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: 6 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

1

Graduate School of Engineering, Osaka Prefecture
University, 1-1 Gakuen-cho, Naka-ku, Sakai-shi,
Osaka 599-8531, Japan

2

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

3

Research Organization for University-Community
Collaborations, Osaka Prefecture University, 1-2 Gakuencho, Naka-ku, Sakai-shi, Osaka 599-8531, Japan

4

Faculty of Chemistry, Vietnam National University, Hanoi,
19 Le Thanh Tong St., Hanoi, Vietnam

5

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 ha 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

1

OH

OH
O

Jatropha factor C1

H3C

19

2

O

16

15

O

OH

H

H3C

4

O
16

13

14

7
8
9
C
10 OH 6

D

4

H3C

13 A

12

13

OH

H3C

O

O
H3C

H

H
OH

20

5

H3C

OH

3

4

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
4

H3C

O
16

OH
H3C

H

H

CH3

O

O

13

4

H3C

H3C O

O

16

OH

H

O

H3C

OH
O
Jatropha factor C3

4

H3C

OH
O
Jatropha factor C6

Fig. 1 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 min 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 4 °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.,

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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 3 lm, 250 mm 9 3 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 min. 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 3 lm, 100 mm 9 3 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 min 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 min according to
the PDA analysis of the HPLC data, and the peaks that
eluted from 40 to 45 min 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 9 0.25 mm i.d., 0.2-lm film thickness) (Supelco,
Bellefonte, PA, USA). The column temperature was held at
50 °C for 1 min, and it was programmed to increase to

123

250 °C at a rate of 10 °C/min and held for 5 min. 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/
min. 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, 1 wt%), and then the mixture was stirred by a magnetic
stirrer for 2 h at room temperature (25 ± 1 °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, 2 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 1 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. 2. 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 min 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 min 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. 2 PDA chromatogram of
the MeOH extract from JCO at
wavelengths ranging from 190
to 300 nm

Fig. 3 HPLC chromatogram of
MeOH extracts a from Jatropha
curcas oil and b authentic
phorbol ester standards. Notes 1
phorbol; 2 PAA, phorbol 12,
13-diacetate; 3 PDBu, phorbol
12, 13-dibutyrate; 4 PDB,
phorbol 12, 13-dibenzoate; 5
TPA, phorbol 12-myristate

13-acetate; 6 PDD, phorbol 12,
13-didecanoate. Each
concentration was ca. 8.0 lg/
mL

a retention time of 58.7 min. 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 min 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 1. Their concentrations ranged from 2 to
6 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 2. 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 1 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 2 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 min
increased. The relationship between the concentrations of
reactants and products in each experiment is shown in
Table 3. 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, 1 mol of phorbol and 2 mol of carboxylic acid
methyl esters were produced from 1 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)

1

Son La

6.2

0.82

2

Binh Phuoc

1.9

0.21

3

Binh Thuan

2.7

0.18

Table 2 Partition coefficients of authentic phorbol esters
No.

Phorbol esters

Partition coefficient K (CV %)

No.

Phorbol esters

Partition coefficient K (CV %)

1

PDA

19.6 (2.6)

4

TPA

5.0 (2.4)

2

PDBu

15.8 (0.7)

5

PEs

2.4 (15)

3

PDB

6.4 (16)

6

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

+

2 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 min 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
do 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 min. 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 4. 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 4. 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. 4 HPLC chromatogram of
transesterification products
a from a MeOH extracts b from
phorbol ester standards

Table 3 Comparison of the concentrations of phorbol and its esters before and after transesterification with KOH/MeOH
Compounds

Experiment 1
Reactant (conc.)

Experiment 2
1

Product (conc.)

1

Reactant (conc.)

Experiment 3
1

Product (conc.)

1

Reactant (conc.)1

Product (conc.)1

1. Phorbol

–

52.5 (14.4)

7.8 (2.1)

33.8 (9.3)

77.8 (21.3)

332 (91.1)

2. PDA

–

–

6.8 (1.5)

–

–

–

3. PDBu

–

–

7.9 (1.6)

–

–

–

4. PDB

–

–

8.1 (1.4)

–

–

–

5. PEs

–

–

–

–

775 (126)

–

6. TPA

85.8 (13.9)

–

7.5 (1.2)

–

–

–

7. PDD

–

–

8.0 (1.2)

–

–

–

Molar ratio2

1.04

1.03

0.62

-5

1 mg/L (mol/L 9 10 ), 2 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 4 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

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