Lipid Classes, Sterols and Tocopherols of Black cumin (Nigella sativa L.), Coriander (Coriandrum...

Lipid Classes, Sterols and Lipid Classes, Sterols and Tocopherols of Black cumin (Tocopherols of Black cumin (Nigella Nigella

sativasativa L.), Coriander ( L.), Coriander (Coriandrum Coriandrum sativumsativum L.) and Niger ( L.) and Niger (Guizotia Guizotia

abyssinicaabyssinica Cass.) Seed oils Cass.) Seed oils

Mohamed Fawzy Ramadan and Jörg-Thomas Mohamed Fawzy Ramadan and Jörg-Thomas MörselMörsel

Technical University of Berlin, Institute of Technical University of Berlin, Institute of Food Chemistry,Food Chemistry,

Gustav-Meyer-Allee 25, TIB 4/3-1, D-13355 Gustav-Meyer-Allee 25, TIB 4/3-1, D-13355 BerlinBerlin

Germany Germany 2525ThTh ISF World Congress, 12-15 October 2003, ISF World Congress, 12-15 October 2003, Bordeaux, FranceBordeaux, France

2525ThTh ISF World Congress, 12-15 October 2003, ISF World Congress, 12-15 October 2003, Bordeaux, FranceBordeaux, France

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

Facts About Oil PlantsFacts About Oil Plants


Murphy DJ (1993)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 Production and Consumption of Vegetable Oils and Fats.Vegetable Oils and Fats.

World production of fats and oils

Animal25%

Vegetable 75%

World consumption of fats and oils

Animal feed7% Chemical

industry 13%

Edible uses80%


Why Nonconventional seed Why Nonconventional seed oils !?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 !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)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, Information on Black Cumin, Coriander and Niger OilseedsCoriander and Niger Oilseeds

Black cumin Black cumin

((Nigella sativaNigella sativa L.) L.)Family Family

RanonculaceaeRanonculaceae

Coriander Coriander

((Coriandrum sativumCoriandrum sativum L.) L.)Family UmbellifereaFamily Umbelliferea

Niger Niger

((Guizotia abyssinicaGuizotia abyssinica Cass).Cass).

Family CompositaeFamily Compositae

Country of source:

Mediterranean countries and India.

Mediterranean countries, Eastern Europe, Russia and India.

-Ethiopia: (50-60% of total 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 and/or seed oils:

1- Edible uses: Sweet dish, pastry, flavoring of food, stomachic, carmanitive, and diuretic agent.2- Medicinal uses: Antibacterial, Antifungal, Antineoplastic, Antihelmenthic.

1- Edible uses: Ingredient of curry powder, Flavoring agent of certain alcoholic beverages.2- Industrial uses: Coriander produce a high petroselinic acid of potential uses (fine chemicals, softeners, soaps, emulsifiers and nylon).

1- Edible uses.2- Manufacture of soap and paints.3- Lubricant of illuminant.4- Protein-rich meal after oil extraction used as feed or fuel.

Total lipids (fresh weight):

40% 30% 30-40%


Black cuminFrom Turkey

Oilseeds

CorianderFrom Hungary

NigerFrom India

Grounding (Particle size = 1-2 mm)

Extraction of crude seed oilsusing n-hexane or

Chloroform:Methanol (2:1, v/v)

Fatty acid methyl estersGLC/FID

Saponification

Phytosterols GLC/FID

Experimental Experimental ProceduresProcedures

Vitamin ENP-HPLC/UV

Open Column Chromatography(Silica gel 60)

Chloroform Acetone Methanol

Neutral lipidsTLC and GLC

Glycolipids HPLC/UV, TLC

and GLC

PhospholipidsHPLC/UV, TLC

and GLC



Oilseeds


NigerFrom India





Saponification


Fatty Acid Fatty Acid AnalysisAnalysis

Vitamin ENP-HPLC/UV





and GLC


and GLC


Levels of Fatty Acids in Crude Levels of Fatty Acids in Crude Seed OilsSeed Oils

Conditions of analysis.Derivatization: Fatty acids were converted to methyl esters by heating in 10% BF3% in methanol. Instrument: Shimadzu GC-14A (Kyoto, Japan) equipped with FID.Column: Supelco SP-2380TM (30 m x 0.25 mm id x 0.2 µm film thickness; Bellefonte, PA, USA).Carrier gas: helium with flow-rate of 0.6 mL/min.Injection: 1 µL of FAME was injected (split, 1:40).Temperature program: *Column temp.: 100 °C programmed by 5 °C/min till 175 °C (10 min), then 8 °C/min till 220 °C (10 min).*FID and injector were set at 250 °C.

C16

:0

C18

:0

C18

:1n-

12

C18

:1n-

9

C18

:2n-

6

C18

:3n-

6

C20

:2n-

6

C22

:0

C22

:1n-

9

C20

:5n-

3

C22

:6n-

3

0

10

20

30

40

50

60

70

Fatty acid

Fatty acid profile of crude seed oils

Black cumin seed oil

Coriander seed oil

Niger seed oilRelative content (%)

Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 202-206.Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Food/Nahrung 46:240-244.Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 215: 204-209.



Oilseeds


NigerFrom India





Saponification


Analysis of Analysis of Lipid ClassesLipid Classes

Vitamin ENP-HPLC/UV

Open Column Chromatography(Silica gel 60, 70-230 mesh)




and GLC


and GLC


Levels of Lipid Classes (g/kg oil) and Levels of Lipid Classes (g/kg oil) and Their Ratios of Saturated to Their Ratios of Saturated to

Polyunsaturated Fatty Acids (S/P)Polyunsaturated Fatty Acids (S/P) a 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

aValues given are the mean of three replicates.

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


Thin-Layer Chromatography of Neutral Lipid Thin-Layer Chromatography of Neutral Lipid Subclasses.Subclasses.

Conditions of analysis.TLC plates: Silica gel F254 (thickness = 0.25 mm; Merck, Darmstadt, Germany).Developing solvents: Hexane: diethyl ether: acetic acid (40:60:1, v/v/v).Sampling: 100 µg lipid for analytical development.25-50 mg lipid for preparative development.Identification: Charring with 40% H2SO4 at 120 °C for 15 min.Quantification: gravimetrically.

Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 202-206.

STE (Rf= 0.95)

TAG (Rf= 0.79)

FFA (Rf= 0.56)

DAG (Rf= 0.39)ST (Rf= 0.37)

MAG (Rf= 0.14)

Niger seed oil NL-

Fraction

Coriander seed oil

NL-Fraction

Black cumin seed oil

NL-Fraction

NL-Standard

Solvent Front

HTGLC/FID


HTGLC/FID Analysis of Triacylglycerols.HTGLC/FID Analysis of Triacylglycerols.


10 20 30 40 50Time (min)

HTGLC/FID Separation of TAG standard compounds

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.Conditions of analysis.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.Injection: 2 µL of purified TAG in dichloromethane (10 mg/mL) was injected (split-splitless was used).Temperature program: 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 Triacylglycerols’ Composition (g/100g TAG) of Seed Oils.Seed Oils.

Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 202-206.Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Czech. J. Food Sci. 20: 98-104.Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 215: 204-209.

INa CN:DBb Black cumin

seed oil

Coriander seed oil

Niger seed 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 numberb Carbon number : Double bondsValues are given as mean of three replicates.



Oilseeds


NigerFrom India





Saponification


Analysis of Analysis of GlycolipidsGlycolipids

Vitamin ENP-HPLC/UV





and GLC


and GLC

NP-HPLC/UV Analysis of Glycolipids’ NP-HPLC/UV Analysis of Glycolipids’ Subclasses.Subclasses.


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

HPLC/UV chromatograms (206 nm) obtained from the glycolipid (GL) standard mixture (A) and the GL subclasses of black cumin

(B), coriander (C) and niger (D) seed oils. A mixture of 2 µg each of standard GL was

injected.

O

HO

HO

HO

CH2OH

O CH2

CH-O-COR

CH-O-COR

O

H O

H O

C H 2O H

O

O O

OH

OH

OHO - C O R 1

O - C O R 2

OH

OO R1

NH-COR2

HOCH2OH

HO

HOHO

O

HOHO

HO

O

OR1

R2

O

H O

OH

C H 2S O 3 H

H O O

O -C O R 1

O -C O R 2

Monogalactosyldiacylglycerol (MGDG)

Digalactosyldiacylglycerol (DGDG)

Cerebroside (CER) R1= H, Steryl glucoside (SG) R1= Acyl, Acylated steryl glucoside (ASG)

Sulfoquinovosyldiacylglycerol (SQD)

1- ESG Rt= 8 min.

2- MGDG Rt= 12 min.

3- SG Rt= 13 min.

4- CER Rt= 15 min.

5- DGDG Rt= 19 min.

6- SQD Rt= 21 min.

http://www.cyberlipid.org/glyt/glyt0008.htmhttp://www.cyberlipid.org/glyt/glyt0008.htm


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

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

Compound Black cumin seed oil

Coriander seed

oil

Niger seed oil

Acylated steryl glucoside (ASG)

9.95 25.1 38.5

Monoglucosyldiacylglycerol (MGD)

7.88 nd nd

Steryl glucoside (SG) 9.45 34.9 29.5

Cerebroside (CER) 11.9 38.4 31.0

Diglucosyldiacylglycerol (DGD)

55.6 nd nd

Sulfoquinovosyldiacylglycerol (SQD)

5.08 nd nd

- Glycolipids represent the major fraction of polar lipids in seed oils.- Plant glycolipids are thought to be nutrients in the human diet, but little is known about their intestinal digestion and absorption in mammals.- The average daily intake of glycolipids in human has been reported to be 140 mg of 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.



Oilseeds


NigerFrom India





Saponification


Analysis of Analysis of PhospholipidsPhospholipids

Vitamin ENP-HPLC/UV





and GLC


and GLC


NP-HPLC/UV Analysis of Phospholipid NP-HPLC/UV Analysis of Phospholipid Subclasses.Subclasses.

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

CH2-O-R

CH-OR1 = -Form O

CH2-O-P-O-R2 = -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 SystemPump: 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)Solvent(B): iso-octane: 2-propanol: water (6:8:0.6, v/v)Flow rate: 0.7 mL/minDetection: UV (205 nm)

0 35

Elution time (min)NP-HPLC/UV chromatogram (205 nm)

obtained from the phospholipid standard mixture


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

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

Compound Black cumin seed oil

Coriander

seed oil

Niger seed oil

Phosphatidylglycerol (PG) 1.51 0.48 2.91

Phosphatidylethanolamine (PE)

25.1 27.1 22.5

Phosphatidylinositol (PI) 9.56 19.2 14.6

Lyso-phosphatidylethanolamine (LPE)

1.20 2.50 Traces

Phosphatidylserine (PS) 12.3 7.20 8.74

Phosphatidylcholine (PC) 46.1 40.3 48.7

Lyso-phosphatidylcholine (LPC)

4.23 3.22 2.55

-The phospholipids are widely distributed in foods and antioxidant effects are attributed to them. -They combine nutritional and technological properties in a single substance class. -This synergistic function makes them ideal candidates for use in functional food.

Values are given as mean of three replicates.



Oilseeds


NigerFrom India





Saponification


Analysis of Analysis of PhytosterolsPhytosterols

Vitamin ENP-HPLC/UV





and GLC


and GLC


GLC/FID Analysis of Phytosterols.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 cultivation of new breeding lines. Therefore, analysis of sterols provides a powerful tool for 1-Quality control of edible oil. 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.Sterols were analyzed after saponification of the oil without derivatization.Instrument: Mega Series high resolution (HRGC 5160, Carlo Erba Strumentazione, Milano, Italy) equipped with FID.Column: DB 5 (30 m x 0.25 mm id, 5% phenylmethylpolysiloxan; J&W , Falsom, CA, USA).Carrier gas: helium with flow-rate of 38 mL/min.Injection: 2 µL of unsaponifiable matter in dichloromethane was injected (split-splitless was used).Temperatures:1- Column temp.: 310 °C isothermal.2- FID and injector temperatures were set at 280 °C.

12

3

4 6

5

7

10 20 30 40 50

Elution time (min)

Sterols Standard


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

Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 214: 202-206.Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Czech. J. Food Sci. 20: 98-104.Ramadan MF, Mörsel J-T (2002)Ramadan MF, Mörsel J-T (2002) Eur. Food Res. Technol. 215: 204-209.

INa Compound Black cumin

seed oil

Coriander seed oil

Niger seed oil

1 Ergosterol nd 0.186 nd

2 Campesterol

0.226 0.735 0.713

3 Stigmasterol

0.314 1.512 0.667

4 Lanosterol 0.106 0.152 0.113

5 -Sitosterol 1.182 1.553 2.035

6 ∆5-Avenasterol

1.025 1.466 0.530

7 ∆7-Avenasterol

0.809 0.365 0.164

Total Sterols

3.662 5.973 4.222a IN= Identification numberValues are given as mean of three replicates.

1

1

2

2

2

2

3

3

3

3

Sterol standard

Unsap. matter of black cumin seed oil

Unsap. matter of coriander seed oil

Unsap. matter of niger seed oil

4

4

4

4

5

5

5

5

6

6

6

6

7

7

7

7

10 20 30 40 50

Elution time (min)



Oilseeds


NigerFrom India





Saponification


Analysis of Analysis of TocopherolsTocopherols

Vitamin ETocopherolsNP-HPLC/UV





and GLC

PhospholipidsHPLC/UV TLC

and GLC


NP-HPLC/UV Analysis of Tocopherols.NP-HPLC/UV Analysis of Tocopherols.

1. α-Tocopherol 2. β-Tocopherol 3. γ-Tocopherol 4. δ-

Tocopherol

OH

O CH3

CH3HHCH3

CH3 CH3

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

HPLC SystemPump: Shimadzu LC-9A (Kyoto, Japan)Column: Zorbax-Sil (5 µm, Knauer, Berlin)Eluent (isocratic): Ethyl acetate / iso-octane (4:96, v/v)Flow-rate: 1.0 mL/minDetection: 295 nm


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

10 20 30

Elution time (min)


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

Black cuminseed oil

Coriander seed oil

Niger seed oil

CompounCompoundd

Black Black cumin cumin

seed oilseed oil

CoriandeCorianderr

seed oilseed oil

Niger Niger seed oilseed oil

αα--TocopheroTocophero

ll

0.284 0.086 0.861

ββ--TocopheroTocophero

ll

0.040 0.672 0.331

γγ--TocopheroTocophero

ll

0.225 0.162 0.570

δδ--TocopheroTocophero

ll

0.048 0.347 0.185

Total Total TocopheroTocophero

lsls

0.597 1.267 1.947

10 20 30

Elution time (min)

1

1

1

2

2

2

3

3

3

4

4

4



ConclusionsConclusions1- 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 & MörselRamadan & Mörsel 2525ThTh ISF World Congress, 12-15 October 2003, Bordeaux, France ISF World Congress, 12-15 October 2003, Bordeaux, France

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Lipid Classes, Sterols and Tocopherols of Black cumin (Nigella sativa L.), Coriander (Coriandrum...

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