LOW TEMPERATURE EXTRACTION OF BULGARIAN ESSENTIAL OIL BEARING PLANTS FROM LAMIACEAE BY LIQUEFIED GASES. 17. SAVORY (SATUREJA HORTENSIS L.)

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www.wjpps.com Vol 3, Issue 6, 2014.
Nenko et al. World Journal of Pharmacy and Pharmaceutical Sciences
LOW TEMPERATURE EXTRACTION OF BULGARIAN ESSENTIAL
OIL BEARING PLANTS FROM LAMIACEAE BY LIQUEFIED GASES
17. SAVORY (SATUREJA HORTENSIS L.)
Nenko Nenov1*, Velizar Gochev2, Ivanka Stoilova3, Tanya Girova2, Teodora
Atanasova4, Albena Stoyanova4
1Department of Thermal Engineering, University of Food Technologies, 26 Maritza Blvd,
4002 Plovdiv, Bulgaria.
2Department of Biochemistry and microbiology, Paisii Hilendarski University of Plovdiv, 24
Tzar Asen str., Plovdiv 4000, Bulgaria.
3Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd, 4002
Plovdiv, Bulgaria.
4Department of Essential Oils, University of Food Technologies, 26 Maritza Blvd, 4002
Plovdiv, Bulgaria.
ABSTRACT
The chemical composition of extract from the Bulgarian essential oil
bearing plants from Lamiaceae family - savory (Satureja hortensis L.)
by extraction with C2H2F4 (1,1,1,2-tetrafluoroethane) was analyzed
using GC and GC/MS. The main compound (concentration higher than
3 %) of extract was thymol (73.91 %). The studied extract
demonstrated antimicrobial activity against Gram-positive, Gram-
negative bacteria and yeasts. The extract has antioxidant activity
against DPPH radical.
KEW WORDS: Savory, liquefied gas extraction, chemical
composition, antimicrobial and antioxidant activities.
INTRODUCTION
Savory (Satureja hortensis L.) is wild growing in Mediterranean and East Asian countries. It
is industrially cultivated in many European countries (France, Germany, Poland, Czech
Republic, Greece, Serbia, Monte Negro and Moldova) and North America. The major
constituents of essential oil are phenol compounds carvacrol (14.3 – 77.6 %) and thymol
(0.01 – 46.0 %), but linalool (to 26.0 %),

-terpinene (7.6 – 52.9 %) and р-cymene (2.8 – 16.0
%) are also distributed [1-5].
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Article Received on
14 April 2014,
Revised on 05 May 2014,
Accepted on 29 May 2014
*Correspondence for Author
Dr. Neno Nenov
Department of Thermal
Engineering, University of
Food Technologies, 26 Maritza
Blvd, 4002 Plovdiv, Bulgaria
nenonenov@abv.bg

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Nenko et al. World Journal of Pharmacy and Pharmaceutical Sciences
In Bulgaria mainly local population of savory is grown as garden plant, nevertheless that
cultivar “Lozenka” was selected and introduced as industrial culture [6-7]. The savory is
rarely processed for producing of essential oil in Bulgaria. Bulgarian savory essential oil
demonstrates antimicrobial activity [8] and its application in perfumery [9], cosmetics [10-
11] and food and flavour industry [6,8] is well known.
Currently in many countries essential oil bearing plants are processed by extraction with
liquefied gases (СО2, halogenated hydrocarbons, etc). The produced extracts are harmless
that’s why they can be widely used in food and flavour industry, cosmetics and medicine.
The usage of liquefied gases overcomes the drawbacks of installations working with volatile
polar and non-polar solvents [12-13]. The installations for extraction with liquefied gases
characterized with high working pressure (0.6 MPa when butane is used, 1.5 MPa when
propane is used and 5-100 MPa when СО2 is used) and increased capital investments are
needed. There are only three extracting installations working with liquefied gases in
Bulgaria. Two of them, working with supercritical СО2 are situated in Dimitrovgrad and
village of Mirkovo and laboratory and semi-industrial installations, working with liquefied
tetrafluoroethane are situated in University of Food Technologies in Plovdiv. These
installations are used for processing of different essential oil bearing plants in Bulgaria [14-
19]. The aim of present study is producing of new plant extract from savory growing in
Bulgaria by using food grade solvent liquefied gas tetrafluoroethane in laboratory installation
and determination of their chemical composition and characteristics for possible application
in pharmaceuticals as well as in natural cosmetics and food products.
MATERIAL AND METHODS
Obtaining of extract
The used solvent is non-polar food grade liquefied gas tetrafluoroethane (CAS number 811-
97-2). The air-plants was ground separately in an attrition mill to a size of 0.15-0.25 mm and
the extract obtained in a 1 dm3 volume laboratory extractor [13] under following conditions:
temperature 20-25 ОC, pressure 5,7-6,5 bar, extraction time 50-70 min. (see Table 1). The
physical-chemical properties of extracts were measured according to [20].
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 m x 0,25 mm; film
thickness 0,25 m); temperature: 70 ОC-10 min, 70-240 ОC-5 ОC/min, 240 ОC-5 min; 240-

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Nenko et al. World Journal of Pharmacy and Pharmaceutical Sciences
250 ОC-10 ОC/min, 250 ОC-15 min; carrier gas helium, 1 ml/min constant flow; injector split,
250 ОC, 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 ОC, MSD, 280 ОC, transfer line.
Determination of antimicrobial activity
Antimicrobial activity of extract was determined against pathogenic and spoilage bacteria and
yeasts from clinical and food isolates and also against reference strains. The used test
microorganisms and their origins are listed in Table 3. The strains are deposited in the
microbial culture collection of Department of Biochemistry and microbiology in Paisii
Hilendarski University of Plovdiv, Bulgaria. Minimal Inhibitory Concentration (MIC, % w/v)
and Minimal Bactericidal Concentration (MBC, %, w/v) of extracts were determined by
reference methods for broth dilution antimicrobial susceptibility tests for bacteria that grow
aerobically [21] and reference method for broth dilution antifungal susceptibility testing of
yeasts [21]. A stock solution to be tested was prepared by diluting the respective extract
sample in 10 % DMSO (SIGMA-ALDRICH CO). Antimicrobial activity of the extract was
determined in concentrations ranging from 0.00025 to 1.6 % (w/v).
Scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
The radical scavenging capacity was determined according to the method described in [22].
1.0 ml from 0.3 mM alcohol solution of DDPH was added to 2.5 ml from the samples with
different concentration of savory extract. The samples were kept at room temperature in the
dark and after 30 min the optic density was measured at 518 nm. The optic density of the
samples, the control and the empty 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 produced extract is mobile liquid with strong characteristic odour. The yield and some of
physical and chemical characteristics of the extract are shown in Table 1. As seen the results
for the yields of extract is comparable with literature data and values of the physical and
chemical characteristics of the extract are almost equal with these for the essential oils.

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Nenko et al. World Journal of Pharmacy and Pharmaceutical Sciences
Table 1. Yield and physical-chemical properties of extract.
Properties Value
Yield, kg raw material/kg extract 80 - 100
Color Dark brown
Volatile oil content, % (v/w) 60-65
Refractive index (n20
D) 1.5060
Specific gravity (d20
20 ) 0.9891
Acid number (mg KOH/g extract) 17.2
Chemical composition of the extract is listed in Table 2. As seen the major constituent (and
only above 3 %) of the extracts is thymol (73.91 %). According to qualitative and
quantitative content of the major constituents the produced extract is equal to the essential
oils, published by above listed authors. Distribution of major groups of aroma substances in
the extract is shown in Fig. 1. Phenyl propanoids are the dominant group in the extract (83.46
%), followed by sesquiterpenes (6.73 %) and monoterpenes (6.41 %).
Table 2. Chemical composition of savory extract.
Compounds RI % Compounds RI %

-pinene 939 2.05 p-methoxy cinnamal 1524 1.04

-pinene 980 1.38

-cadinene 1524 1.39
Myrcene 991 0.58 p-cymene-2,5-diol 1553 1.04
p-cymene 1026 2.10

-cadinol 1653 0.42

-terpinene 1062 1.91 Bisabolol оxide 1744 0.80
Thymol 1290 73.91 Phytol 1949 0.98

-caryophyllene 1418 0.91 palmitic acid 1993 0.58

-bisabolene 1509 2.53 Squalene 2790 0.36

-cadinene 1513 1.38
6
5
4
3
2
1
7
1234567
Figure 1. Group of components in the extract, %.
1- monoterpene hydrocarbons, 2 – oxygen monoterpenes, 3 – sesquiterpene
hydrocarbons, 4 - oxygen sesquiterpenes, 5 – diterpenes, 6 – phenyl propanoids, 7 –
others.

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Nenko et al. World Journal of Pharmacy and Pharmaceutical Sciences
The results of antimicrobial testing are presented in Table 3. As seen the studied extract
demonstrated antimicrobial activity against the tested microbial strains. Gram-positive
bacteria were the most sensitive microbes, followed by yeasts and Gram-negative bacteria.
The less sensitive were both strains of P. auruginosa. Extract characterized with the highest
content of thymol, which probably is the major constituent responsible for antimicrobial
activity of the extract. Antimicrobial activity of the studied extract was equal to the
antimicrobial activity of essential oils from savory from Bulgaria and other countries [23-24].
Тable 3: Аntimicrobial activity of the extract.
Test micro- organisms Origin peppermint extract
MIC, % MBC, %
Staphylococcus epidermidis Clinical isolate 0.05 0.1
Staphylococcus aureus ATCC 6538 0.05 0.1
Escherichia coli Food isolate 0.2 0.2
Escherichia coli ATCC 8739 0.2 0.2
Salmonella abony Clinical isolate 0.2 0.2
Salmonella abony ATCC 6017 0.2 0.2
Pseudomonas auruginosa Food isolate 0.4 0.4
Pseudomonas auruginosa ATCC 9627 0.4 0.4
Candida albicans Clinical isolate 0.2 0.2
Candida albicans ATCC 10231 0.2 0.2
The results of antioxidant testing of the extract are as follows: 83.3 % inhibition of DPPH
radical was reached by extract from savory at concentration 1.2 mg/ml and the IC50 values
was 0.6355 mg/ml (correlation coefficient R2=0.995);
In comparison with other extracts produced by low temperature extraction with
tetrafluoroethane, the extract demonstrated higher antioxidant activity than the extract from:
anise fruits - IC50 е 8.32 mg/ml [17] and coriander fruits - IC50 е 17.74 mg/ml [18], and
almost equal activity in comparison with the extract from cinnamon barks - IC50 0.38 mg/ml
[19].
The results obtained are comparable with the results published by other authors for the same
essential oils and extracts from another geographic origin [25-30]. The similar chemical
composition of the aroma products is the major reason for almost equal antioxidant activity.
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 as

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Nenko et al. World Journal of Pharmacy and Pharmaceutical Sciences
antioxidants, the produced extracts from Bulgarian essential oil bearing plants belonging to
family Lamiaceae, possessed considerably lower antioxidant activity.
CONCLUSION
For the first time in Bulgaria new extract from savory (Satureja hortensis L.) was produced
by extraction with liquefied tetrafluoroethane. The optimal conditions for extraction
procedure were determined. The produced extract was almost equal with essential oils from
the essential oil bearing plants, according to its chemical composition, antimicrobial and
antioxidant activities. The extract is prospective for possible application in different
pharmaceuticals as well as cosmetic and food products, but of course additional
investigations are a must.
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