International Journal of Chemical Studies 2018; 6(5): 3033-3037
P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2018; 6(5): 3033-3037
© 2018 IJCS
Received: 14-07-2018
Accepted: 18-08-2018
Ali Mohammad Abu-Rumman
Department of forensic
chemistry, College of Forensic
Sciences / Naif Arab University
for Security Sciences, Riyadh,
Saudi Arabia
Correspondence
Ali Mohammad Abu-Rumman
Department of forensic
chemistry, College of Forensic
Sciences / Naif Arab University
for Security Sciences, Riyadh,
Saudi Arabia
Analysis of Pimpinella anisum (Yansoon) seeds
using gas chromatography mass spectrometry
Ali Mohammad Abu-Rumman
Abstract
The extraction of milled Pimpinella anisum (Yansoon) seeds were performed using two different
extraction methods; organic extraction with methanol (polar), hexane (nonpolar) and hydrodistillation
method for separation essential oil from Anise seeds. Analysis of Pimpinella anisum (Yansoon) seed
using Gas Chromatography – Electron Ionization / Mass Spectrometry (GC-EI/MS) technique was
applied with full scan mode within mass range from 12 – 500 mass to charge ratio (m/z). The chemical
composition was identified for trace level of 0.01% using Agilent Mass Hunter unknown analysis
software and NIST14 library of mass spectrums. The hydrodistillation extract and hexane extract of
essential oil were revealed the highest abundance for trans-Anethole with 96.54% and 72.15%
respectively, while methanolic extract was given the highest abundance of trans-Pseudoisoeugenyl 2-
methylbutyrate and Butanoic acid, 2-methyl-, 4-methoxy-2-(3-methyloxiranyl) phenyl ester 58.93% and
28.93% respectively. The yield of Anise seed oil was 2.5 – 3.0%.
Keywords: trans-Anethole, hydrodistillation, gas chromatography, methanolic extract, mass spectrometry
1. Introduction
Anise seed is an annual plant belongs to the Apiaceae family, botanically named Pimpinella
anisum which is known as Yansoon in most Arabic countries. Aniseed is an old generic name
for Pimpinella anisum [1]. It is cultivated in Egypt and later in Greece, Rome, and the Middle
East. It has white flowers and yellow-green seeds [2]. Currently, it is cultivated in warmer
climates and Asia [3]. The Pimpinella anisum has common names in different countries such
as: Anis vert (France); Anise seed (Japan); Anise and Star anise (USA); Annesella (Italy);
Anisa, Badian, Kuppi, Muhuri, Saunf and Sop (India); Boucage anis, Petit anise (North Africa)
[4]
. And Anise (England) [3]. Trans-anethole is mainly responsible for distinctive taste and odor
of anise seeds [5].
Anise seed has several properties employed in medicinal, perfume industry and in food
cooking. It is used as flavoring in soups, biscuits, sweets, tarts, baking, cakes [6]. to improve
flavor in cakes and alcoholic beverages in liquors [7]. Also, it is used as anethole flavor in
candy, chewing gum, tobacco, coffee, tea, curries and pickles [8].
The fruit of Pimpinella anisum is used in traditional medicine for treatments of several
diseases. Popularly, in Middle East area, especially in cold weather, anise seeds are consumed
as hot drinks similar as tea, since its positive effect against cold, cough, bronchitis, fever, colic,
mouth and throat inflammation, digestive problems and loss of appetite. It is used as
anticonvulsant, an ingredient in cough medicine, antiseptic, antispasmodic, expectorant,
diaphoretic [9]. An immune stimulant, growth promoter, antifungal, antibacterial [10]. Relaxant
effect, anticholinergic [11]. Stomachic, stimulant, diuretic [12]. Assisted digestion, an appetizer,
diuretic, tranquillizer [13]. Increase milk secretion, encourage menstruation, facilitate birth,
increase libido [14]. Fever, digestive problems and loss of appetite [15]. Anethole is used in
synthesis of pharmaceutical substances such as chloral, an anticonvulsant agent and
phenobarbital [16]. Several studies had described different extraction methods of Anise seed
such as steam distillation [7, 17, 19]. Hydro distillation [20, 22]. Supercritical fluid extraction using
carbon dioxide [23, 25]. Solvent extraction with ethanol, methanol, water [26, 29]. Petroleum ether,
chloroform, ethyl acetate, acetone [30, 31]. And hexane [32, 33].
This aim of this study was to identify the chemical composition of anise seed using solvent
extraction with methanol as polar solvent and non-polar solvent of hexane, and hydro
distillation method. The organic solvent extracts of methanol, hexane and separated volatile oil
from hydrodistillation were analyzed using gas chromatography-mass spectrometry technique
under electron ionization.
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International Journal of Chemical Studies
2. Experimental
2.1 Methods and Materials
The anise seeds (Pimpinella anisum) which cultivated in Syria
were purchased from the local herbs market in Riyadh, Saudi
Arabia. HPLC grade methanol and hexane were obtained
from Fisher Scientific, UK. Sodium sulphate was purchased
from Alpha Chemica, India. MS PTFE syringe filter with pore
size 0.22 µm and diameter 25 mm was purchased from
Membrane Solutions, USA. Orbital shaker model GFL 3017
was obtained from Gesellschaft für Labortechnik (GFL),
Germany. Automated Evaporation/ Concentration System
(XcelVap) was purchased from Horizon Technology, USA.
2.2 Sample preparation
The Pimpinella anisum seeds were milled to fine powder
using electric coffee grinder. 1.0 g of anise seeds powder was
extracted with 25 mL of HPLC methanol and 1.0 g of seeds
was extracted with 25 mL hexane. Both samples were mixed
using orbital shaker at velocity 300 rpm for 3 hrs. Methanolic
and hexane extracts were filtered using syringe filter, 0.22 µm
through sodium sulphate and evaporated under nitrogen till oil
residue. After evaporation, the extracted oil residue was
dissolved with 3 mL ethyl acetate and 1 ml transferred to GC
vial for GC/MS analysis. Essential oil was obtained from 10.0
g of anise powder through extraction via hydrodistillation
using 240 min as distillation time. 100 µL of essential oil was
diluted to 1.0 mL hexane.
2.3 Instrumentation (GC-EI/MS)
Gas chromatograph (7890B GC) coupled with mass
spectrometer (5977A MSD) and auto sampler (7693) from
(Agilent Technologies, USA) were used for identification the
chemical composition of solvent extracts and separated
volatile oil via hydro distillation from anise seeds.
The Gas chromatograph (GC) was equipped with an Agilent
HP-5MS (5% Phenyl methyl siloxane) capillary column (30
m × 0.25 mm I.D × 0.25 µm film thickness). The sample
volume injected 1.0 µL was made in 5:1 split ratio. Helium
with high purity 99.999% was used as carrier gas with
constant flow rate of 1 mL/min. The temperature of injector
and mass selective detector (MSD) transfer line were 250 °C
and 280 °C respectively. Operating GC conditions within run
time 30 min and column temperature was operated as follows:
initial temperature was 50 °C hold time 2 min with multiple
ramp rates of 20 °C/min to 200 °C hold time 2 min and rate of
10 °C/min to 280 °C hold time 10.5 min. The mass
spectrometer (MS) operating parameters were used in electron
ionization (EI) mode with ion source temperature 230 °C and
quadruple temperature 150 °C. The MS acquisition mode was
in Full scan with mass range 12 – 500 mass to charge ratio
(m/z) and electron ionization (EI) voltage was 70 electron volt
(eV).
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3. Results and Discussion
Analysis of anise seeds compounds for three different extracts
present in methanol, hexane and volatile oil obtained from
hydro distillation were performed using GC-EI/MS method.
The GC-MS total ion chromatograms (TIC) which represent
the separated peaks of components according to their
abundance versus the retention time, for three extracts of
hexane, methanol and hydro distillation are illustrated in Fig.
1, Fig. 2 and Fig. 3 respectively.
Agilent Mass Hunter Workstation Unknown Analysis was
used to perform mass spectrum search of components with
database of National Institute Standard and Technology
(NIST) library which contains 242,466 spectra as main
library. Reported results were based on matching mass
spectrums of constituents which including presence of base in
addition to other principal m/z ions as represented in Table 1
(hexane extract), Table 2 (methanol extract) and Table 3
(hydro distillation).
The various results about components for three extracts in
anise (Yan soon) seeds such as retention time (tR), compound
name, chemical formula, molecular weight (MW), chemical
abstract service registry number (CAS No.), retention index
(RI), percentage (%) area and mass spectrum (MS) principal
ions (m/z) are summarized in tables 1-3.
The hydro distillation extract and hexane extract of anise
seeds were revealed the highest abundance for trans-Anethole
with 96.54% and 72.15% respectively, while methanolic
extract was revealed the highest abundance of trans-
Pseudoisoeugenyl 2-methylbutyrate and Butanoic acid, 2-
methyl-, 4-methoxy-2-(3-methyloxiranyl) phenyl ester
58.93% and 28.93% respectively. The content of essential oil
obtained from anise seed was 2.5 – 3.0% via hydro distillation
using 240 min as distillation time. The variation of essential
oil content and composition of anise seed is reliant to
environment, genotype, location, soil, age of trees and
climatic conditions [18]. The specific gravity of essential oil is
0.978 at 25 °C and the refractive index is 1.5530 at 20 °C [34].
According to the solubility rule “like dissolve like”, trans-
Anethole which is p-Propenylphenyl methyl ether, has low
polarity, therefore, it was revealed in non-polar hexane extract
as the major constituent (72.15%) due to its solubility in non-
polar solvents such as hexane, ethyl acetate and ether,
whereas trans-Anethole was not revealed within components
of polar methanol extract. Also, methanol extract recovered
the highest constituents of polar compounds such as trans-
Pseudoisoeugenyl 2-methylbutyrate (58.93%) in comparison
to 12.9% in hexane extract and Butanoic acid, 2-methyl-, 4-
methoxy-2-(3-methyloxiranyl) phenyl ester (28.93%) in
comparison to 6.52% in hexane extract. Hydro distillation was
given the highest abundance for trans-Anethole 96.54%.
International Journal of Chemical Studies

Fig 1: GC-MS total ion chromatogram of anise (Yansoon) seeds hexane extract

Fig 2: GC-MS Total ion chromatogram of anise (Yansoon) seeds methanolic extract

Fig 3: GC-MS total ion chromatogram of hydrodistillation from anise (Yansoon) seeds

Table 1: GC/MS results of chemical composition (%) from hexane extract of Anise (Yansoon) seeds
% Mass spectrum principal
No. tR (Min) Compound name Formula MW CAS # RI
Area ions (m/z)
1 9.05 Benzaldehyde, 4-methoxy- C8H8O2 136 123-11-5 1250 0.17 135, 136, 77, 92, 107
2 9.23 trans-Anethole C10H12O 148 4180-23-8 1301 72.15 148, 147, 117, 77, 105
3 9.92 2-Propanone, 1-(4-methoxyphenyl)- C10H12O2 164 122-84-9 1384 0.12 121, 164, 122, 43, 78
4 10.46 cis-.alpha.-Bisabolene C15H24 204 29837-07-8 1504 0.22 93, 41, 91, 67, 79
5 10.55 Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl- C15H22 202 644-30-4 1483 0.32 119, 132, 41, 105, 55
6 10.63 Aromandendrene C15H24 204 489-39-4 1440 3.74 41, 161, 91, 93, 105
7 10.73 beta.-Bisabolene C15H24 204 495-61-4 1509 0.34 69, 93, 41, 94, 67

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International Journal of Chemical Studies

8 13.54 trans-Pseudoisoeugenyl 2-methylbutyrate C15H20O3 248 58989-20-1 1834 12.9 164, 57, 149, 165, 91
Butanoic acid, 2-methyl-, 4-methoxy-2-(3-
9 14.14 C15H20O4 264 97180-28-4 1892 6.52 57, 29, 137, 41, 164
methyloxiranyl) phenyl ester
10 17.77 Estragole ; Methyl chavicol C10H12O 148 140-67-0 1196 0.15 148, 147, 77, 121, 117
11 22.71 Squalene C30H50 410 111-02-4 2832 0.28 69, 81, 41, 95, 68
12 23.75 Octacosane C28H58 394 630-02-4 2800 1.84 57, 43, 71, 85, 55
13 26.94 Nonadecane C19H40 268 629-92-5 1900 0.6 57, 43, 71, 85, 41
14 26.94 Dodecane, 2-methyl- C13H28 184 1560-97-0 1264 0.66 43, 57, 71, 41, 85

Table 2: GC/MS results of chemical composition (%) from methanolic extract of Anise (Yansoon) seeds
tR % Mass spectrum
No. Compound name Formula MW CAS # RI
(min) Area principal ions (m/z)
1 3.71 Isobutyl acetate C6H12O2 116 110-19-0 771 0.89 43, 56, 73, 41, 39
2 6.64 Benzene, 1-ethyl-2-methyl- C9H12 120 611-14-3 970 0.10 105, 120, 91, 106, 77
3 10.63 Aromandendrene C15H24 204 489-39-4 1440 1.59 41, 161, 91, 93, 105
4 10.75 Benzene, 4-ethenyl-1,2-dimethoxy- C10H12O2 164 6380-23-0 1369 2.83 164, 149, 91, 77, 103
trans-Pseudoisoeugenyl 2-methylbutyrate ; (E)-4-Methoxy-2-(prop-1-en-
5 13.54 C15H20O3 248 58989-20-1 1834 58.93 164, 57, 149, 165, 91
1-yl) phenyl 2-methylbutanoate
6 14.14 Butanoic acid, 2-methyl-, 4-methoxy-2-(3-methyloxiranyl)phenyl ester C15H20O4 264 97180-28-4 1892 28.93 57, 29, 137, 41, 164
7 16.67 Hexanoic acid, 2-propenyl ester C9H16O2 156 123-68-2 1076 0.33 99, 43, 41, 71, 39
8 16.67 Allyl 2-ethyl butyrate C9H16O2 156 7493-69-8 995 0.28 43, 71, 41, 39, 99
9 17.78 Estragole ; p-Allylanisole ; Methyl chavicol C10H12O 148 140-67-0 1196 0.52 148, 147, 77, 121, 117
11 21.56 Undecane, 3,8-dimethyl- C13H28 184 17301-30-3 1228 0.75 57, 43, 71, 85, 70
12 23.75 Octacosane C28H58 394 630-02-4 2800 3.55 57, 43, 71, 85, 55
13 26.96 Decane, 2,5,9-trimethyl- C13H28 184 62108-22-9 - 1.31 57, 43, 71, 41, 56

Table 3: Chemical composition of hydrodistillation extract from Anise seed

tR % Mass spectrum
No. Compound name Formula MW CAS # RI
(min) Area principal ions (m/z)
1 8.54 Estragole C10H12O 148 140-67-0 1196 0.20 148, 147, 77, 121, 117
2 8.93 Benzaldehyde, 4-(1-methylethyl)- C10H12O 148 122-03-2 1239 0.02 133, 105, 148, 119, 77
3 9.29 trans-Anethole ; Anisole, p-propenyl- C10H12O 148 4180-23-8 1301 96.54 148, 147, 117, 77, 105
4 9.60 γ-Elemene C15H24 204 29873-99-2 1433 0.03 121, 93, 41, 107, 67
5 10.33 cis-beta-Farnesene C15H24 204 28973-97-9 1444 0.01 41, 69, 93, 67, 133
6 10.46 α-Himachalene C15H24 204 3853-83-6 1449 0.06 93, 41, 94, 119, 79
7 10.56α-Curcumene ; Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl- C15H22 202 644-30-4 1483 0.02 119, 132, 41, 105, 55
8 10.60 Methyleugenol ; Benzene, 4-allyl-1,2-dimethoxy- C11H14O2 178 93-15-2 1402 0.05 178, 163, 147, 103, 91
9 10.64 γ-Muurolene C15H24 204 30021-74-0 1477 1.51 161, 105, 119, 93, 204
10 10.74 beta.-Bisabolene C15H24 204 495-61-4 1509 0.07 69, 93, 41, 94, 67
11 10.77 α-Longipinene C15H24 204 5989-08-2 1353 0.06 119, 105, 133, 93, 107
14 11.70 Spathulenol C15H24O 220 6750-60-3 1576 0.03 43, 41, 205, 119, 91
16 12.14 alpha-Bisabolol C15H26O 222 515-69-5 1684 0.01 43, 41, 69, 109, 119
trans-Pseudoisoeugenyl 2-methylbutyrate ; (E)-4-Methoxy-2-(prop-1-en-
17 13.55 C15H20O3 248 58989-20-1 1834 0.95 164, 57, 149, 165, 91
1-yl)phenyl 2-methylbutanoate
18 14.15 Butanoic acid, 2-methyl-, 4-methoxy-2-(3-methyloxiranyl)phenyl ester C15H20O4 264 97180-28-4 1892 0.17 57, 29, 137, 41, 164

4. Conclusion 3. Khare CP. Indian Medicinal Plants: An Illustrated

The hydro distillation extraction method for Anise seed was
given the highest recovery of trans-Anethole with 96.54%.
The selection of non-polar solvent such as hexane was
suitable to recover tran-Anethole (72.15%), whereas methanol
solvent was suitable to recover the components such as trans-
Pseudoisoeugenyl 2-methylbutyrate (58.93%). The oil content
obtained of Anise seed was 2.5 – 3.0% and the using of
GC/MS technique was reliable and useful for the analysis.
5. Acknowledgement
The author acknowledges the president of Naif Arab
University for Security Sciences for his support and providing
facilities to carry out this work. I would like to thank all staff
of the forensic chemistry department for providing all the
necessary assistance to perform my research.
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