GC-MS ANALYSIS AND ANTIMICROBIAL ACTIVITY OF ANETHUM GRAVEOLENS (UMBELIFERAE) ESSENTIAL OIL

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Abdel et al. World Journal of Pharmaceutical and Life Science
GC-MS ANALYSIS AND ANTIMICROBIAL ACTIVITY OF ANETHUM GRAVEOLENS
(UMBELIFERAE) ESSENTIAL OIL
Abdel Karim M.1*, Osman A.1, Amira A. E. Satti1,2 and Al-Hafez M.3
1Sudan University of Science and Technology, Faculty of Science (Sudan).
2Qurayat-Jouf University, Faculty of Science and Arts, Dept. of Chemistry (Saudi Arabia).
3King Khalid University, Faculty of Science and Arts, Dept. of Chemistry (Saudi Arabia).
Article Received on 30/12/2019 Article Revised on 20/01/2020 Article Accepted on 10/02/2020
INTRODUCTION
Anethum graveolens (Umbeliferae) is an annual herb
indigenous to southern Europe. It is now cultivated
worldwide for its economic value.[1-4]
Anethum graveolens has a long history of applications in
ethnomedicine including treatment of flatulence,
indigestion and convulsions. Anethum graveolens has
antiemetic and stimulant effects and is said to increase
appetite.[5-7] It has been reported that Anethum
graveolens possesses anti-inflammatory,[8,9]
antihyperlipidemic,[10-12] antimicrobial,[13-17]
antinociceptive properties beside analgesic and smooth
muscle relaxing effects.[18,20] Anethum graveolens
contains, among others, proteins (15.68%),
carbohydrates (36%), fiber (14.80%), moisture (8.39%),
ash(9.8%) beside polyphenols and some minerals.[6,7,21-26]
MATERIALS AND METHODS
Plant material
Seeds of Anethum graveolens were purchased from the
local market-Oswan, Egypt. the plant was identified and
authenticated by direct comparison with a reference
herbarium sample.
Instruments
A Shimadzo GC-MS-QP2010 ultra instrument with
RTX-5MS column (30m, length; 0.25mm diameter;
0.25µm, thickness) was used for GC-MS analysis.
Test organisms
Anethum graveolens oil was screened for antibacterial
and antifungal activities using the standard
microorganisms shown in Table (1)
Table 1: Test organisms.
Ser. No
Microorganism
Type
1
Bacillus sabtilis
G+ve
2
Staphylcococcus aureus
G+e
3
Pseudomonas aeroginosa
G-ve
4
Escherichia coli
G-ve
5
Candida albicans
fungi
Methods
Extraction of oil from Anethum graveolens
Hydrodistillation method was used for extraction of
Anethum graveolens volatile oil.
Esterification of oil
A methanolic solution of sodium hydroxide was prepared
by dissolving (2g) of sodium hydroxide in 100ml
methanol. A stock solution of methanolic sulphuric acid
Research Article
ISSN 2454-2229
wjpls, 2020, Vol. 6, Issue 3, 16-20
World Journal of Pharmaceutical and Life Sciences
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*Corresponding Author: Dr. Abdel Karim M.
Sudan University of Science and Technology, Faculty of Science (Sudan).
ABSTRACT
Information of the constituents of medicinal plant is of great importance since medicinal plants are endowed with
diverse phytochemicals with potential medicinal applications. This study was aimed to investigate the chemical
constituents of the medicinally important Anethum graveolens volatile oil, and to evaluate its antimicrobial activity.
30 components were detected by GC-MS analysis. Major constituents are: D-carvone (37.80%); D-limonene
(18.13%) and apiol (16.16%). The antimicrobial activity of the oil was evaluated via disc diffusion method against
five standard human pathogens (Gram positive: Staphylococcus aureus and Bacillus subtilis; Gram negative:
Escherichia coli and Pseudomonasa aeruginosa and the fungi Candida albicans). The oil showed significant
activity against Escherichia coli, Pseudomonasa aeruginosa and Bacillus subtilis. It also showed good activity
against Staphylococcus aureus.
KEYWORDS: Anethum graveolens, Essential oil, Constituents, Antimicrobial activity.

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Abdel et al. World Journal of Pharmaceutical and Life Science
was prepared by mixing (1ml) of concentrated sulphuric
acid with (99ml) methanol. The oil (2ml) was placed in a
test tube and (7ml) of methanolic sodium hydroxide were
added followed by (7ml) of an alcoholic sulphuric acid.
The tube was shaken vigorously for 5 minutes and left
over night. (2ml) of n-hexane were added and the tube
was vigorously shaken for 5 minutes The hexane layer
was diluted with 5ml diethyl ether. One gram of sodium
sulphate was added as drying agent. The solution was
filtered and the filtrate was transferred to the GC-MS
vial.
GC-MS analysis
The volatile oil from Anethum graveolens was analyzed
by GC-MS. A Shimadzo GC-MS-QP2010 ultra
instrument was used. Chromatographic conditions and
oven temperature program are presented below.
Table 2: Oven Temperature Program.
Rate
Temperature (C)
hold time ( )
-
50.0
0.00
7
180.0
0.00
10
300.0
0.00
Table 3: Chromatographic conditions.
Column oven temperature
c
Injection temperature
c
Injection mode
Split
Flow control mode
Linear velocity
Pressure
100.0 KPa
Total flow
50.0 ml/min
Column flow
1.69 ml/min
Linear velocity
44.7 cm/sec
Purge flow
3.0 ml/min
Split ratio
-1.0
Testing of antimicrobial susceptibility
Bacterial culture was performed on Mueller-Hinton agar,
while fungal cultures were maintained on Sabouraud
dextrose agar. The paper disc diffusion method was used
to screen the antimicrobial activity of the oil. The
experiment was carried out according to.[27] Bacterial
suspension was diluted with sterile physiological
solution to cfu/ml (Turbidity=McFarland standard
0.5). One hundred microliters of bacterial suspension
were swabbed uniformly on surface of MHA and the
inoculum was allowed to dry for 5 minutes. Sterilized
filter paper discs (Whatman No.1.6mm in diameter) were
placed on the surface of the MHA and soaked with
100mg/ml of test sample. The inoculated plates were
incubated at C for 24 hours in the inverted position.
The diameters (mm) of the inhabitation zones were
reorded as average of two replicates.
RESULTS AND DISCUSSION
GC-MS Analysis of Anethum graveolens volatile oil
GC-MS Analysis of Anethum graveolens volatile oil was
performed. The analysis revealed the presence of 30
components (Table 4). The typical total chromatograms
(TIC) is shown in Fig 1.
Fig. 1: Typical total ion chromatograms (TIC).
Table 4: Constituents of Anethum graveolens oil.
No.
Name
Ret. Time
Area%
1.
Bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)-
4.727
0.02
2.
.alpha.-Pinene
4.866
0.25
3.
Camphene
5.152
0.03
4.
3-Carene
5.425
0.06
5.
Bicyclo[3.1.0]hexane, 4-methylene-1-(1-methylethyl)-
5.598
0.25
6.
Bicyclo[3.1.1]heptane, 6,6-dimethyl-2-methylene-, (1S)-
5.680
0.05
7.
.beta.-Myrcene
5.890
0.64
8.
(+)-2-Carene
6.121
0.18
9.
.alpha.-Phellandrene
6.198
1.89
10.
p-Cymene
6.606
1.11
11.
D-Limonene
6.703
18.13
12.
.gamma.-Terpinene
7.307
0.08
13.
o-Isopropenyltoluene
7.979
0.21
14.
Cyclohexanol, 1-methyl-4-(1-methylethenyl)-, cis-
8.529
0.17
15.
trans-p-Mentha-2,8-dienol
8.668
0.11
16.
7-Oxabicyclo[4.1.0]heptane, 1-methyl-4-(1-methylethenyl)-
8.919
0.06

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Abdel et al. World Journal of Pharmaceutical and Life Science
17.
(+)-2-Bornanone
9.197
2.39
18.
Santolina alcohol
9.584
0.06
19.
1-(1,2,3-Trimethyl-cyclopent-2-enyl)-ethanone
9.743
0.17
20.
Terpinen-4-ol
9.878
0.23
21.
3,6-Dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran
10.057
0.57
22.
Cyclohexanone, 2-methyl-5-(1-methylethenyl)-, trans-
10.294
2.20
23.
Cyclodecene, 1-methyl-
10.464
5.01
24.
2-Cyclohexen-1-ol, 2-methyl-5-(1-methylethenyl)-, cis-
10.793
0.17
25.
1-Isopropenyl-3-propenylcyclopentane
10.996
0.24
26.
Carveol
11.063
0.10
27.
D-Carvone
11.366
37.80
28.
2-Cyclohexen-1-one, 3-methyl-6-(1-methylethyl)-
11.551
11.32
29.
Apiol
18.605
16.16
30.
Tolclofos-methyl
22.646
0.34
Major constituents of the oil are:
(i)D-Carvone (37.80%).
(ii)D-Limonene (18.13%).
(iii)Apiol (16.16 %.).
The mass spectrum of D-carvone is shown in Fig. 2. The
molecular ion M+ [C10H14O]+ appeared at
m/z150(RT.11.366). The mass spectrum of D-limonene
is displayed in Fig,3. The signal at m/z136 corresponds
the molecular ion M+[C10H16]+. Fig. 4 shows the mass
spectrum of apiol. The signal which appeared at retention
time 18.605(m/z 222) corresponds the molecular ion
M+[C12H14O4].
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 500.0 525.0 550.0 575.0 600.0
0.0
0.5
1.0(x10,000) 82
54 108
150 161 185 213 244 271 304 336 358 389 416 446 474 503 533 561 590
Fig. 2: Mass spectrum of D-carvone.
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 500.0 525.0 550.0 575.0 600.0
0.0
0.5
1.0(x10,000) 68
67
136
121
166 199 214 242 272 301 329 361 387 419 446 476 506 536 562 592
Fig. 3: Mass spectrum of D-limonene.

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Abdel et al. World Journal of Pharmaceutical and Life Science
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 500.0 525.0 550.0 575.0 600.0
0.0
0.5
1.0(x10,000) 222
177 207
149
121
77
65 242 271 302 342 360 387 416 446 474 510 532 561 590
Fig. 4: Mass spectrum of apiol.
Antimicrobial assay
The oil was screened for antimicrobial activity against
five standard human pathogens, the average of the
diameters of the growth of inhibition zones are depicted
in Table (5). The results were expressed in terms of the
diameter of the inhabitation zone:
<9 mm inactive
9-12 mm partially active
13-18 mm active
>18 mm very active
Ampicilin, gentamicin and clotrimazole were used as
positive control.
Table 5: Inhibition zones of oil.
Sample
Sa
Bs
Ec
Ps
Ca.
Oil (100mg/ml)
17
21
20
20
14
Ampicilin (40mg/ml)
30
15
--
--
--
Gentamicin (40mg/ml)
19
25
22
21
--
Clotrimazole (30mg/ml)
--
--
--
--
38
Ec.: Escherichs coli.
Ps.: Pseudomonas aeruginosa.
Sa.: Staphylococcus aureus.
Bs.: Bacillus subtilis.
Ca.: Candida albicans.
The oil showed significant activity against Escherichia
coli, Pseudomonasa aeruginosa and Bacillus subtilis,
while it showed a good activity against staphylococcus
aureus.
REFERENCES
1. Wren R C. Potter’s new cyclopedia of botanical
drugs and preparations. Saffron Walden, CW
Daniel, 1988.
2. Leung A Y, Foster S. Encyclopedia of common
natural ingredients used in food, drugs and
cosmetics, John Wiley and Sons, New York, 1996.
3. Yazdanparast R, Bahramikia S. Evaluation of the
effect of Anethum graveolens L. crude extracts on
serum lipids and lipoproteins profiles in
hypercholesterolaemic rats, DARU, 2008; 16(2):
88-94.
4. African pharmacopoeia. Vol. 1., Lagos,
Organization of African Unity, Scientific Technical
and Research Commission, 1985.
5. Zargari A. Medicinal Plants. 6th ed. Vol. II, Tehran
University Press, Tehran, 1996; 531-528.
6. Ishikawa T M, Kudo M, Kitajima J. Water-soluble
constituents of dill, Chem Pharm Bull, 2002; 55:
501-507.
7. Kaur G J, Arora D S. Bioactive potential of
Anethum graveolens, Foeniculum vulgare and
Trachyspermum ammi belonging to the family
Umbelliferae -Current status, Journal of Medicinal
Plants Research, 2010; 4(2): 87-94.
8. Valady A, Nasri S, Abbasi N. Anti-inflammatory
and analgesic effects of hydroalcoholic extract from
the seed of Anethum graveolens L. J Med Plants,
2010; 9: 130-124.
9. Naseri M, Mojab F, Khodadoost M. The study of
anti-inflammatory activity of oil-based dill
(Anethum graveolens L.) extract used topically in
formalin-induced inflammation male rat paw,
Iranian Journal of Pharmaceutical Research, 2012;
11(4): 1169-1174.
10. Yazdanparast R, Bahramikia S. Evaluation of the
effect of Anethum graveolens L. crude extracts on
serum lipids and lipoproteins profiles in
hypercholesterolaemic rats, DARU, 2008; 16(2):
88-94.
11. Yazdanparast R, Alavi M. Antihyperlipidaemic and
antihypercholesterolaemic effects of Anethum
graveolens leaves after the removal of
furocoumarins, Cytobios, 2001; 105: 185-191.
12. Hajhashemi V, Abbasi N. Hypolipidemic activity of
Anethum graveolens in rats, Phytother Res, 2008;
22: 372-375.
13. Delaquis P J, Stanich K, Girard B. Antimicrobial
activity of individual and mixed fractions of dill,
cilantro, coriander and eucalyptus essential oils, Int
J Food Microbiol, 2002; 74: 101-109.
14. Singh G, Kapoor I P S, Pandey S K. Studies on
essential oils: part 10: Antibacterial activity of
volatile oils of some spices, Phytother Res, 2001;
16: 680-682.
15. Lobez,P., Sanchez,C., Batlle,R., Nerin,C, Solid and
Vapor Phase Antimicrobial Activities of Six
Essential Oils: Susceptibility of Selected Food –
Borne Bacterial and Fungal Strains, J Agric Food
Chem., 2005; 53(17): 6939-6946.
16. Mohsenzadeh M. Evaluation of antibacterial activity
of selected Iranian essential oils against
Staphylococcus aureus and Escherichia coli in
nutrient broth medium, Pak J Biol Sci., 2007; 10:
3693-3697.

www.wjpls.org
20
Abdel et al. World Journal of Pharmaceutical and Life Science
17. Kaur G J and Arora D S. In vitro antibacterial
activity of three plants belonging to the family
Umbelliferae, Int J Antimicrob Agents, 2008; 31:
393-395.
18. Racz-Kotilla E, Rotaru G, Racz G. Anti-nociceptive
effect of dill (Anethum graveolens L.), Fitoterapia,
1995; 2: 80–81.
19. Hosseinzadeh H, Karimi K R, Ameri M. Effects of
Anethum graveolensL. seed extracts on
experimental gastric irritation models in mice,
Pharmacol, 2002; 2: 21.
20. Harries N, James K C, Pugh W K. Antifoaming and
carminative actions of volatile oils, Journal of
Clinical Pharmacology, 1978; 2: 171-177.
21. The Ayurvedicpharmacopoeia of India. Part I. Vol.
II, New Delhi, Ministry of Health and Family
Welfare, Department of Indian System of Medicine
and Homeopathy, 1999.
22. WHO monographs on selected medicinal plants,
Vol.3. WHO Library Cataloguing in Publication
Data. WHO, 2007; 34-41.
23. Khafagy S M, Mnajed H K. Phytochemical
investigation of the fruit of Egyptian Anethum
graveolens. I. Examination of the volatile oil and
isolation of dillapiole, Acta Pharmaceutica Suecica,
1968; 5: 155–162.
24. Stavri M, Gibbons S. The antimirobial constituents
of Dill (Anethum graveolens), Phytother Res, 2005;
19: 938-941.
25. Radulescu V, Popescu M L, Ilies D C. Chemical
composition of the volatile oil from different plant
parts of Anethum graveolens L. (Umbelliferae),
Farmacia, 2010; 58: 594-600.
26. Yazdanparast R, Bahramikia S. Evaluation of the
effect of Anethum graveolens L. crude extracts on
serum lipids and lipoproteins profiles in
hypercholesterolaemic rats, DARU, 2008; 16(2):
88-94.
27. Ana, C., Ferando, C., Daniel, A., Leila, P., Robert,
R., Amaldo, L., Mycopathologia, 2011; 171:
411-416.