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IJRRAS 19 (1) ● April 2014 www.arpapress.com/Volumes/Vol19Issue1/IJRRAS_19_1_05.pdf

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CHROMATOGRAPHIC SEPARATION AND IDENTIFICATION OF

SOME VOLATILE OILS, ORGANIC ACIDS AND PHENOLS FROM THE

SEEDS OF CUMINUM CYMINUM GROWING IN IRAQ

Fanar Hashum Yousif Al-Hashemi

Department of Horticulture & Landscape Design

College of Agriculture and Forestry, University of Mosul, Iraq

E-mail: [email protected]

ABSTRACT The current study was carried out for separation and identification of some active constituents from Cuminum cyminum fruit

(often called seeds) were collected in unripe, fully ripe stage and investigated of these essential oils (Octanol, Limonene, Thymol,

Anisyl alcohol, Cuminaldehyde, Anethole, Vanillin and also Benzoic acid), organic acids (Aspartic, Citric, Malic, Tartaric,

Propionic, Ascorbic, Oxalic, Maleic and Fumaric acids) and phenols (Salicylic acid, Gallic acid, Cinnamic acid, Hydroquinone,

Resorcinol, P-hydroxybenzoic acid, Rutin, Coumarine, Quercetin). The essential oils were investigated by Gas Liquid

Chromatography (GLC), while quantitative identification of individual target organic acids and phenolic compounds were

achieved by high-performance liquid chromatography (HPLC). Moreover the high percentage oil (10.97%) and the major

components were presented as Cuminaldehyde (14.27%), Vanillin (2.76%) and Anethole (1.93%) in the fully ripe stage of seeds.

Also the essential oil was studied for its physical properties. Many organic acids were identified in fully ripe seeds in eight

compounds comparative with unripe seeds. Salicylic and gallic acids were showed with a highest amounts in both stage of

harvest seeds.

Key words: Cuminum cyminum, volatile oils, organic acid, phenols, GLC, HPLC.

1. INTRODUCTION

Cuminum cyminum L. is an annual plant of the family apiaceae, native from the east Mediterranean to east India,

The word cumin in English is derived from the Latin cuminum, which it self was derived from Greek "Kyminon"

(Nitin et al., 2012). It is widely cultivated in Pakistan, Egypt, Iraq, Turkey, Syria, Sudan (Jalali et al., 2007). Seeds

of Cuminum cyminum are carminative, aromatic, stomachic, stimulant, astringent and cooling in effect, cumin seed

oil is used as multifunctional luminescent paints or in topical clothing ointment, cumin oil effects on carcinogen-

metabolizing enzymes and acid sol sulpfhyd ryls in liver it is also synergistic (Hanif et al., 2012). The cumin seeds is

contained volatile oil (2-5%) and the yellow coloured fresh oil contains Cuminaldehyde as its chief components (El-

Kani et al., 2007). To achieve a better separation of the main organic acids (citric, lactic, formic, acetic, propionic

and butyric) from dairy products various HPLC methods have been reported by (Pelin and Cevdet, 2010). A number

of phenolic compounds were isolated from Cuminum cyminum including phenolic acids, flavonoids, phenolic

diterpenes, in this plant are closely associated with their antioxidant activity, they are also known to play an

important role in stabilizing lipid peroxidation and to inhibit various types of oxidizing enzymes (Gallo et al., 2010).

When cumin seeds are harvested at different times, their physical and chemistry properties may change

considerably.

The aim of this study is investigated the composition of cumin oil at different periods times using GLC analysis and

to identify of many organic acids and phenolic compounds using HPLC method.

2. MATERIAL AND METHODS

PLANT MATERIAL:

C. cyminum seeds were obtained from a local market in mosul and cultured in pots about (15 cm in diameter) in

plastic house of the Department of Horticulture and Landscape Design/ College of Agriculture and Forestry –

University of Mosul/ Iraq. The seeds were cultured in 15 November 2012 and collected in two periods time (unripe

and fully ripe) at 3 week intervals during the harvest season in 2012. The dried seeds in lab temperature were stored

in a dark place until use.

CLASSIFICATION OF C. CYMINUM:

The plant was classified by Mr. Tallal Taha that he is the director of medicinal plant project – Mosul dam, show the

classification according to APG system III, 2009 in (Figure 1).

mailto:[email protected]

IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils

81

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Apiales

Family : Apiaceae

Genus : Cuminum

Species : Cuminum cyminum L.

Figure (1): Classification of Cuminum cyminum according to APG system III, 2009.

EXTRACTION OF ESSENTIAL OIL

Essential oil was extracted by steam distillation using any apparatus of Clevenger type. The extraction took 3hrs. for

mixing 25g of seeds in each stage in 500ml of distilled water, according to the procedure as described in the British

pharmacopeia (1980). After distillation the aqueous phase was extracted with diethyl ether (3x20ml). the organic

phase was extracted with sodium sulphate, and eliminated by pressure distillation reduced in rotary evaporator at

35°C and pure oil was stored at 4°C in obscurity until the beginning of analysis. The physical characteristics of the

cumin oil were studied i.e., percentage ratio of oil, Density, specific gravity, refractive index and colour (Guenther,

1972). Chemical composition of essential oil of Cuminum cyminum L. was analyzed by Gas liquid chromatography

(Satish Kumar, 2010) using analytical conditions in Table (1), Fig. 2(A). The individual standard was also run under

the same conditions.

Table (1): The analytical conditions of volatile oils for GLC analysis

Properties Analytical conditions

Primary temp. of column 100°C

Final temp. of column 300°C

Average height of temp. 10°C/min

Detector temperature 325°C

Flow rate of He 20 ml/min

Column (Length × Internal dimeter) 3% SE-30

Liquid phase 3m×1/8"

Solid phase Teflon (Mesh 100-120)

Attenuation 1mV

Detector type (FID) Flame ionization detector

SOXHLET EXTRACTION:

Soxhlet extraction was carried out with standard apparatus for 8hrs. by using 25g of seeds with 200ml of hexane (El-

Kani et al., 2007) to achieve defatted depending upon to method (Harborne, 1973).

EXTRACTION OF ORGANIC ACIDS:

The seeds of C. cyminum L. (25g) were re-extracted with 200ml of absolute ethanol by using magnetic stirrer for 72

hrs. at 60°C. The mixture is filtered and completed to 10ml in a volumetric flask with ethanol (Grand et al., 1988).

The samples was prepared by using acid hydrolysis with 1N HCL for 1hr in bath water at 100°C and then the

mixture was separation by using ethyl acetate when was added to solution, two layer was shown, the ethyl acetate

layer was kept for other analysis. The compounds Fig. 2 (B) that containing in ethyl acetate were identified by

HPLC – technique (Harborne, 1973).

INSTRUMENTATION AND ANALYTICAL CONDITIONS:

HPLC analysis was using a liquid chromatography (Shimadzu, LC 2010 A/Japan), the column was C18

(4.6×150)mm, at a flow-rate 1ml/min, injection volume was 20 μl, the mobile phase consisting of 40 mM Na2SO4

and the PH was adjusted at 2.68, the column temperature was 30°C, UV absorbance at 210 nm (Dionex, 2004).

EXTRACTION OF PHENOLIC COMPOUNDS:

After the extraction by hexane, the seeds of C. cyminum (25g) were re-extracted with 200ml of absolute ethanol by

using soxhlet apparatus for 72hrs. at 78°C. The extract was filtered and evaporated under vacuum in a rotary

evaporator at 65°C until 20ml. The crude extract after evaporated was carried out for acid hydrolysis (Harborne,


82

1973). While the phenolic compounds were extracted with (2×25ml) ethyl acetate. The compounds were confirmed

by using HPLC-technique Fig. 2 (C).

INSTRUMENTATION AND ANALYTICAL CONDITIONS:

HPLC analysis was performed by using a liquid chromatography (Shimadzu, LC 2010 A/Japan), the column was

C18 (4.6×240)mm, at a flow-rate 1.3ml min-1, injection volume was 50 μl, the mobile phase consisting of

acetonitrile:water (80:20) vlv, the column temperature was 40°C, UV absorbance at 280 nm (Al-Tkay, 2012).

STOCK AND STANDARD SOLUTIONS:

The volatile oils, organic acids and phenols, 0.1g were accurately weighed into volumetric flask, dissolved in diethyl

ether for volatile oils and in ethanol for organic acids and phenols.

CHO

Cuminaldehyde Thymol

OH

CHO

OCH3

OH

Vanillin

CH

OCH3

Anethole

CH2OH

OCH3

Anisyl alcohol

Ch2H3C

CH3

Limonene

CH CH3

A: Chemical structures of volatile oils

OH C C

O O

OH

Oxalic acidTartaric acid

OH

O

OH

OH

O

Malic acid

O

OHOH

C

OCH2OH

H

OH

Ascorbic acid

C

C O

OH

O

HO

Fumaric acid

HO

O

OH

OOH

OH

B: Chemical structures of organic acids

C: Chemical structures of phenolic compounds

Figure (2): The chemical structure of active constituents in Cuminum cyminum.

RESULTS AND DISCUSSION

PHYSICAL PROPERTIES OF CUMIN OIL:

The greatest oil present was 10.97% of cumin seeds, Density and specific gravity reached 0.9023g/cm3 and 0.8329

respectively in the harvesting stage (fully ripe) while the refractive index was 1.3720 comparative with the unripe

seeds Table (2).

COOH

OH

Salicylic acid

OH

OH

Hydroquinon

CH=CHCOOH

Cinnamic acid

O

OH

OH

O

HO

OH

Quercetin

OH

COOH

OH

Gallic acid

OHHO

HO OH

Resorcinole


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Table (2): physical properties of C. cyminum oil

Harvesting

stage Oil %

Density

g/cm3

Specific

gravity

Refractive

index Colour

Unripe 6.64 0.8892 0.8100 1.3210 Light brownish yellow

Fullyripe 10.97 0.9023 0.8329 1.3720

The physical properties of C. cyminum oil have been studied in the seeds in the recently review (Hanif et

al., 2012).

ESSENTIAL OIL CONSTITUENTS:

Many standards of volatile oil was injected by GLC technique Fig. (3). The major components in the samples were

identified as Cuminaldehyde (14.27%) followed by vanillin (2.76%) and Anethole (1.93%) in the oil of fully ripe

seeds Table (3) and Fig. 4 (A). Comparison of the cumin oils of two samples has shown that the Cuminaldehyde

content of the unripe seeds oil was lower than those of fully ripe Fig. 4 (B). The results of our investigation are in

good agreement with those reported by Baser et al. (2000) and Kan et al. (2007).

Harvesting at fully ripe stage caused an increase in the amount of Anethole and Vanillin, but Cuminaldehyde

content decreased when fruit unripe. Our result showed that observed differences in the composition of the oil

besides other factor appear to be the maturity of the cumin fruit.

R

elat

ive

atte

ndan

ce

Anthole

min.

8.73

Rel

ativ

e at

tendan

ce

min.

Cuminaldehyde

8.48

Anisyl alcohol

Rel

ativ

e at

ten

dan

ce

min.

8.07

Limonene

Rel

ativ

e at

ten

dan

ce

min.

4.97

Thymol

Rel

ativ

e at

ten

dan

ce

min.

7.99

Octanol

Rel

ativ

e at

ten

dan

ce

min.

3.40


84

Fig. (3) GLC Chromatograms of standard volatile oil.

Table (3): Chemical composition of the essential oil Cumin fruits at different harvesting times using GLC

Technique.

Volatile oil

compounds

Standard

Rt(min.)

Unripe fruit Fully ripe fruit

Conc. % Components

Rt(min.) Conc. %

Components

Rt(min.)

Octanol 3.404 0.460 2.751 - -

Limonene 4.975 0.943 5.504 - -

Thymol 7.993 - - 0.495 7.063

Anisyl alcohol 8.072 - - 1.934 8.350

Cuminaldehyde 8.489 0.132 8.205 14.279 8.589

Anthole 8.736 0.786 8.994 - -

Vanillin 10.543 0.242 10.525 2.769 10.907

Benzoic acid 20.63 1.652 20.131 0.214 19.850

Benzoic acid

(A)

Vanillin Anthole

Cuminaldehyde Limonene

Octanol

Benzoic acid

Rel

ativ

e at

tendan

ce

min.

20.63

Rel

ativ

e at

tendan

ce

min.

Vanillin 10.54


85

Fig. (4) GLC chromatograms of volatile oils in Cuminum cyminum,

(A) unripe fruits, (B) fullyripe fruits.

IDENTIFICATION OF ORGANIC ACIDS:

From the application of HPLC-technique in the separation of organic acids, PH of the mobile phase and temperature

are crucial parameters. The most suitable mobile phase used for separation of organic acids are aqueous water which

its pH was adjusted 2.68 value with Hydrochloric acid at optimum temperature 30°C, therefore many organic acids

found in the Cuminum seeds were separated. An example of the chromatogram of the organic acid standards is

given in Fig. (5). Eight organic acids could be separated in less than 12 minutes in full ripe seeds but only five

compounds in unripe seeds and the quantities of organic acids in both stage of harvest seeds presented in Table (4)

and Fig. (6). The concentration was very depending upon the harvest stage, among the Organic acids, Propionic acid

showed the highest value in both stage (unripe, fully ripe) of seeds followed by Aspartic acid but a few amount of

Oxalic, Maleic and Fumaric acid in fully ripe seeds. These results were in agreement with the findings of Pelin and

Cevdet, (2010) that oxalic acid showed a few amount comparative with Malic and Citric acids. A recent research of

Aktas et al., (2005) and Al-Ramadhan, (1999) showed to separated many Organic acid compounds such as Oxalic,

Tartaric, Malic, Ascorbic, Fumaric acids by using HPLC technique.

(B)

Benzoic acid

Vanillin

Cuminaldehyde

Anisyl alcohol

Thymol

Aspartic acid

3.107

Tarlaric acid

4.240

Citric acid

3.602

Malic acid

3.639


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Fig. (5) HPLC Chromatograms of standard organic acids.

Table (4): Chemical composition of organic acid for cumin fruits at different harvesting times using

HPLC Technique.

Organic acid

compounds

Standard

Rt(min.)

Unripe fruits Fully ripe fruits

Area % Components

Rt(min.) Area %

Components

Rt(min.)

Aspartic acid 3.107 1.903 2.589 3.016 2.907

Citric acid 3.602 0.056 2.850 1.473 3.072

Malic acid 3.639 0.407 3.003 0.547 3.758

Tartaric acid 4.240 0.589 4.727 0.434 3.993

Propionic acid 5.442 83.734 5.408 73.022 4.713

Ascorbic acid 6.552 - - - -

Oxalic acid 7.553 - - 0.007 7.642

Maleic acid 9.772 - - 0.005 9.716

Fumaric acid 12.375 - - 0.041 11.493

Ascorbic acid

6.552

Maleic acid

9.772 Oxalic acid

7.553

Propionic acid

5.442

Fumaric acid

12.378


87

Fig. (6) HPLC chromatograms of organic acid in Cuminum cyminum


IDENTIFICATION OF PHENOLIC COMPOUNDS:

Phenolic compounds play an important role in defense against cardiovascular disease, aging and cancer, because of

their scavenging ability due to their hydroxyl groups (Karimi and Jaafar, 2011).

Fig. (7), (8) and Table (5) showed the maximum quantities (%) and retention time Rt(min) of 9 standards and two

sample of Cuminum cyminum identified in ethanolic extract by HPLC technique. All phenolic compounds were

identified according to their retention time and spectral characteristics against those of standards. Results confirm a

variation in phenolic content of plant extracts, the highest amount of Salicylic and Gallic acid was appeared in both

stage (unripe and fully ripe).

While P-Hdroxybenzoic acid, Rutin, Coumarine, Quercetin was appeared for a few amount. Bettaieb et al., (2010)

showed that the Cuminum cyminum seeds contain many phenolic compounds such as gallic acid, coumarin,

Quercetin, Resorcinol, Vanilic acid. And a result of Nadeem and Asad (2012) who demonstrated that the

quantitative analysis showed the presence of quercitin in Cuminum cyminum seeds.

Propionic Aspartic

Citric

Malic Tartaric Maleic

Fumaric

Oxalic

Propionic Aspartic

Citric

Malic

Tartaric

(B)

(A)


88

Salicylic acid Gallic acid

Cinamic acid Hydroqunion

Resorcinol

P-Hydroxybenzoic acid

Rutin Coumarine

3.017 3.198

2.849 2.697

2.222 2.530

1.652 2.059


89

Fig. (7) HPLC Chromatograms of standard phenolic compounds.

Fig. (8) HPLC chromatograms of phenolic compounds in Cuminum cyminum


Quercetin

(A)

(B)

Salicylic acid

Gallic acid

Cinamic acid

Hydroqunion

Coumarine Rutin

Quercetin

Salicylic acid Gallic acid

Cinamic acid Hydroqunion

Coumarine

Rutin

Quercetin P-Hydroxyuinon

3.252


90

Table (5): Chemical composition of phenolic compounds for cumin fruits at different harvesting times using

HPLC Technique.

Phenolic compounds Standard

Rt(min.)

Unripe fruits Fully ripe fruits

Area % Components

Rt(min.) Area %

Components

Rt(min.)

Salicylic acid 1.652 63.352 1.674 43.741 1.678

Gallic acid 2.059 21.667 1.842 43.274 1.750

Cinamic acid 2.222 3.647 2.263 3.584 2.249

Hydroqunion 2.530 7.791 2.425 6.757 2.392

Resorcinol 2.697 - - - -

P-Hydroxybenzoic acid 2.849 - - 0.242 2.947

Rutin 3.017 0.359 3.009 0.664 3.062

Coumarine 3.198 0.672 3.135 0.199 3.326

Quercetin 3.252 0.234 3.400 0.240 3.416

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