LOW TEMPERATURE EXTRACTION OF BULGARIAN ESSENTIAL OIL BEARING PLANTS FROM LAMIACEAE BY LIQUEFIED GASES. 16. PEPPERMINT (Mentha piperita (Huds.) L.)

Abstract

The chemical composition of extract from the Bulgarian essential oil bearing plants from Lamiaceae family - peppermint (Mentha piperita L.) by extraction with C2H2F4 (1,1,1,2-tetrafluorethane) was analyzed using GC and GC/MS. The main compounds (concentration higher than 3 %) of extract was as follows: menthone (30.54 %), menthol (21.12 %), menthyl acetate (9.92 %), iso-menthone (4.43 %), cis-sabinene hydrate (3.82 %), 1,8-cineole (3.41 %) and pulegone (3.04 %). The studied extract demonstrated antimicrobial activity against Gram-positive, Gram-negative bacteria and yeasts. The extract has antioxidant activity against DPPH radical.

Full text

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
70
LOW TEMPERATURE EXTRACTION OF BULGARIAN ESSENTIAL
OIL BEARING PLANTS FROM LAMIACEAE BY LIQUEFIED GASES.
16. PEPPERMINT (Mentha piperita (Huds.) L.)
NENKO NENOV2, VELIZAR GOCHEV1, IVANKA STOILOVA3, TANYA GIROVA4, TEODORA
ATANASOVA4, ALBENA STOYANOVA4
1“Department “Biochemistry and microbiology”, Paisii Hilendarski” University of Plovdiv, 24 Tzar Asen str.,
Plovdiv 4000, Bulgaria, vgochev@uni-plovdiv.bg
2Department of Thermal Engineering, University of Food Technologies, 26 Maritza Blvd, 4002 Plovdiv,
Bulgaria, nenonenov@e-xtracts.com
3Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria,
wstoilowa@yahoo.com
4Department of Essential Oils, University of Food Technologies, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria,
alstst@yahoo.com
Abstract: The chemical composition of extract from the Bulgarian essential oil bearing plants from
Lamiaceae family - peppermint (Mentha piperita L.) by extraction with C2H2F4 (1,1,1,2-tetrafluoroethane)
was analyzed using GC and GC/MS. The main compounds (concentration higher than 3 %) of extract was as
follows: menthone (30.54 %), menthol (21.12 %), menthyl acetate (9.92 %), iso-menthone (4.43 %), cis-
sabinene hydrate (3.82 %), 1,8-cineole (3.41 %) and pulegone (3.04 %). The studied extract demonstrated
antimicrobial activity against Gram-positive, Gram-negative bacteria and yeasts. The extract has antioxidant
activity against DPPH radical.
Keywords: Mentha piperita, 1,1,2-tetrafluoroethane, composition, antimicrobial and antioxidant activities.
Introduction
Bulgaria is relatively small country in South-East Europe, which occupies 111 000
km2 and is located in the centre of the Balkan Peninsula, sharing borders with Romania,
Serbia, the Republic of Macedonia, Greece, Turkey and the Black Sea. Besides being very
picturesque and rich in history and culture, Bulgaria possesses a highly varied topography and
a range of micro-climatic areas: mountains, plains, rivers and seacoast valleys. The climate
ranges from moderate continental in the northern part to the Mediterranean/subtropical in
southwest and southeast regions. As a result of these favorable climatic conditions, soils and

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
71
other natural factors, Bulgaria has been a producer of medicinal plants and essential oils for
more than 350 years.
Bulgaria has been renowned as one of the two major global supplier of rose oil (Rosa
damascena Mill.) and, also, as a source of many other essential oils and aromatic products,
including lavender (Lavandula vera D.C.), zdravetz (Geranium macrorrhizum L.), sage
(Salvia sclarea L.) etc. Many essential oil bearing plants, belonging to various genera, are
industrially cultivated and processed in Bulgaria, but the major part belongs to the genera
Lamiaceae (Georgiev and Stoyanova, 2006).
Peppermint (Mentha piperita (L.) Huds.) is annual plant, which is cultivated in many
countries, characterized with moderate and warm climate. The major components of
peppermint oils are: menthol (45 – 50 %), menthon (20 %), limonene, camphene, -myrcene,
etc. (Bauer et al., 2001; Lawrence, 1979, 1981, 2003). The first trials to introduce peppermint
as a crop culture in Bulgaria are recorded in 1905, but with no success. The industrial
cultivation of peppermint begins after 1923, and as soon as 1938. Bulgaria has already
occupied the third place in the world in terms of peppermint oil production. Bulgarian
peppermint oil has gained world popularity under the name Bulgaro-Mitcham oil (after the
Mitcham region, England). The chemical composition of Bulgarian peppermint oil has been a
subject of investigation for many Bulgarian researchers (Ivanoff et al., 1953, Ognyanov et al.,
1961, 1962a, 1962b, 1976; Stoyanova et al., 2000; Vlachov et al., 1964, 1965a, 1965b, 1967a,
1967b, 1967c). Bulgarian mint oil has well-attested antimicrobial (Denkova et al., 2001;
Kumanova, 1988; Pejchev et al., 1973) and insecticide activities (Mateeva and Karov, 1983)
and it is applied in cosmetics – in products for oral hygiene (Atanasov et al., 2002) and in
food products (Hadjikinov et al., 2000).
Currently in many countries essential oil bearing plants are processed by extraction with
liquefied gases (СО2, air, freon and other). 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 apolar
solvents (Nenov, 2006; Nenov et al., 2008). 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 4-7 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

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
72
them, one laboratory and one industrial, working with liquefied СО2 are situated in
Dimitrovgrad and one laboratory installation, working with liquefied tetrafluoroethane is
situated in University of Food Technologies in Plovdiv. These installations are used for
processing of different essential oil bearing plants in Bulgaria (Damianova et al. 2004;
Stoyanova et al., 2005, 2006; Atanasova, 2007; Atanasova et al., 2010; Nenov et al. 2011).
The aim of present study is producing of new plant extracts from peppermint, growing
in Bulgaria, by using liquefied tetrafluoroethane in laboratory installation and determination
of their chemical composition and characteristics for possible application in natural cosmetics,
pharmaceutical and food products
Material and methods
Obtaining of extract
The air-plant was 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-tetrafluoroethane) laboratory-extractor
under following conditions: temperature 20 – 25 ОC, pressure 5,7 - 6,5 bar, time 50 – 70 min.
The physical-chemical properties of extract 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 m x 0,25 mm; film
thickness 0,25 m); temperature: 70 О - 10 min, 70 - 240 ОC - 5 ОC/min, 240 ОC – 5 min; 240
- 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.

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
73
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 “Biochemistry and microbiology”, “Paisii
Hilendarski” University of Plovdiv, Bulgaria. Minimal Inhibitory Concentration (MIC, %
w/v) and Minimal Bactericidal Concentration (MBC, %, w/v) of extract was determined by
reference methods for broth dilution antimicrobial susceptibility tests for bacteria that grow
aerobically (CLSI M07-A8, 2009) and reference method for broth dilution antifungal
susceptibility testing of yeasts (CLSI M27-A3, 2008). 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 by
Mensor et al., 2001. 1.0 ml from 0.3 mM alcohol solution of DDPH was added to 2.5 ml from
the sample with different concentration of mint 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 used solvent 1,1,1,2 tetrafluoroethane is non-polar with dynamic viscosity and
surface tension at 20 оС are small, 198 Рa.s and 8 mN/m, respectively, which allows easier

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
74
penetration into plant cells and benefits extraction of cells associated compounds. Its pressure
at 20 оС is 0.57 MPa, which allows the extraction process to be carried out at acceptable
pressures from 0.2 to 0.7 MPa. Its specific heat of vaporization at the applied thermal
regiment is low (about 200 kJ/kg), which determines small energy consumption for extraction
process (Nenov, 2006). The influence of tetrafluoroethane on the Green House Effect, with
coefficient HGWP = 0.285, and its higher price are its major disadvantages.
The produced extract is liquid with characteristic odour. The yield and some of
physical and chemical characteristics of the extract is shown in Table 1. As seen the results
for the yield 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.
Table 1. Yield and physical-chemical properties of extracts.
Properties
Mint extract
Yield, kg raw/kg extract
280 - 320
Color
Dark green
Dry substance, % (105 OC)
26.3
Refractive index (n
20
D
)
1.4766
Specific gravity (d
20
20
)
0.9361
Acid number (mg KOH/g extract)
17.1
Chemical compositions of the extract are listed in Table 2. As seen the major constituents
(up 3 %) of the extract is as follows: menthone (30.54 %), menthol (21.12 %), menthyl acetate
(9.92 %), iso-menthone (4.43 %), cis-sabinene hydrate (3.82 %), 1,8-cineole (3.41 %) and
pulegone (3.04 %). According to qualitative and quantitative content of the major constituents the
produced extract is equal to the essential oil, published by above listed authors.

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
75
Table 2. Chemical composition of peppermint extract.
Compounds
RI
%
Compounds
RI
%
-pinene
939
0.63
menthol
1173
21.12
sabinene
976
0.30
-terpineol
1198
0.34
-pinene
980
0.51
pulegone
1237
3.04
myrcene
991
0.50
carvone
1242
0.83
p-cymene
1026
0.27
geraniol
1255
0.39
-phellandrene
1031
0.49
thymol
1290
0.57
1,8-cineole
1033
3.41
menthyl acetate
1294
9.92
limonene
1036
0.90
carvacrol
1300
0.57
cis-sabinene hydrate
1068
3.82
-caryophyllene
1418
2.00
linalool
1098
2.04
-farnesene
1458
0.50
piperitone
1152
1.19
germacrene D
1480
2.37
menthone
1154
30.54
caryophyllene oxide
1581
0.26
iso-menthone
1159
4.43
phytol
1949
0.47
menthofurane
1164
0.85
squalene
2790
0.58
neo-menthol
1165
2.49
Distribution of major groups of aroma substances in the extract is shown in Figure 1.
Oxygenated monoterpenes are the dominant group in the extract 88.55 %, followed by
sesquiterpenes 5.11 % and monoterpenes 3.49 %.

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
76
7
6
5
4
3
2
1
1234567
1- monoterpene hydrocarbons, 2 – oxygen monoterpenes, 3 – sesquiterpene hydrocarbons,
4 - oxygen sesquiterpenes, 5 – diterpenes, 6 – phenyl propanoids, 7 – others.
Figure 1. Group of components in the extract, %
The results of antimicrobial testing are presented in Table 3. As seen all of 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. aeruginosa. Antimicrobial activity of the studied
extract was equal to the antimicrobial activity of essential oil from peppermint from Bulgaria
and other countries (Bourel et al., 1995a. 1995b; Jirovetz et al., 2009).
Тable 3: Аntimicrobial activity of the extracts.
Test micro-
organisms
Origin
peppermint extract
MIC, %
MBC, %
Staphylococcus epidermidis
Clinical isolate
0.2
0.2
Staphylococcus aureus
ATCC 6538
0.2
0.2
Escherichia coli
Food isolate
0.4
0.4
Escherichia coli
ATCC 8739
0.4
0.4

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
77
Salmonella abony
Clinical isolate
0.4
0.8
Salmonella abony
ATCC 6017
0.4
0.8
Pseudomonas aeruginosa
Food isolate
0.8
1.0
Pseudomonas aeruginosa
ATCC 9627
0.8
1.0
Candida albicans
Clinical isolate
0.4
0.4
Candida albicans
ATCC 10231
0.4
0.4
The results of antioxidant testing of the extract is as follows: 91.8 % inhibition of
DPPH radical was reached by extract at concentration 1,2 mg/ml and the IC50 values was
0,365 mg/ml (correlation coefficient R2=0,995).
In comparison with other extracts produced by low temperature extraction with
1,1,1,2-tetrafluorethane, the extract demonstrated higher antioxidant activity than the extract
from: anise fruits -IC50 е 8.32 mg/ml (Atanasova, 2007) and coriander fruits - IC50 е 17.74
mg/ml (Atanasova et al., 2010), and almost equal activity in comparison with the extract from
cinnamon barks - IC50 0.38 mg/ml (Nenov et al., 2011).
The results obtained are comparable with the results published by other authors for the
same essential oils and extracts from another geographic origin (Lee and Shibamoto, 2002;
Marinova and Yanishlieva, 1997; Schmidt et al, 2009; Yanishlieva and Marinova, 1995). 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 in
cosmetics and food industry, the produced extracts from Bulgarian essential oil bearing plants
belonging to family Lamiaceae, possessed considerably lower antioxidant activity.

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
78
Conclusion
For the first time in Bulgaria new extract from mint (Mentha piperita Huds. (L.), was
produced by extraction with liquefied tetrafluoroethane. The optimal conditions for extraction
procedure were determined. The produced extract was almost equal with essential oil from the
same essential oil bearing plant, according to its chemical composition, antimicrobial and
antioxidant activities. The extract is prospective for possible application in different
cosmetics, pharmaceutical and food products, but of course additional investigations are a
must.
References
Аtanasov, B., Botushanov, P., Кirova, Е., Аtanasova, Т., Borissova, R., Vladimirov, S., Resources for the
hygiene, prevention and treatment of mouth cavity, Plovdiv, 2002.
Atanasova, T. (2007). Technological investigation for obtaining of the essential and vegetable oil from fruits of
anise (Pimpinella anisum L.). PhD. University of Food Technologies, Plovdiv.
Atanasova, T., Girova, T., Gochev, V., Stoilova, I., Nenov, N., Stoyanova M., Stoyanova, A., Low temperature
extraction of essential oil bearing plants by liquificate gases. 5. Fruits from coriander (Coriandrum sativum L.).
Scientific Works of University of Food Technologies, 2010, 57(1), 363–368.
Bauer, K., Garbe, D., Surburg, H., Common fragrance and flavour materials. Preparation, properties and uses.
IV Compl., Revised Edition Wiley-VCH Verlag GmbH, Weinheim, Germany, 2001.
Bоurrel, C., Vilarem, G., Michel, G., Gaset, A., Etude des proprietes bacteriostatiques et fongistatique en milieu
solide de 24 huiles essentielles prealabeement analysees. Rivista Italiana EPPOS, 1995, 16, 3-12.
Bоurrel, C., Dargent, R., Vilarem, G., Gaset, A., Analyse chimique et properietes fongistatiques de quelques
huiles essentielles en milieu liquide. Effets sur la morphodenes hyphale. Rivista Italiana EPPOS, 1995, 17, 31-
42.
Damianova, S., Stoyanova, A., Konakchiev, A., Djurdjev, I., Supercritical carbon dioxide extracts of spices. 2.
Fennel (Foeniculum vulgare Mill. var. dulce Mill.). Journal of Essential Oil Bearing Plants, 2004, 7(3), 247-
249.
Denkova, Z., Murgov, I., Vasileva, G., Stoyanova, A, Atanasova, T., Djurdjev, I., Inhibition activity of aromatic
products from peppermint. Sci. Works of the Agrarian Academy, 2001, 46(4), 301–305.
Georgiev, E, Genov, N., GC investigation of macro- compounds in the basil oil, Sci. Works of the University of
Food Technologies, 1973, 20(3), 209–17.
Georgiev, E., Stoyanova, A., A guide for the specialist in aromatic industry, Plovdiv, 2006.

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
79
Hadjikinov, D., Hadjikinova, M., Stoyanova, A., Dimitrova, M., Influence of aromatic components upon
antibacterial activity of chewing candies “The Alps”, Sci. Works of the University of Food Technologies, 2000,
44, 172–176.
Ivanoff, D., Ivanov, C., Nicolov, N., Ognianoff, I.m Sur l’acceleration du repos de l’huile brute de menthe-herb
en vue de la stabilization de l’huile rectifiee., Comt. Rend. Chim. Acad., 1953, 6(1), 25-33.
Jirovetz, L, Buchbauer, G., Bail, S., Denkova, Z., Slavchev, A., Stoyanova, A., Schmidt, E., Geissler, M.,
Antimicrobial activity of essential mint and peppermint oils and some of its main compounds. Journal of
Essential Oil Research,2009, 21, 363-369.
Kumanova, R., Dimitrov, D., Todorov, R., Antimicrobial activity of some Bulgarian natural and aromatic
products, In: VІІ Microbiology Congress.Varna. 27 – 29 October, 1989.
Lawrence, B., Essential oils 1976–1978, Wheaton. Allured Publishing Corporation. Carol Stream, USA, 1979.
Lawrence, B., Essential Oils 1979-1980, Wheaton, Allured Publ. Co., 1981.
Lawrence, B., Essential Oils 1995–2000, Allured Publishing Corporation, Carol Stream, USA, 2003.
Lee, K., Shibamoto, T., Determination of antioxidant potential of volatile extracts isolated from various herbs
and spices, Journal of Agricultural and Food Chemistry, 2002, 50, 4947-4952.
Marinova, E., Yanishlieva, N., Antioxidation activity of extracts from selected species of the family Lamiaceae
in sunflower oil, Food Chemistry, 1997, 58(3), 245–253.
Mateeva, A., Karov, S., Investigation on the insecticide action of some essential oils, Sci. Works of the Agrarian
Academy, 1983, 28(3), 129–139.
Mensor, L., Menezes, F., Leitao, G., Reis, A., Santos, T., Coube, C., Leitao, S., Screening of Brazilian plant
extracts for antioxidant activity by the use of DPPH free radical method. Phytotherapy Research, 2001, 15, 127-
130.
National Committee Clinical Laboratory Standards, Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically, Approved Standard, NCCLS Publication M7-A2, Villanova. PA. USA, 1990.
Nenov, N., Low temperature extraction of essential oil bearing plants by liquificate gases. 1. Laboratory
installation, Sci. Works of the University of Food Technologies, 2006, 53(2), 195–200.
Nenov, N, Dichev, S., Аtanasova, T. (2008). Efficient method for obtaining high quality natural additives for
foods, cosmetic and medicinal preparation by liquefied gas extraction, Food and Flavour Industry, 2008, 2, 40-
42.
Nenov, N., Gochev, V., Girova, T., Stoilova, I., Atanasova, T., Stanchev, V., Stoyanova, A., Low temperature
extraction of essential oil bearing plants by liquified gases. 6. Barks from cinnamon (Cinnamomum zeylanicum
Nees), Journal of Essential Oil Bearing Plants, 2011, 14(1), 67–75.
Ognyanov, I., Vlachov, R., Terpene hydrocarbons in Bulgarian peppermint oil, Comt. Rend. Chim. Acad., 1961,
14(5), 459–462.
Ognyanov, I., Vlachov, R., Non-terpene compounds in the terpene fraction of Bulgarian peppermint oil, Comt.
Rend. Chim. Acad., 1962, 15(4), 395–397.

Proceedings of the International Conference
“AGRI-FOOD SCIENCES, PROCESSES AND TECHNOLOGIES” AGRI-FOOD 2014,
May 14-15, 2014, Sibiu, Romania
80
Ognyanov, I., Vlachov, R. (1962b). On the composition of the crude peppermint oils of Bulgarian origin.
Bulletin for the Development in the Essential Oil Industry, 1962, (3), 23-26.
Ognyanov, I., Bulgarian essential oils and natural aromatic products, Medical- Biological Information, 1976, (2),
3–5.
Pejchev, P., Apostolov, G., Nikiforov, N., Topalov, V., Antimycotic and antidermatophitic action of the essential
oils. Bulletin for the Development in the Essential Oil Industry, 1973, (1), 7–15.
Russian Pharmacopoeia, Moscow, 1990.
Schmidt, E, Bail, S., Buchbauer, G., Stoilova, I., Atanasova, T., Stoyanova, A., Jirovetz, L., Chemical
composition, olfactory evaluation and antioxidant effects of essential oil from Mentha x piperita. Natural
Product Communications, 2009, 4(8). 1107–1112.
Stoyanova, A., Paraskevova, P., Anastassov, C., A comparative investigation on the essential oil composition of
two Bulgarian cultivars of Mentha piperita L., Journal of Essential Oil Research, 2000, 12(4), 438-440.
Stoyanova, A., Djurdjev, I., Georgiev E., Super carbon dioxide extracts of spices. 3. Hop (Humulus lupulus L.),
Herba Polonica, 2005, 51(½), 24–8.
Stoyanova, A., Damianova, S., Konakchiev, A., Djurdjev, I., Super carbon dioxide extracts of spices. Part. 1.
Coriandrum (Coriandrum sativum L.). Herba Polonica, 2006, 49(3/4), 235-239.
Stoyanova, A., Denkova, Z., Nenov, N., Slavchev, A., Jirovetz, L., Buchbauer, G., Ho, L., Schmidt, E., Geissler,
M., C2H2F4-Oleoresins of black pepper (Piper nigrum L.) and ginger (Zingiber officinale (L.) Rosc.) from
Vietnam: Antimicrobial testings, gas chromatographic analysis and olfactory evaluation, Electronic Journal of
Enviromental, Agricultural and Food Chemistry, 2006, 5(5), 1615–1623.
Vlachov, R., Ognyanov, I., Oxygene terpene compounds in Bulgarian peppermint oil. Comt. Rend. Chim. Acad.,
1964, 17(2), 145–147.
Vlachov, R. and Ognyanov, I., Composti oessgenati terpenici del’essenza di Mentha piperita di origine Bulgara.
Riv. Ital. Ess. (Milano)., 1965, 47(8), 436–437.
Vlachov, R., Ognyanov, I., Holub, M., Sur certeins hydrocarbures sesquiterpeniques de l’essence Bulgare de
Mentha piperita L., In: Compte rendu du 3-e Congres international des huiles essentielles. Verona. 26 – 28 May,
1965, 243–245.
Vlachov, R., Holub, M., Ognianov, I. and Herout, V., Sesquiterpenic hydrocarbons from the essential oil of
Mentha piperita of Bulgarian origin, Collection of Czechoslov. Chem. Commun (Praha), 1967, 32(2), 808–821.
Vlachov, R., Ognyanov, I., Neue Komponenten des bulgarischen pfefferminzoles. Riechstoffe-Aromen-
Korperflegemittel (Hannover), 1967, 17(8), 315–318.
Vlachov, R., Holub, M. and Herout, V. (1967c). The structure of two hydrocarbons of cadoline type isolated
from Mentha piperita oil of Bulgarian origin. Collection of Czechoslov. Chem. Commun (Praha). 32(2): 822–29.
Yanishlieva, N. and Marinova, E., Antioxidant activity of selected species of the family Lamiaceae grown in
Bulgaria, Nahrung., 1995, 39(5/6), 458–463.