Characterization of benzyl isothiocyanate extracted from mashed green papaya by distillation

Article abstract of DOI:10.1016/j.foodchem.2019.125118

The aim of this study was to extract benzyl isothiocyanate (BITC) from green papaya by distillation apparatus without using organic solvents, and to improve the stability of BITC in aqueous solution. The distillation of mashed green papaya successfully yielded BITC as a water solution with more than 80% purity with good reproducibility. The amount of BITC in the distilled water gradually decreased during its storage at 4 °C, whereas it was not significantly changed at ?20 °C for a few months. Moreover, the addition of L-cysteine ameliorated the BITC decomposition by the 4 °C-storage, but not affected by N-acetyl-cysteine and glutathione. These results suggested that the combination of BITC extraction by distillation and cysteine supplementation as well as frozen storage might be a useful method for the preparation and storage of the safer grade of BITC-containing extract.

Full text of DOI:10.1016/j.foodchem.2019.125118

Food Chemistry 299 (2019) 125118
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Short communication
Characterization of benzyl isothiocyanate extracted from mashed green
papaya by distillation
⁎
Toshiyuki Nakamura, Yoshiyuki Murata, Yoshimasa Nakamura
Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Benzyl isothiocyanate
Papaya
Extraction
Stability
The aim of this study was to extract benzyl isothiocyanate (BITC) from green papaya by distillation apparatus
without using organic solvents, and to improve the stability of BITC in aqueous solution. The distillation of
mashed green papaya successfully yielded BITC as a water solution with more than 80% purity with good
reproducibility. The amount of BITC in the distilled water gradually decreased during its storage at 4 °C, whereas
it was not significantly changed at −20 °C for a few months. Moreover, the addition of L-cysteine ameliorated the
BITC decomposition by the 4 °C-storage, but not affected by N-acetyl-cysteine and glutathione. These results
suggested that the combination of BITC extraction by distillation and cysteine supplementation as well as frozen
storage might be a useful method for the preparation and storage of the safer grade of BITC-containing extract.
1. Introduction
there are only limited reports concerning the stability of ITCs and DTCs
in aqueous solutions.
Isothiocyanates (ITCs) are organic sulfur compounds that are mainly
Based on the chemical characteristics of the ITCs, such as hydro-
phobicity and instability, organic solvents have been used for the ex-
traction of the ITCs from food materials with good yield. Fahey et al. has
shown that 4-methylsulfinylbutyl ITC (sulforaphane) was extracted
from broccoli sprouts using dimethyl sulfoxide (DMSO) and acetonitrile
(Fahey, Zhang, & Talalay, 1997). Nakamura et al. has also reported that
BITC was extracted from papaya seed and edible pulp using methanol
and hexane with a substantive recovery (Nakamura et al., 2007).
However, the extraction of the chemicals using organic solvents is not
suitable for the preparation of food-grade products. In addition, to the
best of our knowledge, there is no report showing that ITCs can be
extracted as aqueous solutions.
The aim of this study is to extract BITC from green papaya, which is
one of the BITC-rich plant materials, by distillation without using or-
ganic solvents, and also to improve the stability of BITC in aqueous
solution. In this study, BITC was successfully extracted from mashed
green papaya by distillation without using an organic solvent with the
relatively high purity. Moreover, the present study demonstrated that
not only frozen storage, but also supplementation of L-cysteine allows
the stability of BITC in the distilled water to be maintained.
contained in cruciferous vegetables. ITCs have been reported to have
human health-promoting properties, such as antioxidative, anti-in-
flammatory, and anti-cancer effects (Mi, Di Pasqua, & Chung, 2011;
Nakamura, Abe-Kanoh, & Nakamura, 2018). Although ITCs have these
effects, ITCs are easily decomposed by the addition of a hydroxyl ion to
the ITC group under a neutral or alkaline condition (Kawakishi &
Namiki, 1969; Pecháček, Velíšek, & Hrabcová, 1997). In addition, the
rearrangement of the ITCs into thiocyanates occurs during the gluco-
sinolate metabolism or storage in aqueous solutions, resulting that the
biological effectiveness of ITCs is considerably reduced. Thus, ITCs in
an aqueous solution might be too unstable to utilize as food additives.
As for in vivo situation, ITCs can quickly conjugate with the reduced-
form glutathione (GSH), the most abundant thiol compound in the cells,
as well as L-cysteine and form dithiocarbamates (DTCs). The DTCs
themselves are also unstable in an aqueous buffer because the con-
jugates can be dissociated into free ITC and thiol compounds. However,
the dissociated ITC can react with a free thiol compound again, and
then DTCs exist in equilibrium with the free form (Jiao et al., 1996).
Thus, the stability of ITC itself is thought to ameliorate by the addition
of the thiol compounds to maintain the conjugated forms. However,
Abbreviations: AITC, allyl isothiocyanate; BITC, benzyl isothiocyanate; DMSO, dimethyl sulfoxide; DTCs, dithiocarbamates; DTNB, 5,5′-dithibis-(2-nitrobenzoic
acid); GC-EI-MS, gas chromatography–electron ionization–mass spectrometry; GSH, glutathione; HPLC-UV, high-performance liquid chromatography with ultraviolet
detection; ITCs, isothiocyanates; NAC, N-acetyl-L-cysteine
⁎
Corresponding author.
E-mail addresses: t-nakamura@okayama-u.ac.jp (T. Nakamura), muta@okayama-u.ac.jp (Y. Murata), yossan@cc.okayama-u.ac.jp (Y. Nakamura).
https://doi.org/10.1016/j.foodchem.2019.125118
Received 21 February 2019; Received in revised form 25 June 2019; Accepted 1 July 2019
Available online 02 July 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

T. Nakamura, et al.
Food Chemistry 299 (2019) 125118
2. Materials and methods
1000000
(A)
2.1. Chemicals
Blank (TIC)
0
BITC was purchased from LKT Laboratories, Inc. (St. Paul, MN,
5
5
5
10
10
10
15
15
15
20
20
20
25
30
USA). 1,2-Benzenedithiol and GSH were obtained from FUJIFILM Wako
Pure Chemical Corporation (Osaka, Japan). L-Cysteine was purchased
from Ishizu Pharmaceutical (Osaka, Japan). N-Acetyl-L-cysteine (NAC)
and 5,5′-dithibis-(2-nitrobenzoic acid) (DTNB) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). S-(N-Benzylthiocarbamoyl)-L-cy-
steine was obtained from Abcam (Cambridge, MA, USA). All other
chemicals were purchased from FUJIFILM Wako Pure Chemical
Corporation (Osaka, Japan) or Nacalai Tesque Inc. (Kyoto, Japan) with
analytical grade.
(
B)
C)
1
000000
Papaya water (TIC)
0
25
30
(
100000
BITC (m/z 149)
2.2. Extraction of BITC from papaya by distillation
0
The green papaya was purchased from a local market in Okinawa,
25
30
Kanagawa and Hiroshima, Japan, in April 2016, June 2018, May 2019
and June 2019. The edible part of the green papaya (250 g) was placed
in a commercial blender (IFM-C20G, Iwatani) and crashed for 1 min
without adding water. After this process was repeated twice, the papaya
pastes (final volume, 500 g) were heated using an oil bath at 100 °C,
and the steam was gathered using a Liebig condenser with coolant
water. The distilled water was collected and the yield was approxi-
mately 300 mL. The distilled water were analyzed by a gas chromato-
graphy–electron ionization–mass spectrometry (GC-EI-MS) system
Time (min)
Fig. 1. Total ion chromatograms of blank (A) and the distilled papaya water
(B), and selected ion monitoring of BITC (C).
2
.5. Analysis of the conjugates of BITC and L-cysteine, NAC and GSH
The conjugation of BITC with L-cysteine, NAC or GSH in the distilled
water was determined by reverse-phase HPLC-UV at 254 nm (Waters
ACQUITY UPLC H-Class) as described previously (Conaway,
Krzeminski, Amin, & Chung, 2001) with some modifications. Briefly,
the HPLC separation was done by a gradient system using solvent A
(
5
Shimadzu GCMS-QP2010 plus) equipped with a 60 m × 0.25 mm Rtx-
MS column. The injector was used in the splitless mode, and the scan
range was from m/z 50 to m/z 1000. Sample injection volume was 1 µL.
The temperature of the column oven was held at 50 °C for 5 min before
being raised to 300 °C at the rate of 10 °C/min, which was then held
constant for 5 min.
(
0.1% formic acid) and solvent B (acetonitrile) and an ACQUITY UPLC
BEH C18 (2.1 × 50 mm) column at the flow rate of 0.4 mL/min with a
column oven temperature of 40 °C. Sample injection volume was 2 µL.
The gradient program was 0 min (A 85%), 0.5 min (A 85%), 4.4 min (A
2.3. Stability of BITC under aqueous conditions
0
%), 4.9 min (A 0%), 5.0 min (A 85%), and 6.0 min (A 85%). The
concentrations of these conjugates were calculated as equivalent to the
authentic standard of S-(N-benzylthiocarbamoyl)-L-cysteine.
The distilled water from the green papaya was stored at 4, −20 and
80 °C in 1.5 mL tubes for the indicated periods. To investigate the
−
effect of the thiol compounds, such as L-cysteine, NAC and GSH, on the
BITC stability, BITC in aqueous solution was prepared as described
previously (Ohta, Takatani, & Kawakishi, 2000) with some modifica-
tions. Briefly, authentic BITC dissolved in DMSO (50 mM) was diluted
in distilled water (the final BITC and DMSO concentrations of 50 µM
and 0.1%, respectively). L-Cysteine, NAC or GSH (1 mM) was added to
this water solution. These samples were stored at 4 °C for the indicated
periods.
2
.6. Measurement of the free thiol groups of L-cysteine, NAC and GSH in the
water containing BITC
The amounts of free thiol in L-cysteine, NAC and GSH in the water
containing BITC were measured using DTNB as described previously
(
Nakamura, Kawai, Kitamoto, Osawa, & Kato, 2009) with some mod-
ifications. Briefly, DTNB (10 mM) was dissolved in 50 mM phosphate
buffer (pH 7.4). The DTNB solution (10 µL) was mixed with 10 µL of L-
cysteine, NAC or GSH in the water containing BITC in 480 µL of the
2.4. Cyclocondensation assay
5
0 mM phosphate buffer (pH 7.4). After incubation for 15 min at room
The concentrations of BITC in the aqueous solutions were measured
by a cyclocondensation assay as previously reported (Zhang, Cho,
Posner, & Talalay, 1992) with some modifications. Briefly, the samples
150 µL) were incubated with 250 µL of 8 mM 1,2-benzenedithiol in
ethanol and 100 µL of 100 mM potassium phosphate buffer (pH 8.5) for
h at 65 °C. After incubation using a water bath, the reaction solutions
temperature in the dark, an aliquot (200 µL) was measured at 412 nm
using a microplate reader (Benchmark Plus, Bio-Rad). The concentra-
tion of free thiol was calculated from the molar extinction coefficient
−
1 −1
(
(
14,100 M
L
).
2
were centrifuged at 19,000×g at 4 °C for 5 min using a high-speed re-
frigerated micro centrifuge (TOMY MX-305). The supernatants con-
taining the reaction product, 1,3-benzodithiole-2-thione, were analyzed
by reverse-phase high-performance liquid chromatography with ultra-
violet detection (HPLC-UV) at 365 nm (Waters ACQUITY UPLC H-
Class). The HPLC separation was done with isocratic elution (0.1%
formic acid/acetonitrile = 20/80) using an ACQUITY UPLC BEH C18
2
.7. Statistical analyses
All values were presented as means ± S.D. Statistical analyses
comparing the thiol compound supplementation groups with the un-
treated group (Day 1) were performed by Student’s t-test, and com-
parison of the BITC purity between the green papaya samples was
evaluated by chi-square test, using Microsoft Excel software (version
(
2.1 × 50 mm) column (Waters, Milford, MA, USA) at the flow rate of
.4 mL/min with a column oven temperature of 40 °C. Sample injection
1
4.7.3).
0
volume was 2 µL. The limit of detection for 1,3-benzodithiole-2-thione
was 100 nM.
2

T. Nakamura, et al.
Food Chemistry 299 (2019) 125118
Fig. 2. Time-dependent changes in the BITC levels in the distilled water during the 4 °C-, −20 °C- or −80 °C-storage. The concentrations of BITC in the distilled water
was measured by a cyclocondensation assay. The formation of the reaction product, 1,3-benzodithiole-2-thione, was determined by reverse-phase HPLC-UV at
365 nm. (A) Standard curve of 1,3-benzodithiole-2-thione and (B) time-dependent changes of the BITC levels in the distilled water. All values were expressed as
means ± SD of four separate experiments.
3
. Results
water from the papaya during storage were determined by HPLC-UV
with the cyclocondensation reaction using 1,2-benzenedithiol. The
standard curve of 1,3-benzodithiole-2-thione, a product of the cyclo-
condensation assay, is shown in Fig. 2A. The calculation data using this
standard curve showed that BITC in the distilled water rapidly dimin-
ished over time, and it was hardly detected 90 days after storage at 4 °C
(Fig. 2B). At 90 days, the BITC level in the distilled water was estimated
at only 2.24 ± 0.12 µM.
BITC is considered to be unstable in an aqueous solution because of
its decomposition by the reaction with a hydroxyl ion (Kawakishi &
Namiki, 1969; Pecháček et al., 1997). Next, the stabilities of BITC in the
water at −20 and −80 °C were investigated. The degradation of BITC
was effectively prevented by each low temperature storage (Fig. 2B).
3.1. Extraction of BITC from mashed green papaya by distillation
The volatile compounds were extracted from the edible part of the
green papaya using a Liebig condenser. The contents of BITC and other
volatiles in the distilled water from the mashed green papaya were
measured by GC–MS. As shown in Fig. 1, from the extract of the green
papaya purchased in Okinawa, BITC was detected as a major peak with
one minor peak, probably due to benzyl nitrile, a degradation product
of BITC. The content percentage of BITC in this green papaya was es-
timated to be 85.6% based on the peak area, and the concentration of
BITC in the distilled water was calculated to be 45 µM. BITC was also
detected as a major peak with almost same purity (85.6 ± 3.0%) from
all the green papaya fruits used in this study, in spite of the purchased
regions and seasons. The statistical analysis by chi-square test revealed
that there were no differences in the purity of BITC between the green
papaya samples.
3.3. Effect of L-cysteine, NAC or GSH on the BITC stability in the distilled
water
To improve the BITC stability, excess amounts of the thiol com-
pounds, L-cysteine, NAC or GSH, were added to BITC in the distilled
water. As shown in Fig. 3A, the addition of L-cysteine effectively pre-
vented BITC from decomposition during 4 °C storage, and the amounts
of BITC were maintained during storage for 14 days. In contrast, the
3.2. Storage time-dependent change in the BITC level in the distilled water
The time-dependent changes in the BITC amount in the distilled
3

T. Nakamura, et al.
Food Chemistry 299 (2019) 125118
Fig. 3. Modulating effects of thiol compounds on the
BITC stability in the distilled water. (A) Effects of L-
cysteine, NAC and GSH on the BITC stability. The
concentrations of BITC in the distilled water were
measured by a cyclocondensation assay with the
HPLC-UV analysis. (B) The conjugate formation of
BITC with L-cysteine, NAC and GSH in the distilled
water. The formed conjugates were analyzed by re-
verse-phase HPLC-UV at 254 nm. Their concentra-
tions were estimated using the standard curve of the
(
A) 50
4
3
2
1
0
0
0
0
0
Day 1
Day 4
Day 7
Day 14
*
*
*
*
*
*
*
authentic
S-(N-benzylthiocarbamoyl)-L-cysteine,
which is equivalent to the other conjugates. (C)
Stability of the free thiol groups in the thiol com-
pounds during storage. The amounts of free thiol
groups of L-cysteine, NAC and GSH in water were
measured by a DTNB assay. All values were ex-
pressed as means ± SD of three separate experi-
ments. An asterisk indicates significant difference
between the thiol compound supplementation
groups and the untreated group (P < 0.05).
untreated
Cont
Cys
NAC
GSH
(
B)
Day 1
4
3
2
1
0
0
0
0
0
Day 4
Day 7
Day 14
Cys
NAC
GSH
(C)
Day 1
Day 4
Day 7
Day 14
1
*
*
*
*
*
0
.5
0
Cys
NAC
GSH
BITC amount gradually declined in the presence of NAC or GSH com-
parable to the control group. The 5-fold higher concentration of NAC or
GSH did not inhibit the BITC degradation (data not shown). HPLC
analysis demonstrated that, in the presence of L-cysteine, BITC existed
as the conjugated form with L-cysteine in the distilled water (Fig. 3B).
On the other hand, NAC and GSH were hardly conjugated with BITC in
the water, even though the free thiol groups of NAC and GSH as well as
L-cysteine were not degraded but still reactive in the distilled water
BITC and other constituents, such as polarity and volatility, might allow
the highly selective extraction for BITC. It was also confirmed that BITC
in the distilled water has a potency to induce the drug metabolizing
enzyme genes, heme oxygenase 1 and NAD(P)H quinone oxidor-
eductase 1, using the cell culture medium (minimum essential medium
alpha powder (Gibco, NY, USA) with 10% fetal bovine serum) made of
the distilled water from green papaya (papaya water medium)
(Supplemental Fig. 1), similar to that of the authentic BITC (Liu et al.,
2017). The stabilities of BITC in the distilled water and in the medium
were determined by not only its biological activity but also the quan-
tification of BITC (Fig. 2B and Supplemental Fig. 2). Expectedly, BITC in
the water solution is very unstable and gradually disappeared during
storage. Although the medium contains some nutritional components,
such as amino acids and proteins, the BITC stability was not changed in
the water and in the medium for 28 days (Supplemental Fig. 2).
Moreover, BITC was more stable at −80 °C than that at −20 °C
(
Fig. 3C).
4. Discussion
In this study, BITC was successfully extracted as a major component
with more than 80% purity from the mashed green papaya without
using an organic solvent. This study is, to the best of our knowledge, the
first report to extract BITC by distillation from plant food materials.
Since several components have been detected in the papaya (Flath &
Forrey, 1977), the difference of physico-chemical properties between
(
Fig. 2B), suggesting that the temperature is one of the most important
determinants for the BITC stability in the water.
4

T. Nakamura, et al.
Food Chemistry 299 (2019) 125118
Interestingly, the decomposition of BITC in the distilled water was
prevented by the addition of L-cysteine, but not NAC and GSH (Fig. 3A).
It has been reported that allyl ITC (AITC) was decomposed by thermal
treatment, which was influenced by the pH condition (Chen & Ho,
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.125118.
1998). Ohta et al. reported that the decomposition of AITC in an aqu-
eous solution was retarded by inclusion complexation within the cav-
ities of α-cyclodextrin (CD) and β-CD (Ohta et al., 2000). The sup-
pression mechanism of the decomposition of BITC and phenyl ITC by
CD might be almost the same as the AITC-α-CD complex (Ohta, Matsui,
Osawa, & Kawakishi, 2004). There is, however, no report on the effect
of thiol compounds on the BITC stability in aqueous solutions, even
though thiol compounds used in this study are well known to primarily
react with BITC in vivo and in vitro (Hong, Freeman, & Liebler, 2005;
Zhang, 2000). As shown in Fig. 3B, the formation of the BITC-cysteine
conjugate in distilled water was observed for 14 days, whereas the
BITC-GSH and BITC-NAC were detected only at a low level at all times.
The much lower levels of the NAC or GSH conjugates might not be due
to lack of thiol groups, because decreases of free thiol groups in L-cy-
steine, NAC and GSH in the distilled water were very slight during the
incubation (Fig. 3C). Although the reason why BITC hardly reacts with
NAC and GSH in an aqueous solution remains to be determined, L-cy-
steine prevents BITC from its decomposition in an aqueous solution,
possibly through the conjugate formation.
The ITC exposure to the cells leads to the rapid and high in-
tracellular accumulation mainly through simple diffusion. The BITC-
GSH conjugate is produced as the primary metabolite after BITC was
absorbed in vivo (Nakamura et al., 2018). BITC-GSH is stepwise meta-
bolized to BITC-cysteinylglycine, BITC-cysteine, and BITC-NAC via the
mercapturic acid pathway. Although BITC is present in our body as
conjugated forms, the conjugates have similar biological activities with
the intact ITCs (Conaway et al., 2005). Taken together with its lower
toxicity than intact BITC, the combination with L-cysteine is one of the
promising strategies to enhance the BITC stability in an aqueous solu-
tion.
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BITC from the mashed green papaya using distillation apparatus. The
volatile BITC can be extracted from food material without using organic
solvents. In addition, the effects of different temperatures and thiol
supplementation on the stability of BITC in aqueous solution were in-
vestigated. Finally, the decomposition of BITC could be inhibited by the
frozen storage or the addition of L-cysteine. The present results support
the idea that the distillation of mashed green papaya might be a useful
method for the preparation of the safer grade of BITC-containing ex-
tract.
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This study was partly supported by MEXT KAKENHI Grant Numbers
7H04725 (TN), 16K14928, and 17H03818 (YN). We thank Ryuji
1
Takata and MANAC Incorporated for their technical supports.
Further reading
Declaration of Competing Interest
Kumar, A., & Sabbioni, G. (2010). New biomarkers for monitoring the levels of iso-
The authors declare no conflict of interest.
thiocyanates in humans. Chemical Research in Toxicology, 23(4), 756–765.
5