Industrial Crops & Products 124 (2018) 941–946

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Industrial Crops & Products

journal homepage: www.elsevier.com/locate/indcrop

An optimized solid-liquid method for rapid extraction of phorbol esters from T

Mozambican Jatropha seeds
Hercílio E. Zimilaa, Jaime S. Mandlatea,b, Rogério S. Chivodzea, Hermínio F. Muiamboa,
⁎
Victor Skripetsa, Amália A. Uamussea,
a
Department of Chemistry, Eduardo Mondlane University, P.O. Box 257, Main Campus, Maputo, Mozambique
b
Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The method of extraction of phorbol esters from Jatropha curcas L. (Jatropha) seed was optimized, using an
Phorbol esters experimental design, and validated in this study. Variables such as extraction time, vortex-stirring rate, sam-
Extraction ple:solvent ratio, solvent type and extraction cycles, were selected and their influence on the extraction yield was
Optimization screened by Plackett-Burman design. Thereafter, the three factors (extraction time, stirring rate and sample:-
Validation
solvent ratio) were optimized by response surface methodology based on Box-Behnken design. The results re-
Experimental design
vealed that the best conditions to achieve optimum response are sample:methanol ratio of 1 mg:50 μL, two
extraction cycles under vortex stirring at 3200 rpm for 3 min/cycle. This method shows good recoveries
(84–95%), repeatability (RSD = 1.89%), linearity (R2 = 0.999), robustness, limits of detection (2.19 ng μL−1)
and quantitation (6.65 ng μL–1). Using this method, phorbol esters can be extracted for quantification purposes,
toxicological studies and application as biopesticides and anti-HIV agents.

1. Introduction
Jatropha curcas L. (Jatropha) belongs to one of the largest and ge-
netically diverse plant family, Euphorbiaceae. It is a drought-resistant
oil-crop that has been gaining more attention in the last few decades as
a promising environmentally friendly biodiesel feedstock (Brittaine and
Lutaladio, 2010). Jatropha is a native plant from South America but
largely distributed and grown in almost all tropical and sub-tropical
areas around the world (Gübitz et al., 1999; Kumar et al., 2016).
De-husked Jatropha seeds contain up to 60% of oil (Makkar et al.,
1997) with very interesting composition and physicochemical proper-
ties for application in diesel engines after transesterification (Gübitz
et al., 1999). Additionally, the direct competition of this oil with food
industry is reduced due to the presence of antinutritional and toxic
substances, which play a crucial role in defence of Jatropha plant
against invaders (Devappa et al., 2010a; Devappa, 2012). Among such
secondary metabolites of Jatropha, phorbol esters (PEs) are the most
investigated. Structures of six different PEs occurring in Jatropha oil
were exhaustively described by Haas et al. (2002). They are in-
tramolecular diesters of the same diterpenic moiety of 12-deoxy-16-
hydroxyphorbol with different dicarboxylic acid. The most abundant
phorbol ester in Jatropha is 12-deoxy-16-hydroxyphorbol-4′-[12′,14′-
butadienyl]-6′-[16′,18′,20′-nonatrienyl]-bicyclo[3.1.0]hexane-(13-O)-
2′-[carboxylate]-(16-O)-3′-]8′-butenoic-10′]ate (DHPB), whose struc-
ture is shown in Fig. 1 (Hirota et al., 1988).
In mammalians, PEs act as tumour-promoters (co-carcinogenic
substances) and skin-irritants (Wink et al., 1997) and can reach the
tissues by several routes, including dermal, ocular, and oral exposures
(Devappa et al., 2013b; Takechi and Imou, 2015). Despite such dele-
terious effects, these compounds seem to have interesting agricultural
and pharmaceutical applications (Devappa, 2012). Indeed, Devappa
et al. (2010b) attributed the molluscicidal, insecticidal and anti-
microbial activities of methanolic extracts of many parts of Jatropha to
PEs. Studies carried out by Devappa et al. (2012) showed that phorbol
ester enriched fraction (PEEF) is a potential biocontrol agent of Spo-
doptera frugiperda insect that commonly attacks crops such as corn,
cabbage and potatoes. In addition, Devappa (2012) reported a method
for conversion of Jatropha PEs into prostratin, a promising adjuvant in
HIV therapy. Therefore, a suitable exploitation of the potential of PEs
would increase both the value-chain and economic viability of Jatropha
products as well as reduce the occupational exposure in handling Ja-
tropha products. To achieve such goal, the development of a rapid and
accurate method for extraction and quantitation of PEs is of paramount
importance.

⁎
Corresponding author.
E-mail address: a.uamusse@uem.mz (A.A. Uamusse).

https://doi.org/10.1016/j.indcrop.2018.08.017
Received 16 May 2018; Received in revised form 8 July 2018; Accepted 7 August 2018
0926-6690/ © 2018 Published by Elsevier B.V.
H.E. Zimila et al. Industrial Crops & Products 124 (2018) 941–946

Japan. Ultra-pure water was obtained from a Milli-Q system (Milli-Q

direct 8.0, Japan).

2.2. Sampling and sample pre-preparation

Jatropha seeds were harvested on June 2016 from Jatropha plants

cultivated in Boane, Maputo, Mozambique (26°02′20.4″ South,
32°21′06.5″ East, annual rainfall 752 mm and 23.7 °C mean tempera-
ture). The seeds were stored in polyethylene bags, transported and air-
dried at room temperature. The seeds were cracked and then the ker-
nels finely grounded using mortar and pestle and stored at –14 °C in
screw-capped polypropylene vials.

2.3. General procedure

The phorbol esters were extracted according to the method de-

Fig. 1. Chemical structure of Jatropha factor 1: 12-deoxy-16-hydroxyphorbol-
4′-[12′,14′-butadienyl]-6′-[16′,18′,20′-nonatrienyl]-bicyclo[3.1.0]hexane-(13-
O)-2′-[carboxylate]-(16-O)-3′-]8′-butenoic-10′]ate (DHPB).
A number of analytical techniques for extraction of PEs from
Jatropha seeds are reported in the literature. Such methods require
large amounts of sample (400 mg to 4 g), solvent (at least 10 mL) as
well as long time for sample preparation (Makkar et al., 1997; Martínez-
Herrera et al., 2006; Devappa et al., 2013a; Baldini et al., 2014).
Thereby, they are unsuitable for daily application in monitoring studies.
While the majority employed methanol (Makkar et al., 1997; Martínez-
Herrera et al., 2006; Devappa et al., 2013a; Baldini et al., 2014; Najjar
et al., 2014), other authors had used dichloromethane (Makkar et al.,
1997) as the extraction solvent. Some approaches applied Soxhlet ex-
tractors (Devappa et al., 2013a; Gogoi et al., 2014), while others as-
sisted the extraction process with ultra-sonic waves (Baldini et al.,
2014; Devappa et al., 2013a), stirring (Devappa et al., 2013a) or their
combination. Inconsistency in extraction conditions (extraction time,
temperature, sample:solvent ratio, and stirring rate) among these
methods is evident, and it rises difficulties for fair comparison of the
results.
Studies focused on the optimization of extraction method of PEs
from Jatropha seeds are scarce. Pereira et al. (2015) developed a su-
percritical method for extraction of PEs from Jatropha seed cake which
is more eco-friendly than solvent extraction. However its availability is
limited for many research laboratories. Some authors had reported that
vortex stirring gives better yields and it is more cost-effective than
Soxhlet and sonication methods (Devappa et al., 2013a), but this pro-
cedure was not yet optimized nor validated.
Given the aspects mentioned above, the present study aimed to
apply multivariate approach to optimize the conditions of extraction of
PEs from Jatropha seeds. The reliability of the results obtained by the
proposed method was ensured carrying out the validation process.
scribed by Devappa et al. (2013a) with some modifications. Briefly,
about 40 mg of sample were placed into 15 mL polypropylene vial,
extraction solvent was added (1, 2 or 3 mL) and the mixture stirred in
vortex (ThermoScientific LP Vortex Mixer 0–3200 rpm). The sample
was allowed to settle until clear phase separation (∼3–5 min) at room
temperature (22 °C) and then the upper liquid layer was transferred into
a clean 15 mL polypropylene vial. The extraction solvents were me-
thanol or 2% tetrahydrofuran:dichloromethane (1:1) in methanol
(THF/DCM/MeOH), which were found to be the best in the study
carried out by Devappa et al. (2010c). The extraction parameters were
adjusted according to the Plackett-Burman and Box-Behnken designs.
The extract was dried at 40 °C under rotary evaporator (with the fol-
lowing model for each part: pump EyelaA−1000S, water-bath EyelaOSB-
2100
, rotator EyelaN-1200A). The dried residue was redissolved in 400 μL
of methanol, filtrated through Nylon syringe filter 0.22 μm (diameter
13 mm) to 600 μL Eppendorf tubes, and analysed by HPLC-UV.
2.4. Experimental design and data analysis
To identify the main factors affecting the yield of PEs, a set of five
factors (extraction time (1, 3, 5 min), stirring rate (600, 1400,
2200 rpm), sample:solvent ratio (1 mg:25 μL, 1 mg:50 μL, 1 mg:75 μL),
extraction cycles (1, 2, 3) and solvent type (MeOH, THF/DCM/MeOH))
were selected and screened by 12-run Plackett-Burman design. Two
central points were considered to investigate a possible non-linear be-
haviour of yield as function of factors levels. Factor levels and the full
design matrix are shown in Tables 1 and 2, respectively. The subsequent
step was done by performing response surface methodology based on
Box-Behnken design to determine the optimal range of the three most
important variables, namely: extraction time (1, 3, 5 min), stirring rate
(1400, 2300, 3200 rpm) and sample:solvent ratio (1 mg:25 μL,
1 mg:50 μL, 1 mg:75 μL). The factor levels and optimization design
matrix are shown in Tables 1 and 4, respectively. A total of 168
Table 1
Factors and levels used in experimental designs.
Factor Plackett-Burman design Box-Behnken design

Factor level Factor level

2. Materials and methods −1 0 +1 −1 0 +1

Extraction time (min) 1.0 3.0 5.0 1.0 3.0 5.0

2.1. Chemicals
(A)
Stirring rate (rpm) (B) 600 1 400 2 200 1 400 2 300 3 200
Methanol (MeOH, CAS # 67-56-1) and acetonitrile (CAS # 75-05-8), Extraction cycles (C) 1 2 3 – – –
both HPLC grade, and dichloromethane (DCM, CAS # 75-09-2) (99%) Sample:solvente ratio 1:25 1:50 1:75 1:25 1:50 1:75
were supplied by SkyLabs, South Africa. Tetrahydrofuran (THF, CAS # (mg:μL) (D)
Solvent type (E) MeOH —— THF/ – – –
109-99-9) (98%) and 4-octylphenol (CAS # 1806-26-4) (99%) were
DCM/
from Fluka Chemica (South Africa) and Sigma-Aldrich (South Africa), MeOH
respectively. DHPB standard were offered by Kurume University,

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Table 2 yield. Those are: (i) two extraction cycles with methanol, sample:sol-
Plackett-Burman design matrix with responses. vent ratio of 1 mg:50 μL, under vortex stirring at 2200 rpm for 5 min;
# A B C D E DHPB (mg g−1) (ii) two extraction cycles with methanol, sample:solvent ratio of
1 mg:50 μL, under vortex stirring at 3200 rpm for 3 min.
1 1 2200 3 75 MeOH 0.6702 ± 0.0797 Recovery was determined by dividing the calculated concentration
2 5 600 1 25 THF:DCM/MeOH 0.0637 ± 0.0035
with the known spiked DHPB concentration in a PEs-free sample. The
3 3 1400 2 50 THF:DCM/MeOH 0.6060 ± 0.1093
4 1 2200 3 25 THF:DCM/MeOH 0.7644 ± 0.0128
highest concentration of DHPB obtained during the robustness assay
5 1 600 1 75 THF:DCM/MeOH 0.0707 ± 0.0078 (1.24 mg g−1) was assumed as the highest level (100%). From this
6 5 2200 1 75 THF:DCM/MeOH 0.6804 ± 0.0271 concentration, levels of 50% and 150% were calculated and the cor-
7 1 600 3 75 THF:DCM/MeOH 0.2839 ± 0.0076 responding values are 0.62 mg g−1 and 1.86 mg g−1, respectively.
8 1 600 1 25 MeOH 0.0951 ± 0.0038
Then, the PEs-free sample was spiked with DHPB at those levels and
9 5 600 3 75 MeOH 0.3948 ± 0.0545
10 5 600 3 25 MeOH 0.3343 ± 0.0497 extracted according to the developed method. The repeatability was
11 5 2200 3 25 THF:DCM/MeOH 0.7224 ± 0.0015 assessed by calculating relative standard deviation (RSD) of the six
12 3 1400 2 50 MeOH 0.6577 ± 0.0090 replicates of the sample.
13 5 2200 1 75 MeOH 0.7108 ± 0.0314
14 1 2200 1 25 MeOH 0.5024 ± 0.0599
3. Results and discussion
Where: A – extraction time (min), B – stirring rate (rpm), C – extraction cycles,
D sample:solvent ratio (1mg: x μL), E – solvent type. 3.1. Screening experiment

experiments were carried out that include 84 for screening by Plackett- A 12-run Plackett-Burman design was performed to identify the
Burman design (12-run, 2 central points and three replicates) and 84 for most important variables (among five, namely extraction time, stirring
Box-Behnken design (14 essays with three replicates for each). rate, sample:solvent ratio, extraction cycles and solvent type) on the
Minitab statistical software version 17.0 was used to generate ex- PEs extraction yield for further optimization. Two centre points were
perimental design matrices as well as to perform data analysis. The added to investigate a possible non-linear relationship between the
whole data evaluation was done at a confidence level of 95%. response and the experimental conditions. The design matrix and re-
spective responses obtained under different experimental conditions are
2.5. Detection and quantification of PEs summarised in Table 2, and are expressed as mean of DHPB (mg
g−1) ± standard deviation.
An aliquot of 20 μL of extracts were manually injected in a high- The results were evaluated by ANOVA (Table 3), in which an effect
performance liquid chromatography (HPLC) with ultraviolet/visible will be significant if p < 0.05. Thus, since p-value of solvent type
detector (UV/Vis), Shimadzu LC-20A. Chromatographic separation was (MeOH and THF/DCM/MeOH) and sample:solvent ratio is greater than
carried out at 40 °C on a Inertsil ODS-4 C18 column (5 μm, 4.6 mm × 0.05, these factors are not statistically significant. Meanwhile, extrac-
150 mm, GL Sciences Inc., Tokyo, Japan) protected by a guard column tion time, stirring rate and extraction cycles affects significantly the
of the same material (4.0 mm × 10 mm). The separation was based on extraction yield of PEs.
isocratic elution of acetonitrile:water (77:23, v/v) at a flow rate of 1 mL Positive slope of the main effects plot (Fig. 2) indicates that the
min−1. PEs were detected at 282 nm and its retention time is between 8 factors have a positive influence on the extraction process. Thus, with
and 13 min. An external calibration method, using standard DHPB, was exception of the solvent type factor, when the tested factors change
employed for quantification of PEs. The calibration curve was built from their lowest to the highest levels, the overall extraction yield tends
preparing five standards of DHPB at 2.5, 5.0, 10.0, 25.0 and 50.0 ng to increase. Important information provided by ANOVA is the sig-
μL−1 in methanol. nificance of curvature, which implies a non-linear relationship of the
extraction yield toward these factors. Indeed, Fig. 2 shows that, with
2.6. Method validation exception of the stirring rate, the highest yield is obtained when the
factors are in its center points.
The method performance was evaluated in terms of linearity, se- The substitution of methanol with the mixture tetrahydrofuran:di-
lectivity, repeatability, recovery, limits of detection (LOD) and quan- chloromethane (1:1) reduce slightly the extraction yield, which is op-
tification (LOQ). All merit figures were evaluated for the optimized posite to the trend observed by Devappa et al. (2010c) in oil. Differ-
method, whose optimum factor levels are 2 extractions with methanol, ences on the matrix composition could be the main reason for this fact.
at sample:solvent ratio of 1mg:50 μL, under vortex stirring at 3200 rpm Besides, this modification may have negatively affected the diffusion
for 3 min. The experiments for LOD, LOQ and each level of recovery rate of methanol through the lipoprotein matrix of the seed kernel.
were done in triplicate, while six replications were performed for re- Hence, methanol was chosen for response surface.
peatability studies. Although the extraction cycles factor is statistically significant, it
The correlation (r) and determination (R2) coefficients of the cali-
bration curve (Section 2.5) were used to evaluate the linearity. A visual Table 3
method was employed for estimation of LOD. For this method, PEs-free ANOVA for Plackett-Burman design.
sample extracts were spiked with DHPB 50 ng μL−1 until observation of Source DF Adj SS Adj MS F-Value P-Value
a peak clearly distinguishable from the baseline at intensity range of 0
to 30 mV. Meanwhile, the LOQ was calculated as LOQ = 10/3xLOD. In Model 7 1.68909 0.24130 58.33 0.000
Blocks 1 0.00018 0.00018 0.04 0.838
order to investigate the method selectivity, the separation factor (α) of
Linear 5 1.56416 0.31283 75.62 0.000
PEs peaks with each other were calculated throughthe Eq. (1). Extraction time (min) 1 0.04502 0.04502 10.88 0.004
t2 − t0 Stirring rate (rpm) 1 1.31420 1.31420 317.69 0.000
α= with t2 > t1 Extraction cycles 1 0.18266 0.18266 44.15 0.000
t1 − t0 (1)
Sample:solvent ratio 1 0.01797 0.01797 4.34 0.050
Where: t2 and t1 are retention times of the desired analyte and the peak Solvent 1 0.00432 0.00432 1.04 0.319
Curvature 1 0.12475 0.12475 30.16 0.000
closest to this; and t0 is the dead time.
Error 20 0.08274 0.00414
Method robustness was assessed by testing the influence of two Total 27 1.77183
operational conditions, within the optimum range, on the extraction

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Table 4 Table 5
Box-Behnken design matrix with responses. ANOVA for Box-Behnken design.
# A B D DHPB (mg g−1) Source DF Adj SS Adj MS F-Value P-Value

1 5 3200 50 0.6588 ± 0.0168 Model 9 0.439766 0.048863 23.51 0.000

2 3 3200 25 0.6390 ± 0.0457 Linear 3 0.296287 0.098762 47.52 0.000
3 3 1400 75 0.3987 ± 0.0443 Stirring rate (rpm) 1 0.234665 0.234665 112.92 0.000
4 1 3200 50 0.6750 ± 0.0482 Extraction time (min) 1 0.050495 0.050495 24.30 0.000
5 3 3200 75 0.6716 ± 0.0225 Samp.:solv. ratio 1 0.011127 0.011127 5.35 0.031
6 5 1400 50 0.6050 ± 0.0312 Square 3 0.107803 0.035934 17.29 0.000
7 3 2300 50 0.6822 ± 0.0265 Stirring rate (rpm)*Stirring rate 1 0.083089 0.083089 39.98 0.000
8 1 2300 25 0.5747 ± 0.0150 (rpm)
9 3 2300 50 0.7282 ± 0.0385 Extraction time 1 0.004695 0.004695 2.26 0.148
10 3 2300 50 0.6139 ± 0.6961 (min)*Extraction time (min)
11 1 2300 75 0.5711 ± 0.0366 Samp.:solv. ratio*Samp.:solv. 1 0.020508 0.020508 9.87 0.005
12 1 1400 50 0.3770 ± 0.0236 ratio
13 3 1400 25 0.2948 ± 0.0133 2-Way Interaction 3 0.035677 0.011892 5.72 0.005
14 5 2300 25 0.6527 ± 0.0290 Stirring rate (rpm)*Extraction 1 0.029801 0.029801 14.34 0.001
time (min)
Where: A – extraction time (min), B – stirring rate (rpm), and D – sample:sol- Stirring rate (rpm)*Samp.:solv. 1 0.002542 0.002542 1.22 0.282
vent ratio (1mg: x μL). ratio
Extraction time 1 0.003335 0.003335 1.60 0.220
(min)*Samp.:solv. ratio
was fixed on two because it cannot have decimal places. On the other Error 20 0.041565 0.002078
hand, sample:solvent ratio was considered for response surface, despite Lack-of-Fit 3 0.018717 0.006239 4.64 0.015
its overall response being insignificant. Its behaviour is similar to the Pure Error 17 0.022848 0.001344
extraction cycles: the highest response is achieved when it is at the Total 29 0.481331

centre point. Nevertheless, it can assume decimal places unlike the

extraction cycles.
sample:solvent ratio (mg:μL)
The determination coefficient (R2), the adjusted determination
coefficient (R2-adj) and the prediction determination coefficient (R2-
3.2. Optimization process
pred) are 0.892, 0.863 and 0.810, respectively. Hence, the empirical
model can explain over 89% of variability of the extraction yield. High
Having selected main variables, response surface methodology
closeness between R2 and R2-adj (< 3%) indicates that the terms of the
based on Box-Behnken design was employed for optimization. Two
model are significant. Moreover, it is expected that this model shall
factors were fixed at convenient levels, namely: solvent type (methanol)
explain about 81% of variability in predicting new observations
and extraction cycles (2 extractions). Table 4 shows the design matrix
(Montgomery, 2013).
and the results, which are expressed as a mean of DHPB ± standard
Fig. 3 shows the contour plots of the interaction between the three
deviation (SD). The levels of extraction time and sample:solvent ratio
tested factors and the relationship between yield and experiment levels.
are the same as those applied in screening experiment, since their op-
Holding sample:solvent ratio at 1 mg:50 μL (Fig. 3a), the degree of
timum range seems to fall in predefined range (1–5 min for extraction
extraction increases as time and stirring rate increase. The highest yield
time and 1mg:25 μL to 1 mg:75 μL for sample:solvent ratio). On the
was achieved when extraction time and stirring rate were in the range
other hand, the stirring rate range was increased to 1400–3200 rpm)
of 2.5–5.0 min and 2000–3200 rpm, respectively. When a short ex-
and its optimum range was found to be beyond 2200 rpm (Fig. 2).
traction time (∼2.5–3.0 min) was applied, the stirring frequency should
Evaluation by ANOVA (Table 5), indicates that the quadratic model
be high to ensure good extraction efficiencies. This is most likely related
is statistically significant, confirming the curvature previously pre-
to the fact that seed samples are rich in oil and, when crushed, they
dicted by Plackett-Burman design. The relationship between the ex-
become pasty which hinders the solvent penetration; and, conse-
traction yield and significant variables is described by regression model
quently, the lower extraction yield. Increasing stirring rate, the inter-
of the Eq. (2).
molecular forces (van der Waals attractions, hydrogen and dipole-di-
DHPB (mg/g) = 0.6753 + 0.0562 A + 0.1211 B + 0.0264 D – 0.1080 pole (cohesive and adhesive) bonds) are weakened (De Castro and
B2 – 0.0546 D2 – 0.0610 AB (2) García, 2002). So, the extraction yield increases as result of increasing
of the surface area. This fact was confirmed by the sample: solvent ratio
Where: A – stirring rate (rpm), B – extraction time (min), C –

Fig. 2. Main effects plot from Plackett-Burman design showing the impact of the factors.

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Fig. 3. Contour plots for average extraction yield of DHPB: (a) Extraction time vs. stirring rate; (b) Sample:solvent ratio vs stirring rate; (c) Sample:solvent ratio vs
extraction time.

vs. stirring rate plot (Fig. 3b), which showed that there is a very close Table 6
relationship between the yield and the stirring rate than the amount of Summary of validation results.
solvent. Fig. 3c shows that high yield was obtained when extraction was Validation parameter Value
performed during 4.0–5.0 min at sample: solvent ratio of 1:30, at least.
However lower ratio hampers the achievement of clear phase separa- LOD (ng μL−1) 4.41
LOQ (ng μL−1) 13.36
tions and it is recommended to work with sample:solvent of 1mg:50 μL.
Repeatability (mg g−1) 1.24 ± 0.02
According to these observations, the extraction was carried out with (RSD = 1.89 %)
methanol, at sample:solvent ratio of 1 mg:50 μL, under vortex stirring at Robustness
3200 rpm for 3 min, with 2 extraction cycles. The content of PEs (mg Condition I (mg g−1) 1.17 ± 0.027
g−1) obtained in these conditions was 1.24 ± 0.024. Condition II (mg g−1) 1.24 ± 0.024
Recovery at spike level of
50 % 87.28 % ± 6.79
100 % 95.54 % ± 4.40
3.3. Analytical performance of the method 150 % 84.09 % ± 3.48
Linearity r = 0.999
This step aimed to ensure that the developed method is suitable for Separation factors
αC2/C1 1.10
the intended use and that the results derived from it can be utilized to
αC3/C2 1.13
determine the reliability and consistency of the analytical data obtained αC6/C3 1.06
(Fuster et al., 2015). The validation parameters are summarized in αC4+C5/C6 1.07
Table 6.
The calibration curve showed a strong and positive (r = 0.999,
R2 = 0.999) relationship between the concentration of DHPB and the
peak area, over all tested analytical range of 2.5–50.0 ng μL−1.
Studies reporting LOD and LOQ of the methods for determination of
PEs are scarce and mostly estimated as equivalent of 12-O-tetra-
decanoylphorbol-13-acetate, which don’t occur in Jatropha (Makkar,
2016). In this study, the LOD and LOQ of the method were found to be
4.41 and 13.36 ng μL−1, respectively. The mean of recoveries at the
three concentration levels tested is 89.0% ± 6.59 and the method also
showed a good repeatability with RSD of 1.89%.
As can be seen in Fig. 4, the typical chromatogram PEs, no inter-
fering peaks in the range of retention of PEs could be detected. Ad- Fig. 4. Chromatogram of methanol extracts of seeds, highlighting the five peaks
ditionally, all separation factors (Table 6) of the nearest peaks of PEs of the PEs at the retention time range of 8.5–13 min, obtained under conditions
described in the Section 2.5.
were found to be greater than 1. These facts are evidence of the

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selectivity of the method.
Extraction time and stirring rate are the factors varied to investigate
the method robustness. The paired t-test (at 95%) was employed for
comparison of the yield means obtained from both conditions. Since
tcrit > |tcalc| (12.71 > 2.13), the differences are not statistically sig-
nificant and, hence, the method is not affected by small and deliberated
changes of the experimental conditions.
4. Conclusions
A method of extraction of PEs from Jatropha seeds was optimized
and validated for the first time for this sample matrix. It is an accurate
and simple procedure that besides requiring shorter time than com-
monly used methods (around 10 min compared to 20–80 min), offers
quantitative recoveries and consumes reduced amount of both sample
and solvent. The analysis can be performed having only 20 mg of
sample and 3 mL of solvent. Plackett-Burman and Box-Behnken designs
showed that the extraction yield is mainly affected by the stirring rate,
presumably due to increasing of the surface area. Extraction time,
sample:solvent ratio, and extraction cycles gave the best yield around
its centre points, while methanol proved to be ideal extraction solvent.
A quadratic empirical model was found to be suitable to describe the
PEs extraction yield as a function of the most important factors and its
interactions. This model covers over 89% of variability in the experi-
mental data and has good predictive capacity. This method is applicable
for extraction of PEs from Jatropha seeds for different purposes, in-
cluding the toxicity screening of the seeds (for breeding purposes), risk
assessment on handling and processing Jatropha products, and isolation
of PEs for application in agriculture and pharmaceutical studies.
Conflict of interests
The authors declare no conflict of interests
Acknowledgements
The authors would like to express their gratitude to Japan Science
and Technology Agency (JST) and the Japan International Cooperation
Agency (JICA), for financial support, and to all members of the Project
Sustainable Production of Biodiesel from Jathropha in Mozambique,
especially from Kurume University.
References
Baldini, M., Ferfuia, C., Bortolomeazzi, R., Verardo, G., Pascali, J., Piasentier, E.,
Franceschi, L., 2014. Determination of phorbol esters in seeds and leaves of Jatropha
curcas and in animal tissue by high-performance liquid chromatography tandem
mass spectrometry. Ind. Crops Prod. 59, 268–276. https://doi.org/10.1016/j.
indcrop.2014.05.034.
Brittaine, R., Lutaladio, N., 2010. Jatropha: A Smallholder Bioenergy Crop – The Potential
for Pro-Poor Development Vol. 8 FAO, Rome.
De Castro, M.D.L., García, J.L.L., 2002. High-pressure, high-temperature solvent extrac-
tion. Acceleration and Automation of Solid Sample Treatment, 1st ed. Elsevier
Science B.V., pp. 233–279. https://doi.org/10.1016/S0167-9244(02)80008-7.
Devappa, R.K., 2012. Isolation, Characterization and Potential Agro-pharmaceutical
946
Industrial Crops & Products 124 (2018) 941–946
Applications of Phorbol Esters From Jatropha curcas Oil. Doctoral thesis. Department
of Aquaculture and Animal Nutrition (480b), University of Hohenheim.
Devappa, R.K., Makkar, H.P.S., Becker, K., 2010a. Biodegradation of Jatropha curcas
phorbol esters in soil. J. Sci. Food Agric. 90, 2090–2097. https://doi.org/10.1002/
jsfa.4056.
Devappa, R.K., Makkar, H.P.S., Becker, K., 2010b. Jatropha toxicity: a review. J. Toxicol.
Environ. Health Part B 13 (6), 476–507. https://doi.org/10.1080/10937404.2010.
499736.
Devappa, R.K., Makkar, H.P.S., Becker, K., 2010c. Optimization of conditions for the
extraction of phorbol esters from Jatropha oil. Biomass Bioenergy XXX, 1–9. https://
doi.org/10.1016/j.biombioe.2010.03.001.
Devappa, R.K., Angulo-Escalante, M.A., Makkar, H.P.S., Becker, K., 2012. Potential of
using phorbol esters as an insecticide against Spodoptera frugiperda. Ind. Crops Prod.
38 (1), 50–53. https://doi.org/10.1016/j.indcrop.2012.01.009.
Devappa, R.K., Bingham, J.-P., Khanal, S.K., 2013a. High performance liquid chromato-
graphy method for rapid quantification of phorbol esters in Jatropha curcas seed. Ind.
Crops Prod. 49, 211–219. https://doi.org/10.1016/j.indcrop.2013.04.044.
Devappa, R.K., Roach, J.S., Makkar, H.P.S., Becker, K., 2013b. Ecotoxicology and en-
vironmental safety occular and dermal toxicity of Jatropha curcas phorbol esters.
Ecotoxicol. Environ. Saf. 94, 172–178. https://doi.org/10.1016/j.ecoenv.2013.04.
021.
Fuster, J., Negro, S., Salama, A., Marcianes, P., Boeva, L., Barcia, E., 2015. HPLC-UV
method development and validation for the quantification of ropinirole in new PLGA
multiparticulate systems: microspheres and nanoparticles. Int. J. Pharm. 491,
310–317. https://doi.org/10.1016/j.ijpharm.2015.06.035.
Gogoi, R., Niyogi, U.K., Tyagi, A.K., 2014. Reduction of phorbol ester content in jatropha
cake using high energy gamma radiation. J. Radiat. Res. Appl. Sci. 7, 2–6. https://doi.
org/10.1016/j.jrras.2014.04.002.
Gübitz, G.M., Mittelbach, M., Trabi, M., 1999. Exploitation of the tropical oil seed plant
Jatropha curcas L. Bioresour. Technol. 67 (1), 73–82. https://doi.org/10.1016/
S0960-8524(99)00069-3.
Haas, W., Sterk, H., Mittelbach, M., 2002. Novel 12-deoxy-16-hydroxyphorbol diesters
isolated from the seed oil of Jatropha curcas. J. Nat. Prod. 65 (10), 1434–1440.
https://doi.org/10.1021/np020060d.
Hirota, M., Suttajit, M., Suguri, H., Endo, Y., Shudo, K., Wongchai, V., Hecker, E., Fujiki,
H., 1988. A new tumor promoter from the seed oil of Jatropha curcas L., an in-
tramolecular diester of 12-deoxy-16-hydroxyphorbol. Cancer Res. 48, 5800–5804.
Kumar, P., Srivastava, V.C., Jha, M.K., 2016. Jatropha curcas phytotomy and applica-
tions: development as a potential biofuel plant through biotechnological advance-
ments. Renew. Sustain. Energy Rev. 59, 818–838. https://doi.org/10.1016/j.rser.
2015.12.358.
Makkar, H.P.S., 2016. State-of-the-art on detoxification of Jatropha curcas products
aimed for use as animal and fish feed: a review. Anim. Feed Sci. Technol. https://doi.
org/10.1016/j.anifeedsci.2016.09.013.
Makkar, H.P.S., Becker, K., Sporer, F., Wink, M., 1997. Studies on nutritive potential and
toxic constituents of different provenances of Jatropha curcas. J. Agric. Food Chem.
45 (8), 3152–3157.
Martínez-Herrera, J., Siddhuraju, P., Francis, G., Dávila-Ortíz, G., Becker, K., 2006.
Chemical composition, toxic/antimetabolic constituents, and effects of different
treatments on their levels, in four provenances of Jatropha curcas L. from Mexico.
Food Chem. 96 (1), 80–89. https://doi.org/10.1016/j.foodchem.2005.01.059.
Montgomery, D.C., 2013. Design and Analysis of Experiments, 8th ed. John Wiley & Sons,
Inc., USA.
Najjar, A., Abdullah, N., Saad, W.Z., Ahmad, S., Oskoueian, E., Abas, F., Gherbawy, Y.,
2014. Detoxification of toxic phorbol esters from Malaysian Jatropha curcas Linn.
kernel by Trichoderma spp. and endophytic fungi. Int. J. Mol. Sci. 15 (2), 2274–2288.
https://doi.org/10.3390/ijms15022274.
Pereira, C., de, S.S., Pessoa, F.L., Mendonca, S., Ribeiro, J.A., de, A., Mendes, M.F., 2015.
Technical and economic evaluation of phorbol esters extraction from Jatropha curcas
seed cake using supercritical carbon dioxide. Adv. Chem. Eng. 5 (3), 1–7. https://doi.
org/10.4172/2090-4568.1000132.
Takechi, S., Imou, K., 2015. Sustainable energy production system from Jatropha in
Mozambique project. 22nd International Conference on Production Research.
pp. 1–6.
Wink, M., Koschmieder, C., Sauerwein, M., Sporer, F., 1997. Phorbol esters of J. curcas –
biological activities and potential applications. In: Gubitz, G.M., Mittelbach, M.,
Trabi, M. (Eds.), Biofuels and Industrial Products from Jatropha curcas. DBV Graz,
Germany, pp. 160–166.