption coefficient of the chromophore in the molecule and, as can be seen in the HPLC chromatogram shown in Fig 3b, an approximately three-fold higher sensitivity (per gram) of PDB was estimated in comparison with that of TPA As shown in Fig 4a, small three peaks in the range from 50 to 52 near the phorbol ester peaks obtained by analysis of transesterification products of MeOH extract are detected The retention times and the pattern of these peaks not overlap with those of phorbol esters By GC-FID analysis, these peaks are assigned as BDF produced from JCO, of which contents are methyl palmitate (16.3 wt%), methyl stearate (7.3 wt%), methyl oleate (35.7 wt%), and methyl linoleate (36.7 wt%) A certain amount of JCO is participated into MeOH phase during MeOH extraction; therefore, the same components of FAME are produced in the process of transesterification This result indicates that small three peaks are not the products from phorbol esters after transesterification Jatropha curcas oil In this section, the behaviors of phorbol esters contained in the large matrix of JCO were examined in the process of BDF production The transesterification of JCO was conducted with a KOH/MeOH solution using a mechanical stirring method After the reaction was completed, the reaction products were neutralized by phosphoric acid, and then allowed to separate into the FAME and glycerin phases The FAME and glycerin phases were dissolved in THF and MeOH solvents, respectively, and an aliquot was analyzed by HPLC The chromatogram of the FAME and glycerin products was similar to that of the transesterification products of the MeOH extract (Fig 4a) The three main peaks of FAME in the glycerin phase were observed in the range from 50 to 52 The contents of phorbol esters were less than the detection level in both of the FAME and glycerin phases The transesterification of JCO was further conducted using the co-solvent method with a co-solvent of acetone and THF (Thanh et al 2013; Luu et al 2014) The results were the same as those observed for the mechanical stirring method These results indicated that phorbol esters contained in the large matrix of JCO were completely converted into skeletal frame of phorbol and the corresponding carboxylic acid methyl esters After transesterification, the contents of phorbol in the crude BDF (BDF1) and glycerin phases are shown in Table Phorbol mostly participates into the glycerin phase (1.4–1.7 mg/g), but only small amount distributes into the FAME phase (0.0032 mg– 0.0046 mg/g) because of a polar property of phorbol Clean-up process After transesterification, the reaction mixture was separated into the glycerin and BDF1 phases, and then a final product of BDF (BDF2) was obtained by cleaning-up BDF1 with water to improve the BDF quality The distributions of phorbol in the FAME and aqueous phase were examined The results are shown in Table Phorbol, the content of which was 0.0037–0.0046 mg/g remained in FAME, was washed out with water and participated into the aqueous phase (0.0045–0.0064 mg/L) As a result, the level of phorbol in BDF2 was reduced to the non-detectable level 123 H H Duong et al Fig HPLC chromatogram of transesterification products a from a MeOH extracts b from phorbol ester standards Table Comparison of the concentrations of phorbol and its esters before and after transesterification with KOH/MeOH Compounds Experiment Reactant (conc.) Experiment Product (conc.) Reactant (conc.) Experiment Product (conc.) Reactant (conc.)1 Product (conc.)1 Phorbol – 52.5 (14.4) 7.8 (2.1) 33.8 (9.3) 77.8 (21.3) 332 (91.1) PDA – – 6.8 (1.5) – – – PDBu – – 7.9 (1.6) – – – PDB – – 8.1 (1.4) – – – PEs – – – – 775 (126) – TPA 85.8 (13.9) – 7.5 (1.2) – – – PDD – – 8.0 (1.2) – – – Molar ratio2 1.04 1.03 0.62 -5 mg/L (mol/L 10 ), The ratio of the molar concentration of phorbol in product to those of the reactants, PDA, phorbol 12, 13-diacetate; PDBu, phorbol 12, 13-dibutyrate; PDB, phorbol 12, 13-dibenzoate; PEs, a group of phorbol esters extracted from JCO; TPA, phorbol 12-myristate 13-acetate; PDD, phorbol 12, 13-didecanoate As for the transesterification using acetone as a co-solvent, it was impossible to determine the content of phorbol because of overlapping with large peak of acetone The toxic components of phorbol ester and phorbol contained in BDF2 are less than the detection level, and the BDF is safe to use, although further purification is needed 123 for production of the commercial product On the contrary, the wastewater, emitted from cleaning-up process containing not only toxic phorbol but also other chemicals such as solvents, alkali, and oily products, deteriorates the aqueous environmental quality when discharged without any treatment The fates of phorbol and phorbol esters in Fate of toxic phorbol esters in Jatropha curcas oil by a biodiesel fuel production process Table Concentrations of phorbol in the clean-up process Transesterification method After reaction In BDF1 (mg/g) Clean-up with water In glycerin (mg/g) In BDF2 (mg/g) In the water layer (mg/L) Conventional (no solvent) 0.0032 1.7 ND 4.5 Co-solvent (acetone) – – ND – Co-solvent (THF) 0.0046 1.4 ND 6.4 BDF1 crude biodiesel fuel after phase separation; BDF2 a final biodiesel fuel after clean-up with water; ND not detected; –, impossible to determine because of the overlap with acetone solvents; THF, tetrahydrofuran; no solvent, without co-solvent the process of wastewater treatment should be investigated to estimate their impact to aqueous environment On the other hand, in case of by-product of glycerin that is the useful natural resource of medicines and cosmetics, the detoxification of phorbol in glycerin obtained from JCO is strongly required for avoiding the direct human health effects before use Conclusions The behaviors of toxic phorbol esters during BDF production were investigated A group of phorbol esters in JCO in the experiment were assigned to be a tigliane type The partition coefficient (KMeOH/oil) of these phorbol esters was 2.4 Accordingly, it is necessary to perform at least three MeOH extractions to remove more than 95 % of the phorbol esters from the oil In the transesterification with KOH/MeOH, BDF and glycerin, as a by-product, were produced, and simultaneously, the phorbol esters were converted into the skeletal frame of phorbol and the corresponding carboxylic acid methyl esters Notably, most of phorbol partitioned into the glycerin phase The small amount of phorbol residue in the BDF1 could be removed by washing with water because of its high polarity These results suggest that the BDF product produced by the transesterification reaction followed by the purification process is safe to use In case of by-product of glycerin produced from JCO, the detoxification of phorbol is strongly required for avoiding the direct human health effects when it is used by cosmetics and medical products Acknowledgments The authors thank the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) for their support of the Science and Technology Research Partnership for Sustainable Development (SATREPS) project titled ‘‘Multi-Beneficial Measure for Mitigation of Climate Change in Vietnam and Indochina Countries by the CultivationProduction-Utilization of Biomass Energy.’’ Compliance with ethical standards Conflict of Interest of interest The authors declare that they have no conflict References Ahmed WA, Salimon J (2009) Phorbol 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