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ISSN: 1314-6246 Gochev et al. J. BioSci. Biotech. 2012, 1(2): 135-139.
RESEARCH ARTICLE
http://www.jbb.uni-plovdiv.bg
135
Velizar Gochev 1
Tanya Girova 1
Ivanka Stoilova 2
Teodora Atanasova 3
Neno Nenov 4
Veselin Stanchev 5
Albena Soyanova 3
Low temperature extraction of essential oil
bearing plants by liquefied gases. 7. Seeds from
cardamom (Elettaria cardamomum (L.) Maton)
Authors’ addresses:
1 Department of Biochemistry and
Microbiology, Faculty of Biology,
Plovdiv University, Plovdiv, Bulgaria.
2 Department of Biotechnology
3 Department of Essential Oils
4 Department of Heating Technology
5 Department of Automatic, informatics
and managing engineering,
University of Food Technologies,
Plovdiv, Bulgaria.
Correspondence:
Velizar Gochev
Faculty of Biology,
Plovdiv University
24, Tsar Assen Str.
4000 Plovdiv, Bulgaria
Tel.: +359 32 261493
e-mail: vgochev@uni-plovdiv.bg
Article info:
Received: 9 August 2012
In revised form: 2 September 2012
Accepted: 3 September 2012
ABSTRACT
The chemical composition of extract from the seeds of cardamom (Elettaria
cardamomum (L.) Maton, obtained by extraction with tetrafluoroethane was
analyzed using GC and GC/MS. The major compounds (concentration higher
than 3%) of extract were: terpinyl acetate (36.8%), 1,8-cineole (29.2%), linalyl
acetate (5.2%), sabinene (3.9%) and linalool (3.1%). The studied extract
demonstrated antimicrobial activity against pathogenic species Staphylococcus
aureus, S. epidermidis, Salmonella abony and was inactive against Pseudomonas
aeruginosa. The extract possessed low antioxidant activity against DPPH
radicals.
Key words: antimicrobial activity, antioxidant activity, cardamom, extraction
with tetrafluoroethane
Introduction
Cardamom is produced from cultivated or wild plants in
the mountainous regions of southern India, Sri Lanka,
Indonesia, and Guatemala. It has been used in the traditional
Chinese medicine and Indian Ayurvedic medicine for
thousands of years, mainly for treating respiratory diseases,
fevers and digestive complaints.
The cardamom essential oil is obtained by steam
distillation from the ripe and dried seeds of the tropical grass
Elettaria cardamomum (L.) Matori (Zingiberaceae). It is a
colorless or very pale yellow liquid with an aromatic,
penetrating, slightly camphoraceous odor and a persistent,
pungent, strongly aromatic taste. The physico-chemical
properties (ISO 4733:1981) were: relative density at 20/20°C:
0.191 - 0.936; refractive index at 20°C: 1.4620 - 1.4680;
optical rotation at 20°C: range from +22 to +41; solubility: 1
vol. in max. 5 vol. 70% ethanol (Bauer et al., 1997; Georgiev
& Stoyanova, 2006).
The major components of the cardamom oil are 1,8-
cineole (21-41%) and
-terpinyl acetate (21-35%). The
cardamom oil also contains the following components:
-
terpineol (0.8-6.2%, and to 11.5% in oil from Pakistan),
limonene (1.7-3.7%), sabinene+
-pinene (0.3-2.4%), borneol
(0.1-1.2%), linalool (0.4-8.7%), linalyl acetate (1.6-2.4%),
nerol (0.6-1.6%), geraniol (1.1-3.7%), neryl acetate (0.8-
1.2%), farnesol (up to 12.5% from the total isomers),
nerolidol (0.2-6.7%), isosafrole (3.8%) and other minor
compounds. Trace constituents like unsaturated aliphatic
aldehydes and
-terpinyl acetate may be important for the
ISSN: 1314-6246 Gochev et al. J. BioSci. Biotech. 2012, 1(2): 135-139.
RESEARCH ARTICLE
http://www.jbb.uni-plovdiv.bg
136
typical aroma of the oil (Bauer et al., 1997; Laurence, 2004;
Georgiev & Stoyanova, 2006).
The oil possesses antioxidant activity (Misharina et al.,
2009). It is used primarily for seasoning foods, alcoholic
beverages, the applied doses in foods varied from 0.20 to
0.50 mg %, and the minimal notable doses varied in the range
0.04 - 0.05 mg % (Bauer et al., 1997; Georgiev & Stoyanova,
2006). In small dosages, the oil is also used in perfumery and
cosmetics. For example, cardamom oil is a constituent of
mouthwash to treat bad breath and in creams, because the oil
has diuretic properties and may alleviate fluid retention and
cellulite (Rose, 2002; Georgiev & Stoyanova, 2006).
Cardamom oil is mainly used in aromatherapy as a
digestive remedy to alleviate flatulence, heartburn, nausea,
indigestion and colic. It acts as a general tonic for the
digestive system and speeds up sluggish digestion. In India,
cardamom oil is believed to have aphrodisiac qualities and is
used to reduce the feelings of stress and tension that may be
inhibiting sexual fulfillment. Its restorative properties make it
effective in treating physical and mental fatigue (Rose, 2002;
Georgiev & Stoyanova, 2006).
Different extracts are produced from cardamom seeds by
using various solvents such as diethyl ether (Ağaoğlu et al.,
2005; Syed Abdul Rahman et al., 2010), water (Suneetha &
Krishnakantha, 2005), ethanol (Nanasombat &
Lohasupthawee, 2005) and methanol (El-Segaey et al., 2007).
Chemical compositions and antimicrobial properties of the
extracts depend mainly on the type of the used solvent. The
cardamom fruits can also be extracted with liquefied gases by
supercritical carbon dioxide (Hamdan et al., 2008;
Gopalakrishnan 1994; Marongui et al., 2004) and sub-critical
propane (Hamdan et al., 2008) extractions. Propane was
found to be more capable than carbon dioxide to recover seed
oil at sub-critical condition with lower ratio of solvent/solid
and better quality attributes (Hamdan et. al., 2008). The
major constituents of both extracts were 1,8-cineole and
-
terpinyl acetate and the content of these compounds in the
final extract depend on the extraction conditions such as
temperature, working pressure and solvent.
Currently C2H2F4 (1,1,1,2-tetrafluorethane) is prospective
liquefied gas, which is licensed for producing of extracts for
application in food and flavour industry. Unfortunately there
are no publications for its application for extraction of
cardamom seeds.
The aim of present study was to produce cardamom
extract by 1,1,1,2-tetrafluorethane and to characterize the
obtained product according to its chemical composition,
antimicrobial and antioxidant properties.
Materials and Methods
Plant material
Cardamom seeds (Elettaria cardamomum (L.) Maton)
were obtained from trade market, origin Guatemala harvest
2009, humidity 8% (Russian Pharmacopoeia, 1990).
Obtaining of extract
The air-seeds of cardamom were ground separately in an
attrition mill to a size of 0.15-0.25 mm and the extract
obtained by a 1 dm3 volume C2H2F4 (1,1,1,2-tetrafluorethane)
laboratory-extractor (Nenov, 2006) under following
conditions (continuous flow and evaporation of solvent):
pressure 0.5 MPa; temperature 18-20oС and extraction time
60 min. The physico-chemical properties were measured
according to Russian Pharmacopoeia (1990).
Determination of chemical composition
GC analysis was performed using an Agilent 7890A gas
chromatograph equipped with FID detector and HP-
INNOWAX Polyethylene Glycol column (60 mm x 0.25
mm; film thickness 0.25 m); temperature: 70°C - 10 min,
70-240°C - 5°C/min, 240°C - 5 min; 240-250°C - 10°C/min,
250°C - 15 min; carrier gas helium, 1 ml/min constant flow;
injector split, 250°C, split ratio 50:1.
Gas Chromatography-Mass Spectrometry Analysis:
GC/MS analysis was carried out on an Agilent 5975C gas
chromatograph, carrier gas helium, column and temperature
as for GC analysis, FID 280°C, MSD 280°C, transfer line.
Determination of antimicrobial activity
Antimicrobial activity of the cardamom extract was
determined against pathogenic and spoilage bacteria from
clinical and food isolates and also against reference microbial
strains. The used test microorganisms and their origins are
listed in Table 2. The strains are deposited in the Microbial
Culture Collection at the Department of Biochemistry and
Microbiology”, Plovdiv University, Bulgaria. Minimal
Inhibitory Concentration (MIC) and Minimal Bactericidal
Concentration (MBC) of cardamom extract were determined
by serial broth dilution method in accordance with CLSI
reference method (CLSI Standards, 1990) A stock solution to
be tested was prepared by diluting the respective cardamom
extract sample in DMSO (Sigma-Aldrich Co.). Antimicrobial
activity of the extract was determined in concentrations
ranging from 0.00025 to 1.6% (w/v).
ISSN: 1314-6246 Gochev et al. J. BioSci. Biotech. 2012, 1(2): 135-139.
RESEARCH ARTICLE
http://www.jbb.uni-plovdiv.bg
137
Scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical
The radical scavenging capacity was determined
according to the method described by Mensor et al. (2001).
1.0 ml from 0.3 mM alcohol solution of DDPH was added to
2.5 ml from the samples with different concentration of
cardamom extract. The samples were kept at room
temperature in dark and after 30 min the optical density was
measured at 518 nm. The optical density of the working
samples, the positive controls and the blank samples were
measured in comparison with ethanol. The IC50 value
represented the concentration of the compounds that caused
50% inhibition of radical formation.
All experiments were done in triplicate and the results
were statistically evaluated using a level of confidence
γ=0.95.
Results and Discussion
The obtained extract is yellow mobile liquid with strong
characteristic for the plant material odour and taste. The yield
of cardamom extract is 2.7-3.0% (v/w).
Physicochemical properties were as follows: dry
substance (105°C): 9.70%, refractive index (20°C): 1.4647,
specific gravity (20°C): 0.9430, acid number: 3.6.
The chemical composition of the extract is presented in
Table 1. Twenty-seven components representing 94.5% of
the total content were identified. Ten of them were in
concentrations over 1% and the rest 17 constituents were in
concentrations under 1%. The major constituents (over 3%)
were terpinyl acetate (36.8%), 1,8-cineole (29.2%), linalyl
acetate (5.2%), sabinene (3.9%) and linalool (3.1%).
Results from the tests for antimicrobial activity are
presented in Table 2.
Antioxidant activity of the cardamom extract is presented
in Figure 1. As seen from the figure, 55.2% inhibition of
DPPH radical was reached at concentration 100 mg/ml and
the IC50 value was 63.3 mg/ml (correlation coefficient
R2=0.995).
According to physicochemical properties, the produced
cardamom extract is almost equal to the cardamom essential
oil. 90.7% of the identified substances in the extract belong to
the group of monoterpenes, followed by phenyl propanoides
(2.2%), sesquiterpenes (1.5%) and others compounds (0.1%).
Oxygenated monoterpenes (80.0%) are the major group.
According to the content of major constituents, the
produced freon extract of cardamom seeds is similar to the
published in the literature (Hamdan et al., 2008). The
qualitative differences in the rest of the constituents are due
to the type of the used solvent and the process parameters.
Table 1. Chemical composition of the cardamom extract.
Components
%
RI
MONOTERPENES
Hydrocarbons
Sabinene
3.9
973
d-Limonene
2.3
1026
Мyrcene
1.9
990
-Pinene
1.9
939
-Pinene
0.4
981
-Terpinene
0.3
1059
Oxygenated monoterpenes
Terpinyl acetate
36.8
1340
1,8-Cineole
29.2
1032
Linalyl acetate
5.2
1254
Linalool
3.1
1093
-Terpineol
1.6
1167
Geraniol
0.9
1240
Geranial
0.8
1225
Terpinen-4-ol
0.6
1182
Sabinene hydrate
0.3
1054
Fenchone
0.6
1090
Neral
0.3
1253
Geranyl acetate
0.3
1378
Carvyl acetate
0.2
1362
Terpinyl propionate
0.1
1430
SESQUITERPENES
Hydrocarbons
-Selinene
0.3
1458
-Cadinene
0.2
1522
Germacrene
0.1
1468
Oxygenated sesquiterpenes
Nerolidole
0.9
1534
PHENYL PROPANOIDS
Anethole
2.1
1269
p-Cymene
0.1
1020
OTHERS
Decenal
0.1
1260
The cardamom extract demonstrated antimicrobial
activity against Gram-positive and Gram-negative bacteria,
belonging to species S. epidermidis, S. aureus, E. coli and S.
abony. The extract was inactive against both strains of P.
aeruginosa, which belong to the group of the most resistible
bacterial strains. The ability of P. aeruginosa to produce
extracellular polysaccharides increased antimicrobial
resistance of these bacteria mainly through permeability
barrier.
ISSN: 1314-6246 Gochev et al. J. BioSci. Biotech. 2012, 1(2): 135-139.
RESEARCH ARTICLE
http://www.jbb.uni-plovdiv.bg
138
Table 2. Аntimicrobial activity of extract from cardamom..
№
Test microorganisms
Origin
MIC, % (v/v)
MBC, % (v/v)
1
Staphylococcus epidermidis
Clinical isolate
0.4
0.8
2
Staphylococcus aureus
ATCC 6538
0.4
0.8
3
Escherichia coli
Food isolate
0.8
0.8
4
Escherichia coli
ATCC 8739
0.8
0.8
5
Salmonella abony
Clinical isolate
0.8
0.8
6
Salmonella abony
ATCC 6017
0.8
0.8
7
Pseudomonas aeruginosa
Food isolate
inactive
8
Pseudomonas aeruginosa
ATCC 9627
inactive
These strains also produced two types of soluble pigments,
pyoverdin and pyocyanin, which probably participate in cell
defense against antimicrobials.
Figure 1. Antiradical activity of cardamom extract against
DPPH.
In comparison with strong antioxidants such as ascorbic
acid (4.20 g/cm3), rutin (14.65 g/cm3), BHT (1.12 g/cm3)
and BHA (4.41 g/cm3), which are traditionally used in
cosmetics and food industry, the produced cardamom extract
possesses considerably lower antioxidant activity. In
comparison with other extracts produced by low temperature
extraction with 1,1,1,2-tetrafluorethane from anise fruirts
(IC50=8.32 mg/ml – Atanasova, 2007), coriander fruits
(IC50=17.74 mg/ml – Atanasova et al., 2010) and cinnamon
barks (IC50=0.38 mg/ml – Nenov et al., 2011), the cardamom
extract also demonstrates lower antioxidant activity.
Conclusion
The extract from cardamom (Elettaria cardamomum (L.)
Maton) seeds produced by low temperature extraction with
tetrafluoroethane characterized with higher content of
terpinyl acetate (36.8%), 1,8-cineole (29.2%), linalyl acetate
(5.2%), sabinene (3.9%) and linalool (3.1%) with
characteristic odour and taste. The produced extract
demonstrated antimicrobial activity against some of the most
widely spread pathogenic and spoilage bacteria in foods and
characterized with low antioxidant activity in comparison
with other extracts produced by low temperature extraction.
Currently, the experiments for application of the produced
cardamom extract in cosmetic and food products are in
progress.
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ISSN: 1314-6246 Gochev et al. J. BioSci. Biotech. 2012, 1(2): 135-139.
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http://www.jbb.uni-plovdiv.bg
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