IJRRAS 19 (1) ● April 2014 www.arpapress.com/Volumes/Vol19Issue1/IJRRAS_19_1_05.pdf
80
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).
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,
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
83
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
86
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
87
Fig. (6) HPLC chromatograms of organic acid in Cuminum cyminum
(A) unripe fruits, (B) fullyripe fruits.
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)
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
89
Fig. (7) HPLC Chromatograms of standard phenolic compounds.
Fig. (8) HPLC chromatograms of phenolic compounds in Cuminum cyminum
(A) unripe fruits, (B) fullyripe fruits.
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
IJRRAS 19 (1) ● April 2014 Al-Hashemi ● Chromatographic Separation & Identification of Volatile Oils
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
REFERENCES [1]. Aktas, A. H.; S. Songul; Y. Mustafa; E. Cubuk (2005). Determination of carboxylic acids in apple juice by HPLC. Iran. J.
Chem. & Chem. Eng.
[2]. Al-Ramadany, T. R. (1999). Chemical study on Foeniculam Vulgare Mill growing in north of Iraq. M. Sc. Thesis,
Science college, Department of Chemistry.
[3]. Al-Tkay, T. (2012). Secondary chemical components and some anatomical properties of tree trunks of Melia azedarach
L. Ph. D. thesis, Forestry/ Wood Science, College of Agriculture and Forestry, University of Mosul.
[4]. APGIII, (2009). "An update of the Angiosperm phylogeny group classification for the orders and families of flowering
plants". Botanical J. of the linnean Society. 161: 105-121.
[5]. Baser, K. C.; S. H. Beis; N. Azcan and M. Kara (2000). Production of essential oil from cumin seeds. Chem.. Nat. Comp.,
36(3): 265-268.
[6]. Bettaieb, I.; S. Bourgou; W. Aidi; L. Ferid and B. Marzouk (2010). Essential oils, phenolics, and antioxidant activities of
different part of cumin (Cuminum cyminum L.). Journal of Agricultural and Food Chemistry, 58(19): 104-108.
[7]. British Pharmacopoeia (1980). "The pharmaceutical press". London App. XIF. 1273.
[8]. Dionex (2004). Acclaim organic acid (OA) HPLC column. www.dionex.com
[9]. El-Kani, M.; G. Fereshteh; M. Mehdi and R. Soosan (2007). Extraction of volatile oil from cumin (Cuminum cyminum L.)
with superheated water. Journal of food process engineering, 30(2): 255-266.
[10]. Gallo, M.; R. Ferracane; G. Giulia; A. Ritieni and V. Fagliano (2010). Microwave assisted extraction of phenolic
compounds from four different spices. Molecules, 15(3): 6366-6374.
[11]. Grand, A.; R. Verpoort; P. A. Liowndoryram and J. L. Poussel (1988). Anti-infections phytotherapies of the II.
Antimicrobial activity of 33 species, J.
[12]. Guenther, E. E. (1972). "Essential Oils". Vol. 1. R. E. Krieger publishing Company, Huntington, New York, USA. 8-87.
[13]. Hanif, C.; T. Ayesha; S. Adila; M. Saeed; A. Tanveer and M. Ashfaq (2012). Physico-chemical Investigation and
antimicrobial activity of essential oil of Cuminum cyminum L. World applied Sciences Journal, 19(3): 330-333.
[14]. Harborne, J. B. (1973). "Phytochemical Methods: A Guide to Modern Technique of Plant Analysis". 1st ed., Cox and
Wyman, London. 52-73.
[15]. Jalali, M.; B. Zekavat and H. Sereshti (2007). Use of gas chromatography-mass spectrometry combined with resolution
methods to characterize the essential oil components of Iranian cumin and caraway. J. Chromatography A. 11(43): 216-
226.
[16]. Kan, Y.; M. Kartal; T. Ozek; S. Aslan and K. Husnu (2007). Composition of essential oil of Cuminum cyminum L.
According to harvesting times. Turkish J. Pharm. Sci., 4(1): 25-29.
[17]. Karimi, E. and Z. E. Jaafar (2011). HPLC and GC-MS determination of bioactive compounds in microwave obtained
extracts of three varieties of Labisia pumila Benth. Molecules, 16(2): 6791-6805.
[18]. Nadeem, M. and R. Asad (2012). Cumin (Cuminum cyminum L.) as a potential source of antioxidants. PAK. J. Food. Sci.,
22(2): 101-107.
[19]. Nitin, R.; S. Yaclav; A. K. Verma; T. Lalit and R. Sharma (2012). A monographic profile on quality specifications for a
herbal drug and spice of commerce Cuminum cyminum L. International Journal of Advanced Herbal Science and
Technology, 1(1): 1-12.
[20]. Pelin, E. and N. Cevdet (2010). Determination of organic acids in olive fruit by HPLC. Zech. J. Food. Sci., 28(3): 202-
205.
[21]. Satish Kumar, K. (2010); "Extraction of essential oil using steam distillation". Ph. D. Thesis, Department of chemical
engineering, NIT, Rourkela.