Isothiocyanate content in mustard oils from

white, brown and black mustard after

conventional and modern extraction
techniques

Ivana Vrca, PhD student

University of Split

Faculty of Chemistry and Technology

 PLANT MATERIAL
•  Brassicaceae family
•  Mostly used for mustard producCon
•  Contains sulfur compounds (glucosinolates)

A sharp flavor of mustard leads to

an explanaCon of what
glucosinolates are!

BLACK MUSTARD SEEDS

Brassica nigra L.
WHITE MUSTARD SEEDS
Sinapis alba L. BROWN MUSTARDS SEEDS
Brassica juncea L.
And glucosinolates
are…

GLUCOSINOLATES AND THEIR PRODUCTS
•  Glucosinolates are natural organic compounds (secondary plant metabolites)

•  In plant Cssues, the glucosinolates are present at the same Cme as the
enzyme myrosinase
•  Myrosinase is distributed in myrosin cells that do not contain glucosinolates

DegradaCon of glucosinolates Myrosinase cells

The first glucosinolates
are isolated from white
and black mustard 1830.!
 Isothiocyanates

AnHcancer AnHmicrobial AnHfungal AnHoxidant

acHvity acHvity acHvity acHvity
 CONVENTIONAL METHODS OF
EXTRACTION OF VOLATILE COMPOUNDS
FROM SAMPLES OF MUSTARD SEEDS

•  ExtracHon with organic solvent •  HydrodisHllaHon (apparatus by Clevenger)

aMer autolysis
•  DisCllaCon with solvent (water)
•  DisClled water
•  Uneven warming
•  Dichloromethane
•  Slow method
•  Slow method
MODERN METHOD OF EXTRACTION OF
VOLATILE COMPOUNDS FROM SAMPLES
OF MUSTARD SEEDS
Microwave-assisted disHllaHon Microwave-assisted extracHon (flavors)
(fragrance) Microwave oven
Glass reactor
Cooling system

EssenCal oil
Aqueous phase
Cooling system

Water reflux
Glass reactor
Aqueous phase
Microwave oven
Dichloromethane volaCle extract

Microwave extracHon (ETHOS X)

•  No added solvents
•  Uniform heaCng
•  Fast method
 EXPERIMENTAL PART

1. Screening of surface mustard seeds morphology by SEM (scanning electron

microscopy) and opCcal microscopy
2. ApplicaCon of convenConal and modern extracCon techniques in order to
extract volaCle sulfur-containing compounds from mustard seeds
3. Analysis of volaCle sulfur-containing compounds by GC-MS technique

 EXPERIMENTAL PART

SEM microscope Sample (mustard seed) OpHcal microscope

ExtracHon with organic solvent

aMer autolysis HydrodisHllaHon in Clevenger MAE
type apparatus

Sample concentraHon by
GC- MS analysis nitrogen evaporator
SEM MICROSCOPE
1) White mustard seed 2) Brown mustard seed 3) Black mustard seed
(whole and crushed) (whole and crushed) (whole and chrushed)
OPTICAL MICROSCOPE

4) Brown mustard seed (whole and crushed)


 Working condiHons

Autolysis

HydrodisHllaHon Microwave GC-MS analysis

(ExtracHon with (Apparatus by Clevenger) Mobile phase:Helium
organic solvent) extracHon StaHonary phase: Nonpolar
Colon: VF-5MS

Crushed seeds, 10 g, Crushed seeds, 50 g, 2.5 h,

24 h, 37 °C 100 °C MW hydrodisHllaHon
Powder, 20 g, 6 h, 37 °C Crushed seeds (100 g), 1 h soaking,
500 W, 30 min
Powder (70 g), 30 min soaking,
500 W, 30 min


MW extracHon
Crushed seeds (100 g), 3 h soaking,
500 W, 15 min
Powder (70 g), 30 min soaking,
500 W, 15 min

 GC-MS RESULTS
White mustard
Myrosinase
GLS Precursor Chemical composiCon R.T.

Sinigrin Allyl isothiocyanate

5.83 min

Where: CondiHons:
MW extract 12.68 % Powder, 70 g, 30 min soaking, 500 W, 15 min
Clevenger hydrodisCllate - Tr Crushed seeds, 50 g, 100 °C

§  Tr-traces, less than 0,5%

White mustard
 GC-MS RESULTS
Brown mustard
Myrosinase
GLS Precursor Chemical composiCon R.T.

Sinigrin Allyl isothiocyanate 5.83 min

Gluconapin But-3-enyl isothiocyanate 9.23 min

Brown mustard
 GC-MS RESULTS
Brown mustard
Allyl isothiocyanate
•  Where: CondiHons:
•  MW disCllate 80.30 % Powder (70 g), 30 min soaking, 500 W, 30 min
•  Clevenger hydrodisCllate 95.94 % Crushed seeds, 50 g, 100 °C
•  MW extract 86.71% Powder (70 g), 30 min soaking, 500 W, 15 min

But-3-enyl isothiocyanate

•  Where: CondiHons:
•  Clevenger hydrodisCllate 1.02 % Crushed seeds, 50 g, 100 °C
 GC-MS RESULTS
Black mustard
Myrosinase
GLS Precursor Chemical composiCon R.T.

Sinigrin Allyl isothiocyanate 5.83 min

But-3-enyl isothiocyanate 9.23 min

Gluconapin

Black mustard
 GC-MS RESULTS
Black mustard
Allyl isothiocyanate
•  Where: CondiHons:
•  MW disCllate 19.60%
Crushed seeds (100 g), 1 h soaking, 500 W, 30
•  Clevenger hydrodisCllate 9.55% min
Crushed seeds (50 g), 100 °C
•  MW extract 6.72% Crushed seeds (100 g), 3 h soaking, 500 W, 15 min

But-3-enyl isothiocyanate
•  Where: CondiHons:
•  MW disCllate 55.17% Crushed seeds (100 g), 1 h soaking, 500 W, 30 min
•  Clevenger hydrodisCllate 82.58 % Crushed seeds (100 g), 3 h soaking, 500 W, 15 min
•  MW extract 10.31 % Crushed seeds (50 g), 100 °C
•  CH2Cl2 extract aeer autolysis 0.82 % Powder (20 g), 6 h soaking, 37 °C
 GC-MS ANALYSIS
White mustard Brown mustard Black mustard
RelaCvni intenzitet

RelaCvni intenzitet

RelaCvni intenzitet
Time/min Time/min Time/min

1) CH2Cl2 extract aMer autolysis; 2) Clevenger hydrodisHllate; 3) MW disHllate; 4) MW extract;

 Allyl
isothiocyanate

But-3-enyl
isothiocyanate
 CONCLUSIONS
•  Microwave extracCon decrease the Cme of extracCon (15 - 30 min) in relaCon to
Clevenger (2.5 h) and extracCon with organic solvent aeer autolysis (6 and 24 h)

•  The presence of targeted sulfur compounds and its content depends on applied
extracCon method

•  Allyl isothiocyanate was predominant compound in brown mustard seeds oil in

MW disCllate and Clevenger hydrodisCllate (80.30% and 95.94%) and MW extract
(86.71%), while its concentraCon was lower in black mustard seeds oil (19.60% in
MW disCllate and 9.55% in Clevenger hydrodisCllate) and in white mustards seeds
oil (12.68% in MW extract)
 CONCLUSIONS

•  But-3-enyl isothiocyanate was predominant compound in black mustard seeds oil

(82.58% in Clevenger hydrodisCllate and 55.17% in MW disCllate), while its content
was the lowest in CH2Cl2 extract aeer autolysis (0.82%)

•  But-3-enyl isothiocyanate was detected in brown mustard seeds oil only in

Clevenger hydrodisCllate (1.02%)

•  But-3-enyl isothiocyanate was not detected in white mustard seeds oil

 CONCLUSIONS

•  Surface morphology scanned by SEM shows that tested sample has a compact pore
microstructure throughout the whole sample

•  Pore of white mustard seeds are significantly less than pore of brown and black
mustard seeds

Yields

•  Clevenger hydrodisCllate 0.003 – 0.07%

•  MW disCllate 0.0001 – 0.002%

•  MW extract 0.003 - 0.013%

•  CH2Cl2 extract aeer autolysis 0.001 – 0.024%

 PERSPECTIVES

•  Use of modern extracCon of volaCle sulfur-containg compounds with supercriCcal
CO2 from various mustard seeds

•  EvaluaCon of biological acCviCes of extracted volaCle sulfur-containing

compounds from mustard seeds

- microbiological acCvity

- anCproliferaCve acCvity on different cancer cell lines

- hypoglycemic acCvity (inhibiCon of α-amylase and α-glucosidase)

•  SEM and opCcal microscopy screening of samples surface aeer different extracCon
treatments

•  DeterminaCon of gastrointesCnal stability of volaCle sulfur-containing compounds

from mustard seeds by two-phase in vitro digesCon model using human digesCve
enzymes
I want to thank
you…
•  CroaHan Science FoundaHon (HRZZ)

a) Research project: „Plants as source of bioacCve sulfur compounds and their

ability to hiperacumulate metals”, prof. Ivica Blažević (Faculty of Chemistry and
Technology, University of Split)

b) PhD project: „Young researchers’ career development project –training of

doctoral students”, supervisor prof. Tea Bilušić (Faculty of Chemistry and
Technology, University of Split)

•  PhD student Azra Đulović (Faculty of Chemistry and Technology, University of

Split)

•  Assistant professor Franko Burčul (Faculty of Chemistry and Technology,

University of Split)

•  IUPAC and HSKIKI – scholarship for parCcipaCon in 26 HSKIKI

The End…

Hippocrates recommends white mustard

for internal use against gastrointes5nal
problems and for externally, mixed with
vinegar to extract inflamma5on!

Thank you for your attention!