Lipid Classes, Sterols and Tocopherols of Black cumin (Nigella sativa L.), Coriander (Coriandrum sativum L.) and Niger (Guizotia abyssinica Cass.) Seed oils

Lipid Classes, Sterols and Tocopherols of Black
cumin (Nigella sativa L.), Coriander (Coriandrum
sativum L.) and Niger (Guizotia abyssinica Cass.)
Seed oils

Mohamed Fawzy Ramadan and Jörg-Thomas Mörsel
Technical University of Berlin, Institute of Food Chemistry,
Gustav-Meyer-Allee 25, TIB 4/3-1, D-13355 Berlin
Germany

25Th ISF World Congress, 12-15 October 2003, Bordeaux, France

Facts About Oil Plants

1- The production of oil plants takes the third place in world
production after starch plants and fruits.

2- More than 90% of the oil plants are produced in the tropical and
subtropical areas.

3- The cultivation of oil plants plays a major role in development
politics (ca. 30% of the crops are exported).

4- The major oil crops of international importance: i.e., soybean
(Glycine max), oil palm (Elaeis guineenis), rapeseed (Brassica spp.),
sunflower (Helianthus annuus), cottonseed (Gossypium spp.), and
groundnut (Arachis hypogaeo), together accounting for about 84% of
world vegetable oil production.

Rehm S, Espig G (1991) Oil plants. In: The cultivated plants of the tropics and
subtropics, Verlag Josefmargraf, Weikersheim, pp. 76-119.
Murphy DJ (1993) The major oil crops. In: Designer oil crops: breeding, processing
and biotechnology, VCH Verlag, Weinheim, pp. 5-72.

Production and Consumption of Vegetable
Oils and Fats.

World consumption of fats and oils
World production of fats and oils
Animal feed
Animal 7% Chemical
industry
25% 13%

Edible uses
80%

Vegetable
75%

Murphy DJ (1993) The major oil crops. In: Designer oil crops: breeding, processing
and biotechnology, VCH Verlag, Weinheim, pp. 5-72.


Why Nonconventional seed oils !?

1- The increasing human population made it necessary to use as
many agricultural resources as possible for the cultivation of
oilseeds.

2- Nonconventional oilseeds may contain novel components
and unusual fatty acids in their chemical makeup.

3- It is expected that the production costs for these seed oil will
drop after further yield increases are gained through plant
breeding efforts and development of new processing
technology.
For example, the production of oil palm was the principle
motivation for growing and marketing.


Marketing of New Oilseeds !

„Create a customer“ is the only definition of
business purpose. So, marketing of new oilseeds
will depend on the answer of the following
questions.

1- Who is the customer?
2- Where is the customer?
3- Why does the customer buy?
4- What is value in the eyes of the customer
(what the product will do for the customer)???

Endrea JG (1992) Niche marketing of new oilseeds: An industrial
perspective. In: Seed oils for the future, AOCS press, Champaign,
pp. 1-8.


Information on Black Cumin, Coriander and
Niger Oilseeds

Black cumin Coriander Niger
(Nigella sativa L.) (Coriandrum sativum L.) (Guizotia abyssinica Cass).
Family Ranonculaceae Family Umbelliferea Family Compositae

Country of source: Mediterranean countries and Mediterranean countries, Eastern Europe, -Ethiopia: (50-60% of total
India. Russia and India. production), India (2% of total
production).
- Minor oilseeds crop in some other
African countries.

Production: Not available 90-100 ton per year 400.000 ton per year (not involved in
the world oilseeds trade).

Uses of oilseeds 1- Edible uses: Sweet dish, pastry, 1- Edible uses: Ingredient of curry powder, 1- Edible uses.
flavoring of food, stomachic, Flavoring agent of certain alcoholic 2- Manufacture of soap and paints.
and/or seed oils: carmanitive, and diuretic agent. beverages. 3- Lubricant of illuminant.
2- Medicinal uses: Antibacterial, 2- Industrial uses: Coriander produce a high 4- Protein-rich meal after oil
Antifungal, Antineoplastic, petroselinic acid of potential uses (fine extraction used as feed or fuel.
Antihelmenthic. chemicals, softeners, soaps, emulsifiers and
nylon).

Total lipids (fresh 40% 30% 30-40%
weight):


Experimental Oilseeds
Procedures

From Turkey From Hungary From India

Grounding
(Particle size = 1-2 mm)

Fatty acid methyl esters Extraction of crude seed oils
Vitamin E
GLC/FID using n-hexane or
NP-HPLC/UV
Chloroform:Methanol (2:1, v/v)
Saponification

Phytosterols Open Column Chromatography
GLC/FID (Silica gel 60)

Chloroform Acetone Methanol

Glycolipids Phospholipids
Neutral lipids HPLC/UV, TLC HPLC/UV, TLC
TLC and GLC
and GLC and GLC


Fatty Acid Analysis Oilseeds


Grounding

Vitamin E
NP-HPLC/UV
Saponification



TLC and GLC
and GLC and GLC


Levels of Fatty Acids in Crude Seed Oils
Conditions of analysis.
Derivatization: Fatty acids Fatty acid profile of crude seed oils
were converted to methyl
esters by heating in 10% BF3%
70 Black cumin seed oil
in methanol.
Instrument: Shimadzu GC- 60
14A (Kyoto, Japan) equipped Coriander seed oil
with FID. 50
Column: Supelco SP-2380TM Relative Niger seed oil
(30 m x 0.25 mm id x 0.2 µm content (%) 40
film thickness; Bellefonte, PA,
USA).
30
Carrier gas: helium with flow- 20
rate of 0.6 mL/min.
Injection: 1 µL of FAME was 10
injected (split, 1:40).
0
Temperature program:
C16:0

*Column temp.: 100 °C
C18:0
C18:1n-12
programmed by 5 °C/min till C18:1n-9
C18:2n-6
C18:3n-6

C22:0
C20:2n-6
175 °C (10 min), then 8

C22:1n-9
C20:5n-3
C22:6n-3
°C/min till 220 °C (10 min).
*FID and injector were set at
250 °C.
Fatty acid

Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 202-206.
Ramadan MF, Mörsel J-T (2002) Food/Nahrung 46:240-244.

Analysis of Lipid Oilseeds
Classes


Grounding

Vitamin E
NP-HPLC/UV
Saponification

GLC/FID (Silica gel 60, 70-230 mesh)


TLC and GLC
and GLC and GLC


Levels of Lipid Classes (g/kg oil) and Their Ratios
of Saturated to Polyunsaturated Fatty Acids (S/P) a

Lipid class Black cumin seed oil Coriander seed oil Niger seed oil

Neutral lipids 972 960 970

S/P 0.246 0.264 0.359

Glycolipids 21.8 23.9 19.0

S/P 0.383 0.506 0.496

Phospholipids 3.20 8.50 2.80

S/P 0.571 0.714 0.477

a
Values given are the mean of three replicates.

Ramadan MF, Kroh LW, Mörsel J-T (2003) J. Agric. Food Chem. Jf0346713 In press.


Thin-Layer Chromatography of Neutral Lipid
Subclasses.
Solvent Front STE (Rf= 0.95)
TLC plates: Silica gel F254 TAG (Rf= 0.79)
(thickness = 0.25 mm; Merck,
Darmstadt, Germany).
Developing solvents: Hexane:
HTGLC/FID
diethyl ether: acetic acid
(40:60:1, v/v/v).
Sampling: FFA (Rf= 0.56)

100 µg lipid for analytical
development.
DAG (Rf= 0.39)
25-50 mg lipid for preparative ST (Rf= 0.37)
development.
Identification: Charring with
40% H2SO4 at 120 °C for 15 min.
Quantification: gravimetrically. MAG (Rf= 0.14)
Niger seed oil Coriander seed oil Black cumin seed oil NL-Standard
NL-Fraction
NL-Fraction NL-Fraction



HTGLC/FID Analysis of Triacylglycerols.
Importance of TAG analysis.
TAG comprise the main fraction (>90% of total lipid)
in seed oils. Therefore, the availability of such data
would facilitate the:
1- Understanding of seed oil structural
composition.
2- Understanding of TAG biosynthesis and
deposition of plant cells.
Purification: TLC.
Instrument: Mega Series high resolution (HRGC 4160, Carlo Erba
Strumentazione, Milano, Italy) equipped with FID.
Column: RTX-65TG (30 m x 0.25 mm id, 65% diphenyl-35%
dimethylpolysiloxa; Restek Gmbh, Sulzbach, Germany).
Carrier gas: H2 with flow rate of 10 mL/min. 10 20 30 40 50
Injection: 2 µL of purified TAG in dichloromethane (10 mg/mL) was Time (min)
injected (split-splitless was used). HTGLC/FID Separation of TAG standard
Temperature program:
compounds
1- Column temp.: 260 °C (5 min) then programmed by 5 °C till 360 °C
(25 min).
2- FID temp.: 360 °C.
3- Injector temp.: 340 °C.


Triacylglycerols’ Composition (g/100g TAG) of
Seed Oils.
INa CN:DBb Black cumin Coriander seed Niger seed
seed oil oil oil
1 30 : 0 (tricaprin) nd nd nd
2 36 : 0 (trilaurin) nd nd nd
3 42 : 0 (trimyristin) nd nd nd
4 48 : 0 (tripalmitin 3.50 0.15 9.80
5 48 : 3 (tripalmitolein) nd nd nd
6 50 : 1 (1,3-dipalmitoyl-2-oleoyl glycerol) 1.50 0.16 13.5
7 51 : 0 (trimargrin) nd nd nd
8 52 : 2 (1,2-dioleoyl-3-palmitoyl glycerol) 17.0 10.3 10.8
9 54 : 0 (tristearin) 3.80 3.30 4.50
10 54 : 3 (triolein, tripetroselinin) 42.9 54.5 20.4
11 54 : 6 (trilinolein) 31.3 2.97 21.9
a
IN= Identification number
b
Carbon number : Double bonds
Values are given as mean of three replicates.

Ramadan MF, Mörsel J-T (2002) Czech. J. Food Sci. 20: 98-104.

Analysis of Oilseeds
Glycolipids


Grounding

Vitamin E
NP-HPLC/UV
Saponification



TLC and GLC
and GLC and GLC


NP-HPLC/UV Analysis of Glycolipids’
Subclasses.
CH 2 OH C H 2O H
O
http://www.cyberlipid.org/glyt/glyt0008.htm
O
HO
O CH 2
HO
OH
CH-O-COR O
HO CH-O-COR HO
O O
1- ESG Rt= 8 min.
OH
HO O -C O R 1 2- MGDG Rt= 12 min.
OH O -C O R 2

Monogalactosyldiacylglycerol (MGDG) 3- SG Rt= 13 min.

R2
OH
4- CER Rt= 15 min.
Digalactosyldiacylglycerol (DGDG)
5- DGDG Rt= 19 min.
6- SQD Rt= 21 min.
HO
CH2 OH
R1 O
O
O R1
O
O
NH-COR2

HO HO
HO
HO

HO
HO
Cerebroside (CER)
R1= H, Steryl glucoside (SG)
R1= Acyl, Acylated steryl glucoside (ASG)
C H 2S O 3H
O

Sulfoquinovosyldiacylglycerol (SQD)
HO
OH
O HPLC/UV chromatograms (206 nm) obtained from the
HO
O -C O R 1 glycolipid (GL) standard mixture (A) and the GL subclasses
O -C O R 2 of black cumin (B), coriander (C) and niger (D) seed oils. A
mixture of 2 µg each of standard GL was injected.
Ramadan MF, Mörsel JT (2003) Food Chem. 80: 339-345.


Levels of Glycolipids (g/100g total glycolipids)
in Seed Oils.

- Glycolipids represent the Compound Black Coriander Niger
major fraction of polar lipids cumin seed oil seed oil
in seed oils. seed oil
- Plant glycolipids are
thought to be nutrients in the Acylated steryl glucoside (ASG) 9.95 25.1 38.5
human diet, but little is Monoglucosyldiacylglycerol (MGD) 7.88 nd nd
known about their intestinal
digestion and absorption in Steryl glucoside (SG) 9.45 34.9 29.5

mammals. Cerebroside (CER) 11.9 38.4 31.0
- The average daily intake of
Diglucosyldiacylglycerol (DGD) 55.6 nd nd
glycolipids in human has
been reported to be 140 mg of Sulfoquinovosyldiacylglycerol (SQD) 5.08 nd nd
ASG, 65 mg of SG, 50 mg of
CER, 90 mg of MGD and 220
mg of DGD. Glucose was the only sugar detected.
Values are given as mean of three replicates.

Ramadan MF, Mörsel JT (2003) Food Chem. 80: 339-345.


Phospholipids


Grounding

Vitamin E
NP-HPLC/UV
Saponification



TLC and GLC
and GLC and GLC


NP-HPLC/UV Analysis of Phospholipid
Subclasses.
CH2-O-R

CH-OR1 = β-Form
O

CH2-O-P-O-R2 = α-Form

OH

R2 = CH2-CH2-N+(CH3)3 Phosphatidylcholine.
R2 = CH2-CH2-NH+ Phosphatidylethanolamine.
R2 = CH2-CH(NH3)+ CO2H Phosphatidylserine.
R2 = (CH)6 (OH)5 Phosphatidylinositol.
HPLC System
Pump: Shimadzu LC-9A (Kyoto, Japan)
Column: LiChrosorb-Si 60 (5 µm, Knauer, Berlin)
Eluent (Programm):
Solvent (A): iso-octane: 2-propanol (6:8, v/v) 0 35
Solvent(B): iso-octane: 2-propanol: water (6:8:0.6, v/v) Elution time (min)
Flow rate: 0.7 mL/min
Detection: UV (205 nm)
NP-HPLC/UV chromatogram (205 nm) obtained
from the phospholipid standard mixture

Ramadan MF, Mörsel J-T (2003) Phytochem. Anal. In press.


Levels of Phospholipids (g/100g total phospholipids)
in Seed Oils.

-The phospholipids are Compound Black Coriander Niger
cumin seed oil seed oil
widely distributed in
seed oil
foods and antioxidant
effects are attributed to Phosphatidylglycerol (PG) 1.51 0.48 2.91
them. Phosphatidylethanolamine (PE) 25.1 27.1 22.5
-They combine
Phosphatidylinositol (PI) 9.56 19.2 14.6
nutritional and
technological properties Lyso-phosphatidylethanolamine (LPE) 1.20 2.50 Traces
in a single substance 12.3 7.20 8.74
Phosphatidylserine (PS)
class.
-This synergistic Phosphatidylcholine (PC) 46.1 40.3 48.7

function makes them Lyso-phosphatidylcholine (LPC) 4.23 3.22 2.55
ideal candidates for use
in functional food. Values are given as mean of three replicates.

Ramadan MF, Mörsel J-T (2003) Lebensm.-Wiss. U. Technol. 36:273-276.


Phytosterols


Grounding

Vitamin E
NP-HPLC/UV
Saponification



TLC and GLC
and GLC and GLC


GLC/FID Analysis of Phytosterols.
Importance of sterol analysis.
Concentration of sterols has been reported to be little
affected by environmental factors (soil or climate) or Sterols Standard
cultivation of new breeding lines. Therefore, analysis of
sterols provides a powerful tool for
1-Quality control of edible oil. 3
2-Detection of oil and mixtures otherwise not recognized
by the fatty acid profile.
Recently, phytosterols are added to vegetable oils spreads as
an example of a successful functional food. These products
are now available in the market and have been scientifically
proven to lower blood LDL by around 10-15% as part of a
healthy diet.
Conditions of analysis. 5
2
Sterols were analyzed after saponification of the oil without derivatization. 1
Instrument: Mega Series high resolution (HRGC 5160, Carlo Erba Strumentazione, Milano, Italy)
equipped with FID. 4 6
Column: DB 5 (30 m x 0.25 mm id, 5% phenylmethylpolysiloxan; J&W , Falsom, CA, USA). 7
Carrier gas: helium with flow-rate of 38 mL/min.
Injection: 2 µL of unsaponifiable matter in dichloromethane was injected (split-splitless was
used). 10 20 30 40
Temperatures: 50
1- Column temp.: 310 °C isothermal.
2- FID and injector temperatures were set at 280 °C.
Elution time (min)


Levels of Phytosterols (g/kg) in Seed Oils.

INa Compound Black cumin Coriander Niger Sterol standard
seed oil seed oil seed oil 3

1 Ergosterol nd 0.186 nd 1
2 5
4 7
6
2 Campesterol 0.226 0.735 0.713 5
Unsap. matter of
7
black cumin seed 6
3 Stigmasterol 0.314 1.512 0.667
oil 2 3
4 Lanosterol 0.106 0.152 0.113 4
Unsap. matter of 3 5
5 β-Sitosterol 1.182 1.553 2.035 coriander seed oil
6
2
6 ∆5-Avenasterol 1.025 1.466 0.530 1 4
7

Unsap. matter of 5
7 ∆7-Avenasterol 0.809 0.365 0.164
niger seed oil 2 3
Total Sterols 3.662 5.973 4.222
4 6
7

a
IN= Identification number 10 20 30 40
Values are given as mean of three replicates. 50

Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 202-206. Elution time (min)
Ramadan MF, Mörsel J-T (2002) Czech. J. Food Sci. 20: 98-104.

Tocopherols


Grounding

Fatty acid methyl esters Extraction of crude seed oils Vitamin E
GLC/FID using n-hexane or Tocopherols
Chloroform:Methanol (2:1, v/v) NP-HPLC/UV
Saponification



Neutral lipids HPLC/UV, TLC HPLC/UV TLC
TLC and GLC
and GLC and GLC


NP-HPLC/UV Analysis of Tocopherols.

OH
1. α-Tocopherol
C3
H
C3
H C3
H H 2. β-Tocopherol
C3
H H 3. γ-Tocopherol

O C3
H 4. δ-Tocopherol

5,7,8-Trimethyl (α-Tocopherol )
5,8-Dimethyl (β-Tocopherol)
7,8-Dimethyl (γ-Tocopherol )
8-Methyl (δ-Tocopherol)

Importance of Tocopherols.
1- Great importance in nutrition.
2- Improving the stability of the oil (antioxidant properties).

HPLC System
Pump: Shimadzu LC-9A (Kyoto, Japan)
Column: Zorbax-Sil (5 µm, Knauer, Berlin)
Eluent (isocratic): Ethyl acetate / iso-octane (4:96, v/v)
10 20
Flow-rate: 1.0 mL/min
30
Detection: 295 nm
Elution time (min)


Levels of Tocopherols (g/kg) in Seed Oils.

1
3
Black cumin
Compound Black cumin Coriander Niger seed seed oil
seed oil seed oil oil 2 4

α-Tocopherol 0.284 0.086 0.861 2
4
1
β-Tocopherol 0.040 0.672 0.331 3
Coriander
seed oil
3
γ-Tocopherol 0.225 0.162 0.570
1

δ-Tocopherol 0.048 0.347 0.185
2 Niger seed oil

4
Total 0.597 1.267 1.947
Tocopherols
10 20
30
Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 521-527. Elution time (min)


Conclusions
1- Although black cumin, coriander and niger seed oils have been part of
human diet in many parts of the world and their consumption is also
becoming increasingly popular in the non-producer countries,
information on the phytochemicals in these oils is limited. Yet these
phytochemicals may bring nutraceutical and functional benefits to food
systems.

2- As part of the effort to assess the potential of these seed oils, the
information is also of importance in processing and utilizing the crude
oils and their by-products.

3- High levels of oils recovered from the studied oilseeds and their
superior level of TAG (ca. 90% of total lipid) make them a suitable
source of vegetable oils production and encourage their commercial
manufacture.

4- High amounts of essential fatty acids and fat-soluble bioactives
(tocopherols, phytosterols and polar lipids) in the examined seed oils
may contribute to
A- High nutritional value.
B- Great stability toward oxidation.


Ramadan & Mörsel

Lipid Classes, Sterols and Tocopherols of Black cumin (Nigella sativa L.), Coriander (Coriandrum sativum L.) and Niger (Guizotia abyssinica Cass.) Seed oils

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Lipid Classes, Sterols and Tocopherols of Black cumin (Nigella sativa L.), Coriander (Coriandrum sativum L.) and Niger (Guizotia abyssinica Cass.) Seed oils