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Thin-layer chromatography—fingerprint, antioxidant activity, and gas

chromatography—mass spectrometry profiling of several Origanum L.
species

Article  in  JPC - Journal of Planar Chromatography - Modern TLC · September 2017

DOI: 10.1556/1006.2017.30.5.7

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Thin-Layer Chromatography–Fingerprint, Antioxidant
Activity, and Gas Chromatography–Mass Spectrometry
Profiling of Several Origanum L. Species
Tomasz Baj, Elwira Sieniawska*, Agnieszka Ludwiczuk, Jaros∞aw Widelski, Anna Kie∞tyka-Dadasiewicz,
Krystyna Skalicka-Woźniak, and Kazimierz G∞owniak

Key Words:

Origanum
Thin-layer chromatography–fingerprint
DPPH bioautography
Chemotypes

Summary cymyl, acyclic (linalool/linalyl acetate), and other (mostly

Essential oils obtained from Origanum species are characterized
by high diversity in composition. In this work, thin-layer
chromatography (TLC)–fingerprinting of Origanum species was
performed for the first time and enabled the discrimination of
three chemotypes (carvacrol, caryophyllene oxide, and terpineol/
sabinyl). Gas chromatography–mass spectrometry and statistical
analyses confirmed the presence of the mentioned chemotypes
and deepened the characterization of the studied essential oils.
TLC–bioautography showed that thymol and carvacrol are the main
antioxidant compounds in Origanum sp. essential oils (EOs).
1 Introduction
Origanum species can be found in many herbal products used
for treating respiratory and stomach ailments due to its anti-
microbial and spasmolytic properties. These Lamiaceae plants
have also traditional culinary applications. Their flavoring
properties are attributed mainly to the essential oil contain-
ing carvacrol, thymol, γ-terpinene, p-cymene, linalool, terpi-
nen-4-ol, and sabinene hydrate as the major compounds [1].
The essential oil is usually obtained by hydrodistillation of
the aerial parts or separated flowers of Origanum. The previ-
ous research on Origanum species from different regions of
Europe indicated high diversity in the essential oil compo-
sition [2], and several chemotypes were described: sabinyl,
sesquiterpene-rich) [2]. Origanum essential oil and its main
constituents exhibited good antioxidant activities in a variety
of in vivo and in vitro models [3] including thin-layer chroma-
tography (TLC)–bioautography [4].
Thin-layer chromatography is generally considered as a repro-
ducible and authentic method for rapid qualitative analysis and
bioactivity testing. It is also very often the method of choice for
testing the identity and purity of drugs and for detecting adul-
terations [5]. Therefore, TLC is very suitable for fingerprint-
ing of species/varieties resulting in chemical profiles enabling
the discrimination of chemotypes. From the agrotechnological
point of view, knowing the chemotypes of the cultivated plants
is critical to increase technological and economical effective-
ness in the uses of essential oils and their production. Therefore,
in this work, we aimed to perform (1) TLC‒fingerprinting and
gas chromatography‒mass spectrometry (GC–MS) analysis
of essential oils obtained from selected Origanum species and
cultivars grown mainly in Poland; (2) antioxidant 2,2-diphenyl-
1-picrylhydrazyl (DPPH) bioautography to characterize the
antioxidant properties of the studied essential oils. TLC‒fin-
gerprinting of Origanum species was done for the first time.
2 Experimental
2.1 Sample Preparation

The studied plant samples were obtained from the Garden of

T. Baj, E. Sieniawska, A. Ludwiczuk, J. Widelski, and K. Skalicka-Woźniak,
Chair and Department of Pharmacognosy with Medicinal Plant Unit, Medical
University of Lublin, Chodzki 1, 20-093 Lublin, Poland; A. Kiełtyka-Dadasie-
wicz, Department of Plant Production Technology and Commodities, Univer-
sity of Life Sciences, 15 Akademicka, 20-954 Lublin, Poland and Garden of
Cosmetic Plants and Raw Materials, Research and Science Innovation Center,
Tarasowa 4/96, 20-819 Lublin, Poland; and K. Głowniak, Department of Cos-
metology, University of Information Technology and Management in Rzeszow,
Sucharskiego 2, 35-225 Rzeszow, Poland.
E-mail: esieniawska@pharmacognosy.org
Research and Science Innovation Center in Wola Zdybska near
Lublin (Poland) (51° 44′ 49″ N 21° 50′ 38″ E) with the excep-
tion of one sample which was bought from a regional market
in Arachova, Greece (Table 1). The aerial parts and flowers
were collected in June 2016, and authentication was done by
A. Kiełtyka-Dadasiewicz. Voucher specimens were deposited
in the Research and Science Innovation Center. The essential
oils (EOs) of the air-dried plant material were obtained by
hydrodistillation in a Deryng-type apparatus for 3 h. The oils
were stored in tightly sealed 1.5 mL amber vials at 4°C prior to
analyses.

386 Journal of Planar Chromatography 30 (2017) 5, 386–391

0933-4173 © Akadémiai Kiadó, Budapest
DOI: 10.1556/1006.2017.30.5.7
Journal of Planar Chromatography 30 (2017) 5
 TLC–Fingerprint, Antioxidant Activity, and GC–MS Profiling of Origanum L. Species

Table 1 for variables (compounds of essential oils) and cases (plant

The plant material used for EO distillation. samples) was obtained by PCA 1 vs. PCA 2 and PCA 1 vs. PCA
3 correlation.
No. Code Species/varieties Origin Part used

Origanum vulgare spp.

1 A Poland Aerial parts
hirtum (Link) Ietsw. 3 Results and Discussion
2 B Origanum vulgare L. Poland Aerial parts
The fingerprinting of essential oils (EOs) obtained from seven
3 C Origanum majorana L. Poland Aerial parts Origanum specimens (Table 1) was done by thin-layer chroma-
tography. Visualization of the volatile components present in
Origanum vulgare L
4 D
“Hot and Spicy”
Poland Aerial parts all EOs was performed by derivatization with Godin reagent.
The bioautography with DPPH was also done. The results are
Origanum vulgare L presented in Figures 1 and 2. The TLC–fingerprint analysis
5 E Poland Aerial parts
“Aureum” revealed that EO hydrodistilled from the aerial parts of O. vul-
Origanum vulgare L. gare L. (B) is the most abundant in chemical constituents. The
6 F Greece Aerial parts main compounds present in this EO were also visible in the
“Greek Kaliteri”
EO obtained from the flowers (G); however, their abundance
7 G Origanum vulgare L. Poland Flowers was different. Essential oils from the Polish O. vulgare L. spp.
hirtum (A) and O. vulgare L. “Hot and Spicy” (D) as well as
EO from the Greek O. vulgare L. “Greek Kaliteri” (F) showed
2.2 TLC–Fingerprinting and DPPH Bioautography similar TLC profiles with dominating pink spot, which was
identified as carvacrol. The same compound was also present in
Chromatographic separation was carried out on silica gel 60
EO obtained from the cultivar “Aureum” (E) but was invisible
F254 chromatographic plates of 20 cm × 10 cm with 3-mm thick-
or barely visible in O. majorana L. (C) and O. vulgare L. (B and
ness. The essential oils were dissolved in n-hexane and spotted
G), respectively. The TLC–fingerprint of O. majorana L. (C)
on the chromatographic plate, then developed using a mixture
EO was different to the most of the O. vulgare specimens, but
of toluene and ethyl acetate (95:5, v/v). Half of the chromato-
similar, especially in regards to the compounds with Rf values
graphic plate was derivatized with Godin reagent (A: 1% van-
below 0.4, to O. vulgare L. “Aureum”.
illin solution in ethanol and B: 5% H2SO4 solution in ethanol)
and then heated in 105°C, and another half was sprayed with
DPPH (0.2%). After derivatization of the TLC plate, a picture
was made using a videoscanner (Reprostar 3, CAMAG, Mut-
tenz, Switzerland). The *.jpg file was processed by means of
Photoshop graphical software (Adobe, USA) plug-in filter.

2.3 Gas Chromatography–Mass Spectrometry Analysis

GC–MS separation was carried out on the Shimadzu

GC-2010 Plus coupled to a QP2010 Ultra mass spectrometer
using a Phenomenex capillary column ZB-5 MS (30 m, with
inside diameter of 0.25 mm, and coating thickness of 0.25
μm). The initial column temperature was set at 50°C, held
for 3 min, and then heated to 250°C at a rate of 8°C min−1; Figure 1
this temperature was held for 2 min. The temperatures 250, TLC–fingerprint (left side: A–G) and DPPH bioautography (right
250, and 220°C were kept for the injector, interface, and ion side: A′–G′) of the studied essential oils.
source, respectively. The split ratio after injection of 1 μL
was 1:20, and helium was the carrier gas with a flow rate of
1 mL min−1. Ionization was performed by electron impact at
70 eV. Mass spectral data were acquired in the scan mode in
the m/z range of 40–500 with the scan rate 0.20 s per scan.
The identification of the separated compounds was based on
the comparison of their mass spectra with those of Mass-
Finder and NIST mass spectra libraries as well as in rela-
tion to a homologous series of n-alkanes (C8–C24) under the
same operating conditions.

2.4 Statistical Analysis

Multivariate statistical analysis was performed using Statistica

software version 12 (StatSoft, Palo Alto, CA, USA). Cluster Figure 2
analysis (CA) was done using the Ward method and the Euclid- Visualization of TLC plate after applying the plug-in filter in Photo-
ean distance measure. The principal component analysis (PCA) shop.

Journal of Planar Chromatography 30 (2017) 5 387

TLC–Fingerprint, Antioxidant Activity, and GC–MS Profiling of Origanum L. Species

Table 2
The chemical composition of the studied essential oils.

No. RI Compound A B C D E F G

1 927 α-Thujene 0.2 0.4 0.2 0.1 0.2 0.1

2 934 α-Pinene 0.3 0.4 0.1 0.2 0.3 0.1

3 952 Camphene 0.1

4 974 Sabinene 11.3 2.3 0.4 0.2 1.7

5 979 β-Pinene 0.6 0.1 0.1 0.2

6 982 1-Octen-3-ol 0.2 1.9 0.2 1.0 0.2 0.5

7 985 3-Octanone 0.5 0.1 0.2

8 989 β-Myrcene 1.0 0.5 0.7 0.6 0.5 0.1

9 999 3-Octanol 0.6 0.1

10 1008 α-Phellandrene 0.1 0.1 0.1

11 1019 α-Terpinene 1.0 0.6 4.6 0.5 0.3 1.3 1.2

12 1027 p-Cymene 2.4 1.3 0.8 1.8 5.0 1.2

13 1032 Limonene 0.1 0.4 0.8 0.3 0.1

14 1034 β-Phellandrene 0.1 0.9 2.5

15 1034 1.8-Cineole 0.4 0.1

16 1037 trans-β-Ocimene 1.9 0.1 0.3 0.1

17 1047 cis-β-Ocimene 0.3 0.9 0.1 0.1

18 1060 γ-Terpinene 6.5 1.4 9.5 2.4 1.2 3.3 2.6

19 1074 trans-Sabinene hydrate 0.1 0.6 6.7 0.3 1.4 0.4 8.2

20 1087 α-Terpinolene 0.2 2.0 0.3 0.7

21 1102 Linalool 0.2 0.7 3.6 1.1 0.5 0.2

22 1105 cis-Sabinene hydrate 0.2 0.4 16.9 0.2 18.7 1.6 0.9

23 1130 trans-p-2-Menthen-1-ol 0.2 1.5 0.4 0.3 0.5

24 1153 Camphor 0.2

25 1180 Borneol 0.3 0.2 0.2 0.3 0.8

26 1186 Terpinen-4-ol 0.4 1.8 26.9 0.4 6.1 4.3 6.3

27 1194 p-Cymen-8-ol 0.1 0.1

28 1201 α-Terpineol 0.5 4.7 2.5 1.0 0.7

29 1210 cis-Piperitol 0.1

30 1215 trans-Piperitol 0.3 0.1

31 1232 Tymol methylether 0.1 0.1

32 1241 Carvacrol methylether 0.2 0.1 0.3 0.3 0.1 0.1

33 1249 Linalyl acetate 0.7 2.5 0.3 0.5 0.1

34 1252 Neryl formate 0.5

35 1253 Geraniol 0.7 0.1

36 1287 Bornyl acetate 0.2

37 1299 Terpinene-4-acetate 0.1 0.4

38 1300 Thymol 0.2 12.2 6.2 0.2

39 1309 Carvacrol 82.8 6.9 0.6 89.3 7.0 66.6 0.6

388 Journal of Planar Chromatography 30 (2017) 5

 TLC–Fingerprint, Antioxidant Activity, and GC–MS Profiling of Origanum L. Species

Table 2 (continued)

No. RI Compound A B C D E F G

40 1337 Bicycloelemene 0.2

41 1349 α-Terpinyl acetate 0.1

42 1357 Neryl acetate 0.2

43 1367 Carvacryl acetate 0.1

44 1376 Geranyl acetate 0.3 0.1

45 1382 α-Copaene 0.2 0.3 0.2

46 1391 β-Bourbonene 2.9 0.5 1.1

47 1395 β-Elemene 0.5 0.4 0.2

48 1429 (E)-β-Cayophyllene 0.9 5.5 3.8 1.1 4.8 1.1 2.2

49 1438 β-Copaene 0.5 0.2

50 1448 Aromadendrene 0.2 0.1

51 1465 α-Humulene 0.1 0.9 0.2 0.1 1.0 0.1 0.6

52 1470 allo-Aromadendrene 0.7 0.8 1.5

53 1490 Germacrene D 0.1 0.1 16.5 1.0

54 1498 α-Guaiene 0.1 0.2

55 1504 Bicyclogermacrene 3.8

56 1506 (E,E)-β-Farnesne 0.8 2.6 1.5 7.7 0.6

57 1511 β-Bisabolene 0.6 0.6 0.4 0.7 0.5

58 1525 δ-Cadinene 0.1 1.1 0.2 2.7 0.4

59 1539 trans-Calamenene 0.6

60 1543 α-Bisabolene 0.1 1.3 0.6

61 1562 Salviadienol 1.4 0.8

62 1590 Spathulenol 5.5 0.2 0.4 11.5

63 1595 Caryophyllene oxide 15.5 0.1 0.4 0.6 21.4

64 1608 Viridiflorol 0.4 0.8

65 1627 Humulene epoxide II 2.7 3.2

66 1651 Cedr-8-en-15-ol 0.6

67 1655 γ-Cadinol 0.4 1.4

68 1657 α-Cadinol 0.5 2.2 0.3

69 1683 Eudesma-4(15),7-dien-1β-ol 0.4 1.2

70 1962 n-Hexadecanoic acid 0.1 0.7 0.8 0.8

A, Origanum vulgare spp. hirtum (Link) letsw.; B, Origanum vulgare L.; C, Origanum majorana L.; D, Origanum vulgare L. “Hot and Spicy”;
E, Origanum vulgare L. “Aureum”; F, Origanum vulgare L. “Greek Kaliteri”; G, Origanum vulgare L. RI, retention indices

Based on the TLC–fingerprints, the analyzed EOs can be
divided into three chemotypes. The first one is composed of
samples A, D, and F, the second one of samples B and G, while
the third one of samples C and E. The use of a plug-in filter
gave a similar effect to the Sabattier effect, which facilitated the
visualization and interpretation of the TLC results (Figure 2).
To confirm the data obtained from TLC analysis, GC–MS fin-
gerprinting was performed. The data are presented in Table 2.
The GC–MS data support the observations of the TLC–fin-
gerprint analysis. O. vulgare L. spp. hirtum (A), O. vulgare L.
“Hot and Spicy” (D), and Greek O. vulgare L. “Greek Kaliteri”
contained carvacrol as dominating compound (66–89%). Addi-
tionally, the Greek sample was characterized by the presence
of noticeable amounts of p-cymene, terpinen-4-ol, and thymol.
EOs from O. vulgare L. obtained from the aerial (B) parts as
well as from the flowers (G) were abundant in caryophyllene

Journal of Planar Chromatography 30 (2017) 5 389

TLC–Fingerprint, Antioxidant Activity, and GC–MS Profiling of Origanum L. Species
oxide (15.5% and 21.4%, respectively). A high amount of ter-
pinen-4-ol (27%) and cis-sabinene hydrate (17%) was present
in O. majorana L. (C) EO. The essential oil obtained from
O. vulgare L. “Aureum” (E) contained mainly cis-sabinene
hydrate, germacrene D, and thymol (18.7%, 16.5%, and 12.2%,
respectively).
The antioxidant activity of the studied EOs may be attributed
mainly to the compounds visible as pink spots. The highest
decolorization of violet background (Figures 1 and 2) was
obtained for samples rich in thymol and carvacrol, suggesting
that the antioxidant activity of the samples may be exerted by
these monoterpene alcohols. This conclusion is supported by
previously published data on the antioxidant activity of thymol
and carvacrol [6–8].
TLC–fingerprint (Figures 1 and 2) indicated the presence of
three different chemotypes among oregano samples. The most
noticeable is the TLC similarity of samples A, D, and F (O.
vulgare L. spp. hirtum, O. vulgare L “Hot and Spicy,” and
Greek O. vulgare L. “Greek Kaliteri”), which in GC–MS were
described as belonging to carvacrol chemotype. The subspecies
O. vulgare L. spp. hirtum was characterized by the presence
of 83% of carvacrol, and this is consistent with the literature
showing that the EO of this plant may contain 67–85% of car-
vacrol [9]. Carvacrol being a dominating compound in O. vul-
gare L “Hot and Spicy” is understandable since this cultivar
is propagated from O. vulgare L. spp. hirtum. From a chem-
ical profile (TLC–GC/MS fingerprint), also a Greek oregano
is a strain of O. vulgare L. spp. hirtum. The true O. vulgare L.
EO represents sesquiterpenes chemotype [2], and our results
obtained for samples of flowers and herb of O. vulgare L. of
Polish origin showed caryophyllene oxide as being the pre-
dominant compound. This sesquiterpene was accompanied
mainly by (E)-β-caryophyllene and spathulenol, confirming
the indicated chemotype. The other interesting observation of
the TLC–fingerprint was the profile of the cultivar O. vulgare
Figure 3
Principal component analysis (PCA) – factor scores plot. A-D-F, carvacrol chemotype; B-G, caryophyllene oxide chemotype, and C-E, terpin-
eol/sabinyl chemotype. A, PC1 vs. PC2; B, PC1 vs. PC3.
390
L. “Aureum” (E). This sample revealed the presence of com-
pounds characteristic for marjoram EO as well as for O. vulgare
L. spp. hirtum EO. The GC–MS analysis confirmed that the
studied marjoram EO (C) belongs to the mixed terpinen-4-ol/
cis-sabinene hydrate chemotype. However, besides terpin-
en-4-ol, the other terpinene alcohols were also present in sig-
nificant amounts accounting for 45% in this EO. Hence, this
chemotype may also be described more generally as terpineol/
sabinyl. This chemotype was previously described as charac-
teristic for species sold in Polish herb markets [10] and also for
plants cultivated in other European and African countries [10].
The other mixed chemotype described for marjoram cultivated
in Cyprus was sabinyl/α-terpineol chemotype, and, in this case,
both dominating constituents were present almost in the same
amount (40%) [11]. The observations described above were fur-
ther supported by statistical analysis.
PCA allowed for the determination of different chemotypes of
the examined oils. As a criterion for selecting the reduction of
the number of the main components, the cumulative percentage
of the explained variance of the analyzed variables was chosen.
On this basis, it was determined that the first three main com-
ponents represented a total of 89.12% variance of the primary
variables. The first two components described a total of 69.86%
variance of the primary variables. PCA1 vs. PCA2 analysis has
shown that the main discriminating components of the essential
oils were carvacrol and caryophyllene oxide. It allowed for the
division of the examined oils into two chemotypes: carvacrol
(A-D-F) and caryophyllene oxide (B-C-E-G). When analyzing
the vectors of primary variables, they differed in their length on
the coordinate system of the first components, suggesting that
there may be an additional discriminative relationship. There-
fore, further study was conducted on PCA1 vs. PCA3 analysis.
PCA 1 vs. PCA 3, factor scores plot, enabled the identification
of three groups of samples confirming the presence of differ-
ent chemotypes: carvacrol chemotype (A-D-F), caryophyllene
Journal of Planar Chromatography 30 (2017) 5
 TLC–Fingerprint, Antioxidant Activity, and GC–MS Profiling of Origanum L. Species
Figure 4
Principal component analysis (PCA) – factor loadings plot. 22 (cis-sabinene hydrate), 26 (terpinen-4-ol), 39 (carvacrol), and 63 (caryophyllene
oxide). A, PC1 vs. PC2; B, PC1 vs. PC3.
oxide chemotype (B-G), and terpineol/sabinyl chemotype
(C-E) (Figure 3). PCA, factor loadings plot, showed four com-
pounds as the main chemotype discriminating factors: cis-sa-
binene hydrate (22), terpinen-4-ol (26), carvacrol (39), and car-
yophyllene oxide (63) (Figure 4). The results of this analysis
suggest some similarity between marjoram EO and O. vulgare
L. “Aureum” EO. Although the dominating compounds in
these EOs were different (terpinen-4-ol > cis-sabinene hydrate
> γ-terpienene and cis-sabinene hydrate > germacrene D > thy-
mol, respectively), TLC–fingerprint confirmed the partial sim-
ilarity in the chemical profiles and explained statistic resem-
blance. The close chemical relationship in a group A-D-F and
the separateness of this group from other samples can be seen
on dendrogram (Figure 5). Group A-D-F is a carvacrol chemo­
type originating from Origanum vulgare L. spp. hirtum. The
other EOs are more differentiated; however, the dendrogram
confirms again the presence of caryophyllene oxide (B-G) and
terpineol/sabinyl (C-E) chemotypes.
Figure 5
Hierarchical dendrogram representing the chemical composition
similarity relationship among the analyzed Origanum specimens.
For abbreviations, see Table 1.
Journal of Planar Chromatography 30 (2017) 5
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4 Conclusion
TLC–fingerprinting of Origanum species was performed for
the first time and enabled the discrimination of three chemo-
types (carvacrol, caryophyllene oxide, and terpineol/sabinyl).
GC–MS and statistical analyses confirmed the presence of
the mentioned chemotypes and deepened the characterization
of the studied essential oils. TLC–bioautography showed that
thymol and carvacrol are the main antioxidant compounds in
Origanum sp. EOs.
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Accepted: June 28, 2017
391