Scientific Report Report_2016.pdfInvestigation of glandular hairs from species of the genus Thymus...
Transcript of Scientific Report Report_2016.pdfInvestigation of glandular hairs from species of the genus Thymus...
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Scientific Report
regarding the project implementation «Glandular hairs of some Thymus species: micromorphological,
biochemical and microbiological features » (PN-II-RU-PD-2012-3-0307) during the whole period of
implementation
(For online version of the report,
studies that have been or are being published were metion only, to protect information)
Abstract: In this project inter-disciplinary researches on several spontaneous species of Thymus are carried
out. The study is focused on micromorphology and ultrastructure of the glandular hairs, biochemistry and
microbiology of the volatile oils depending on environmental factors (altitudinal and seasonal gradients) and
development stage of the plant. The researches are regarding the following: 1. The structure, ultrastructure,
the distribution and frequency of glandular hairs on vegetative plant organs; 2. The chemical composition
and the biological activity (antibacterian and antifungical) of the volatile oils. The investigations are carried
out using actual methods and techniques in plant biology (fotonic and electronic microscopy; CG-MS gas
chromatography; microplates assay, Kirby-Bauer diffusimetric method). By correlation of all the results, the
finallity of the study will provide significant original data regarding the aromatic value and the therapeutic
role of the analyzed species, which can be served as promotors for other complex investigations with
technological transfer.
Objectives:
1. Investigation of glandular hairs from species of the genus Thymus from the Romanian flora
1.1. Collecting the biological material from different areas of the country, according to: altitude and
vegetative season
1.2. Identifying and characterizing the morphological types of glandular
1.3. Revealing the distribution and frequency of the glandular hairs on the surface of the plant organs
using photonic microscopy and statistically processing the obtained data.
2. Study of the volatile oils obtained from species of the genus Thymus - biochemical researches
2.1. Collection of biological material from different areas of our country depends on altitude,
vegetative season and plant ontogenetic stage
2.2. Extraction of volatile oils by hydrodistilation
2.3. Analysis of the chemical composition of volatile oil
3. Study of the volatile oils obtained from species of the genus Thymus - microbiological researches
3.1. Documentation using existing literature about the importance of testing essential oils on
microorganisms studied (Staphyloccocus aureus, Escherichia coli, Candida albicans)
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3.2. Testing the antibacterial activity of essential oils on Staphylococcus aureus
3.3. Testing the antibacterial activity of essential oils on Escherichia coli
3.4. Testing the antifungal activity of essential oils on Candida albicans
4. Specializing training - information and documentation on the domain of the project
4.1. Bibliographical information in various universities centres
4.2. Specialized training in biological research institutes and universities in the country (Institute for
Biological Research Stejarul Piatra Neamt, Research Centre for the Study of Quality of Horticultural
Products, University of Agricultural Sciences and Veterinary Medicine)
5. Dissemination of research results
5.1. Participation in a number of national and international scientific manifestations
5.2. Publication of scientific papers in national and international journals recognized CNCS
indexed/top rated ISI.
5.3. Making a WEB site to provide data obtained aiming to establish international collaboration in the
field.
As part of the Lamiaceae family, the genus Thymus can be considered one of the most important
genera of this family, due to the large number of species of which is formed (Morales, 2002). In Romania
vegetate 17 species of the genus Thymus, 16 being spontaneous species and one (Thymus vulgaris L.)
cultivated (Ciocârlan, 2009). In general, the most studies on the genus Thymus grant a special attention of
the species Thymus vulgaris, known for brass and the characteristic smell and also due to the
pharmacological activities. The project proposes an interdisciplinary analysis of those Thymus species that
grow in Romania and which were less or not at all study, so the completion of the survey project to bring as
many data on the structure of glandular hair, chemical composition of the essential oil produce and their
antimicrobial activities. Existing data together with those who will be obtained will be the subject of a
forthcoming monograph on the genus Thymus from Romania.
1. Investigation of glandular hairs from species of the genus Thymus from the Romanian flora
1.1. Collecting the biological material from different areas of the country, according to: altitude and
vegetative season
1.2. Identifying and characterizing the morphological types of glandular (Plate I, II, III)
3.1. Determining the distribution and frequency of secretory hairs using photon microscopy and
statistical processing of data obtained
Lamiaceae family contains many species of herbs and medicinal herbs, with a great economic
importance due to the volatile oils they produce. Glandular hairs are considered exclusive sites of
biosynthesis of volatile oils and therefore their number is directly proportional to the quantity of volatile oil
produced by the plant. Analyzing the actual bibliography available, it came to my attention that there is a
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small number of works dedicated to the study of secretory structures from an ultrastructural point of view
and also on their frequency and distribution in plant organs depending on environmental factors.
A special attention will be granted to the study of glandular hair number depending on the stage of
ontogenetic development of the plant, altitudinal gradient and the seasonal growth of the period, which
represents an element of originality of the theme proposed. It is known that altitudinal gradient is associated
with changes of environmental factors such as temperature, rainfall, air etc. On the other hand, the gradient
of the seasonal growth is associated with changes of parameters, such as photoperiod, the air temperature
and water availability. The combination of these factors exerts a pressure on the plant, which is expressed by
changes in the morphology, anatomy, physiology and its productivity.
During the years 2013-2014 the plant material, represented by species of the genus Thymus L. was
collected from different geographical areas, depending on the growing season and altitude (Table 1, 2;
Figure 1, 2). Species identification was performed by Dr. Ioan Sârbu from Botanic Garden “Anastasie Fătu”,
Iași and by prof. Dr. Nicolae Stefan, taxonomist at Faculty of Biology, University "Al. I. Cuza "University
of Iasi.
The identification of taxa has been done using the following papers: Flora Europaea, vol. 3 and Flora
ilustrată a României – Pteridophyta et Spermatophyta (Ciocârlan, 2009). The collected material was
registered and stored in „Alexandru Ioan Cuza” Universityʼs Herbarium from Iaşi.
Table 1: Collection of biological material according to altitude and phenophases
Species Locality Altitude Phenophases
Thymus praecox sp. polytrichus (A.
Kern. Ex Borbas) Jalas
Novaci, Mountains Parâng,
Gorj
950 m vegetative anthesis fruiting
Thymus praecox sp. polytrichus (A.
Kern. Ex Borbas) Jalas
Rânca, Mountains Parâng,
Gorj
1600m vegetative anthesis fruiting
Thymus praecox sp. polytrichus (A.
Kern. Ex Borbas) Jalas
Peak Dengheru, Mountains
Parâng, Vâlcea
2069 m vegetative anthesis fruiting
Thymus praecox sp. polytrichus (A.
Kern. Ex Borbas) Jalas
Pasul Urdele, Mountains
Parâng, Vâlcea
2145 m vegetative anthesis fruiting
Table 2: Collection of biological material according to geographical location and phenophases
Species Locality Phenophases
Thymus alternans Klokov Baia de Fier, Gorj vegetative anthesis fruiting
Thymus dacicus Borbás Novaci, Gorj vegetative anthesis fruiting
Thymus dacicus Borbás Valea Lotrului, Vâlcea vegetative anthesis fruiting
Thymus comosus Heuff. ex Griseb. &
Schenk
Jina, jud. Sibiu vegetative anthesis fruiting
Thymus pannonicus sp. auctus All. Hill Șorogari, Iași vegetative anthesis fruiting
Thymus pannonicus sp.pannonicus
(Lyka) Soo
Hill Șorogari, Iași vegetative anthesis fruiting
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Figure 1. Thymus praecox sp. polytrichus in diferent phenophases (vegetative, anthesis, fruiting)
Figure 2. Thymus alternans in diferent phenophases (vegetative, anthesis, fruiting)
For the identification and characterization of morphological types of secretory hairs photonic and
electronic microscopy have been used. For photon microscopy plant material was originally fixed and
preserved in 70% alcohol. Subsequently, the biological material was cut using a razor botanical and a
microtome hand. The sections thus obtained were subjected to double staining, after being incorporated in
the glycero-gelatin (Plate II, Fig. 4-8). For transmission electron microscopy plant material was prefixed in
2.7% glutaraldehyde, dehydrated in successive, increasing in concentration, acetone solutions. The samples
were embedded in Epon 812 epoxy resin and polymerized at 600C. The blocks were sectioned with a Leica
Ultramicrotome, to obtain semithin and ultrathin sections, for analyses under the optical microscope and
electron microscope respectively. The obtained sections were stained with uranyl acetate and lead citrate and
observed with a Jeol 1010 TEM (Plate I, II Fig. 1-4, III).
After using microscopy techniques, two morphological types of secretory hairs, located on the surface
of vegetative organs were identified:
- Peltate hair formed from a unicellular base, a pedicel and a gland comprised of more than two cells
(Plate I, Fig 4-5; Plate II, Fig. 5, 8);
- Capitate hairs formed from a unicellular base, a single or bicellular pedicel and a gland of 1 (max 2)
cells (Plate I, Fig. 3, 7, 8; Plate II, Fig. 2-4).
In all analyzed samples, secretory hairs are present throughout the length of the stem, their frequency
increases from the base to top of the organ. They are formed from a unicellular base implanted between
epidermal cells, a unicellular pedicel and a gland of 1, 2 or more cells secreting. The most frequent are
unicellular secretory hairs.
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1.
2.
3. 4.
5. 6.
7. 8.
Plate I: Semithin sections made on Thymus species studied. 1. Transversal section through the foliar blade of Th. alternans; 2. Superficial section through the stem of Th. alternans; 3. Secretory hair from the stem of Th. alternans; 4. Cross section through the foliar blade of Th. dacicus; 5. Peltate
secretory hair from the level of foliar blade of Th. dacicus; 6. Highlighting the capitates and peltates hairs from the level of foliar blade of Th. dacicus;
7. Capitate hair from the inferior epidermis of foliar blade of Th. dacicus; 8. Capitate hair from the superior epidermis of foliar blade of Th. dacicus.
Plate I
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1. 2.
3. 4.
5. 6.
7. 8.
Plate II: Cross sections made on Thymus species studied. 1. Cros section through the foliar blade of Thymus praecox sp. polytrichus; 2. Secretory hair
from upper epidermis of foliar blade of Thymus praecox sp. polytrichus; 3. Superficial sections through the stem of Thymus praecox sp. polytrichus; 4. Secretory hair from the stem of Thymus praecox sp. polytrichus; 5.Peltate secretory hair from the foliar blade Th. pannonicus sp. auctus; 6. Capitate
hair from the foliar blade of Th. pannonicus sp. auctus; 7. Epidermises of foliar blade of Th. pannonicus sp. auctus with secretory hairs; 8. Peltate hair
from the upper epidermis of foliar blade of Th. pannonicus sp pannonicus.
Plate II
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Regarding the effect of altitude and the growing season is easily observed that plants growing at
higher altitudes (2069m and 2145m) are significantly lower compared with plants that grow at lower
altitudes. At the plants that grow at high altitudes the leaf lamina surface is much lower, so the number of
secretory hairs is much smaller. Following studies conducted we found that the density of capitate secretory
hairs tends to increase from spring to autumn, in both sides of the leaf lamina in all populations analyzed.
Peltate secretory hairs are present in both the epidermis of the leaf lamina, without fluctuations over periods
1. 2.
3. 4.
5. 6.
Plate III: Semithin sections made on Thymus species studied. 1. Secretory hair sectioned longitudinally from the foliar blade of Th. alternans; 2.
Secretory hair transverse cut from the level of foliar blade of Th. alternans; 3. Secretory hair wit bicellular pedicel from the foliar blade of Th. dacicus; 4-6. Secretory hairs from the stem of Th. dacicus.
Plate III
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of vegetation. Both capital and peltate secretory hairs are more numerous in populations growing in areas
with lower altitudes.
The secretory hairs density and frequency is not uniform over the whole surface of the leaf lamina, the
highest number recorded on basal and middle regions of abaxial face. On the mature plant, the secretory hais
are located in special concavities from the epidermises, being dispersed (Bosabalidis and Skoula, 1998).
Early termination of the initiating secretory hairs in the apical region reflects the complet differentiation of
epidermis in basipetally sense, coinciding with the general pattern of leaves forming from dycotiledonatae,
described by Esau (1977).
For Thymus praecox sp. polytrichus were evaluated what are the effects of altitude and phenophase on
the secretory in both epidermis of the leaf lamina. For this purpose it was considered the evaluation of the
number of secretory hairs per unit area, the middle area of the leaf lamina (Figure 3 a, b; Figure 4).
Figure 3. Graphical representation of the variation in the number of hairs secretory, on the level of upper
epidermis (a) and lower epidermis (b), on Thymus praecox sp. polytrichus, depending on altitude and
phenophase.
Figure 4. The variation of secretory hairs, comparative in both epidermis, on Thymus praecox sp.
polytrichus, depending on altitude and phenophase.
Analyzing the results we can say that the number of secretory hairs slightly decreases, in both
epidermis of the leaf lamina, with increasing altitude. Also, it is noted that the number of secretory hairs is in
generally higher in the vegetative stage and anthesis, compared to the fruiting stage, in both the epidermis.
a. b.
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In the lower epidermis there was a slight increase in secretory hairs, especially at altitudes of 950 m,
1600 m and 2145 m.
For a more complete analysis of the effectiveness of essential oils secretory structures we considered
necessary also, the evaluation of number of secretory hairs from other Thymus species that were studied (Th.
alternans, Th. dacicus, Th. comosus, Th. pannonicus sp. pannonicus, Th. pannonicus sp. auctus) (Figure 5
and 6).
Figure 5. Graphical representation of the variation in the number of hairs secretory on the level of upper
epidermis (a) and lower epidermis (b), on Thymus sp., depending on altitude and phenophase.
Figure 6. The variation of secretory hairs, comparative in both epidermis, on Thymus sp., depending on
altitude and phenophase.
The largest number of hairs secretory per unit area, was found in Th. alpestris in anthesis stage, at the
level of the lower epidermis and the upper epidermis level. By correlating the result with the amount of
essential oil obtained by hydrodistillation, we noticed that on Th. alpestris we obtain the largest amount of
volatile oil, per 100g plant material. It can be said that the number of secretory hairs is directly proportional
with the amount of volatile oil produced by the plant. Taking this into account, we can identify spontaneous
populations with highly aromatic value (which resides from the frequency and morphological types of
a. b.
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secretory hairs and chemical composition of volatile oil) depending on altitude, time of harvest during the
growing season, the ontogenetic stage of the plant.
The lowest number of secretory hairs was found in Thymus dacius, phase of fruiting.
Correlating the results of the distribution and frequency of the secretory hairs, quantity and quality of
essential oil, it can be concluded that the optimal time to harvest Thymus species is the period of flowering
(anthesis) or its preceding period.
2. Study of the volatile oils obtained from species of the genus Thymus - biochemical researches
2.1. Collection of biological material from different areas of our country depends on altitude and
vegetative season plant
2.2. Extraction of volatile oils by hydrodistilation
2.3. Analysis of the chemical composition of volatile oil
The plant material is mentioned in point Investigation of glandular hairs from species of the genus
Thymus from the Romanian flora: micro-morphological researches. After collection it was weighed and
dried at room temperature. Generally need 300g fresh plant material for biochemical studies. The volatile oil
was extracted from aerial plant organs using a Clevenger type apparatus according to the European
Pharmacopoeia standards, in the Laboratory of Plant Physiology, Faculty of Biology, Iaşi. The compounds
of the volatile oil was separated using the gas chromatographic method coupled with a mass spectrometry
detector, using a GC-MS Agilant Technologies 6890N device coupled with mass detector (MSD) 5975 inert
XL Mass Selective Detector. The chromatography conditions are: HP 5MS column external size 30 m x 0.25
mm – internal size 0.25 μm (5% Phenylmethylsiloxane); mobile phase: Helium – flow: 1 mL/min; injector
temperature: 250°C; detector temperature: 250°C; temperature regimen: initialy from 40°C (10
degrees/minute) to 280 degrees (5.5 min constant); injected volume: 0.1-0.3 µl; splitting rate -1:100.
Thymus alternans Klokov is a relatively new species for Romania, being first mentioned in 2002 in
the Romanian flora (Ciocârlan, 2002). This taxon was described in 1954 by M. Klokov and published in
Need. Syst. (Leningrad). In the same year, the species was published in Flora of the USSR, XXI, by the
same author who processed this genus (Ciocârlan, 2002). In 1996 this taxon was described by Martonfi in
the paper “Thymus alternans Klokov – a new species from Slovak flora”, paper published in Biologia,
Bratislava.
The species is quite similar to Th. pulegioides L., shows hairy stems opposite sides, alternating from
one node to another, leaves are oblong elliptical, ciliate on margins, 2-4 pairs of lateral veins weak
prominent and calyx tube is hairy (Ciocârlan, 2002). In Romania the species grows in mountain meadows at
low altitudes 500-1100m in the north and northeast, but we found the species in the Central Southern part of
Romania, in Baia de Fier, Gorj County.
After analyzing the essential oils 51 chemical compounds were identified, representing between
90.585% and 99.249% of the total compounds identified (Table 1). The main compounds identified were
thymol, carvacrole, o-geraniol, followed by methyl-thymol, o-cimene, p-cimene, γ-terpinene, terpinyl
acetate, geranil acetate and β-bisabolene. The largest number of constituents (34 compounds) was identified
in the volatile oil extracted from the individuals collected in 2013 in vegetative and anthesis phenophases.
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The smallest number of compounds (26 compounds) was identified in the volatile oil extracted from plants
collected in 2013 in vegetative phenophase.
The studies have revealed that phenolic monoterpenes (thymol and carvacrol), specific to Thymus
genre, in general, are present in large amounts in all phases of vegetation in both years. Another compound
found in large quantities was geraniol, present in all phenophases. The carvacrol, one of the main
constituents of the essential oil of Th. alternans, has anthelmintic activity (Harborne and Baxter, 1983),
antibacterial activity (Russell, 1986), antitussive and carminative activity (Leung A. Y. and Foster S. 1995).
The thymol has many biological activities, being considered analgesic (Madaus G., 1976), anthelmintic
(Mocanu Ş. and Răducanu D., 1983) and also exhibits a choleretic activity (stimulates bile of the liver),
colagog activity (stimulates contraction of the gallbladder and bile in the gut evacuation) and stimulates
kidney functions (Mocanu Ş. and Răducanu D., 1983).
Another compound identified in all three phenophases, in both year, was the geraniol. The geraniol
has numerous biological implications, it can be used as: anthelminthic (Madaus, 1976), antibacterial (Muroi
and Kubo, 1993), antispasmodic (Buchbauer et al., 1989), an expectorant (Madaus, 1976). Also it is
suggested that geraniol presents anticancer properties
(http://www.ars.rin.gov/cgibin/duke/chemical.pl?ALPHATERPINENE).
Regarding the chemical composition of essential oils from other species of Thymus, we can affirm
that a high chemical variability was observed. Thus, Maksimovic Z. and collaborators in 2008 identified in
the volatile oil Th. pannonicus. All., harvested in northern Serbia, a total of 33 constituents, the main being
geranial (41.42%) and neral (29.61). These compounds were not identified in the volatile oil Th. pannonicus
collected in Romania (Boz I. et al., 2009), but traces of geraniol were found, a compound that forms geranial
by oxidation. Other researchers have identified in the volatile oil of this species large amounts of thymol
(25-41%) and p-cimen (17-38%) (Pluhar Z. et al., 2007).
Table 1. The composition of essential oils extracted from Thymus alternans, plants collected in
different phenophases, two consecutive years
Compound
Vegetative Anthesis Fruiting
Year Year Year
2013 2014 2013 2014 2013 2014
α-Thuyen 0.231 0.41 0.236 0.656 0.252 0.349
α-Pinene 0.214 0.187 0.255 0.294 0.174 -
Camphene 0.256 - 0.31 0.159 0.115 -
Octen-3-ol 0.425 0.614 0.316 0.67 0.234 0.733
3-Octanone 0.205 0.174 - - - 0.292
β-Pinene 0.203 0.39 0.274 0.485 0.23 0.351
3-Octanol 0.14 - - - - -
o-Cimene 11.556 - 4.505 - 4.544 -
γ-Cimene - - - - - 10.719
α-Terpinene - 0.58 - 0.662 0.254 -
p-Cimene - 8.818 - 10.506 - -
Silvestren - - - 0.295 - -
Limonene 0.177 0.167 - - 0.104 -
Eucalyptol 0.79 0.362 0.495 0.463 0.43 0.568
γ-Terpinene 1.983 5.164 0.71 4.231 1.153 2.544
cis-Sabinene hydrate 0.496 0.528 0.531 0.177 0.171 0.736
Isopropyl methyl cyclohexene- - - - 0.14 - -
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1-ol
Linalool 0.22 0.179 0.588 - 0.555 0.318
Camphor 0.159 - - - - -
Borneol 1.657 0.333 0.738 0.69 0.478 0.767
Terpinen-4-ol 0.221 0.107 0.563 0.174 0.124 0.175
α-Terpineol 0.258 0.263 0.208 - -
Nerol 0.427 0.259 1.623 0.227 1.276 0.888
Methyl thymol isomer 2.184 - 0.83 0.922 0.626 1.216
Methyl thymol 6.261 3.666 2.537 6.274 3.239 5.889
Geraniol 10.863 27.729 29.04 7.25 29.845 12.523
Geranial - - 1.178 - 0.259 0.454
Bornyl acetate - - 1.103 - - -
Thymol 11.224 4.308 15.262 8.826 7.001 8.252
Carvacrol 27.764 29.768 10.066 40.638 18.476 34.73
α- Terpinyl acetate 4.056 - 0.205 3.632 - -
Neril acetate - - 0.556 - - -
Geranil acetat 4.39 0.848 13.037 2.549 17.859 3.788
β-Burbonen 0.206 - 0.269 0.14 6.268 0.346
α- Caryophyllene 1.361 - 2.731 - 1.937 -
β- Caryophyllene - 1.102 - 1.429 - 2.437
γ-Murolene 0.132 - 0.191 - 0.138 -
Germacrene D 0.498 0.595 1.295 1.439 1.885 1.437
Methyl-isoeugenol 0.086 - 0.353 - - -
γ-Elemene - - - 0.283 0.39 0.585
Leden - 0.17 - - - -
β-Bisabolene 5.818 3.19 5.562 3.836 - 5.778
β-Cadinene - 0.213 - 0.586 - -
tau Cadinene - - - - 0.377 -
γ-Cadinene 0.241 - 0.426 - 0.18 -
Germacrene D-4-ol - - 0.553 - - -
Spathulenol 0.313 0.156 - - 0.328 0.395
Caryophyllene oxide 1.68 0.568 0.993 0.478 0.347 1.313
tau Murolol - - 0.303 - - -
TOTAL % 96.985 90.585 97.897 98.319 99.249 97.583
Another species analyzed is Thymus dacicus, the species being collected from Novaci (jud. Gorj) in
different phenological phases during the years 2013-2014.
Following the analysis of essential oils have been identified a total of 56 chemical compounds,
representing between 84.228% and 99.128%, of the total number of identified compounds on the column
(Table 4). The highest number of chemicals (38 compounds) was identified in the volatile oil derived from
taxa collected in 2013, the stage of fruition. The lowest number of chemical compounds (25 compounds)
was identified in the volatile oil derived from taxa collected in 2013 in the vegetative stage.
Tabel 4. The chemical composition of essential oil of Thymus dacicus, collected in various
phenophases, in two consecutive years (2013-2014), from Novaci, jud. Gorj
Compound
Vegetative Anthesis Fruiting
Year Year Year
2013 2014 2013 2014 2013 2014
α-Pinene - 0.53 - - 0.809 -
Camphene - 0.842 - - 0.361 -
Octen-3-ol - 0.395 - 0.445 0.808 0.577
Mircene - 2.276 - - - -
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β-Pinene - - - - 1.145 0.188
p-Cimene 7.466 - - 4.698 - 10.832
α-Terpinene - - - - 0.297 0.258
β –Cimene - 0.378 - - 0.897 -
Limonene - 0.328 - 0.25 0.236 -
Eucaliptol 0.661 0.527 - 0.492 - 0.623
cis-β-ocimene - 0.372 - - - -
γ-Terpinene - 0.278 - 1.313 0.923 2.119
cis-Sabinen hidrate 2.022 1.824 - - 2.057 -
Linalool - 0.702 11.44 1.928 11.667 0.359
Camphor - 0.943 0.552 - 1.254 -
Borneol 0.842 2.632 2.636 0.598 1.95 1.043
Terpinene-4-ol 1.642 0.487 1.197 - 6.206 -
α-Terpineol - 3.819 9.879 0.248 3.568 -
Nerol 2.568 2.628 1.305 1.489 1.051 1.893
Linalyl acetate - - 1.822 - - -
Neral 0.811 0.349 0.665 0.456 - 1.015
Metyl timol isomer 0.523 - - - - -
Metyl timol 2.14 5.407 - 3.057 0.401 4.78
Neral - - - - 1.213 -
Geraniol 18.376 5.696 2.724 33.025 1.39 28.81
Geranial 1.974 - 1.162 - 1.891 0.769
Citral - - - 0.76 - -
Bornil acetate 1.798 7.894 1.741 - 5.106 -
Thymol 2.314 5.397 - 4.124 - 4.697
Carvacrol 6.025 0.365 - 12.477 - 16.045
α-Terpinyl acetate - - - 7.904 - 0.174
Neril acetate - 1.543 0.383 - 1.39 -
Lavandulol acetate - - 1.027 - - -
Geranil acetate 18.489 1.589 - 12.683 1.126 11.044
α-Burbonen 0.728 - - - 0.301 0.486
β-Burbonen - 0.324 - 0.517 9.7 1.799
α-Cariophyllene - 1.435 - - 0.584 -
Alloaromadendren - - 1.047 - 1.275 -
β-Cariophyllene 1.299 5.333 2.518 2.49 - 1.788
Farnesen 0.811 0.33 - - - -
Germacrene D - 2.166 0.808 3.065 1.125 -
γ-Elemen - 1.546 0.537 0.586 3.355 0.729
β-Bisabolen 3.136 1.377 2.052 5.283 - 4.972
β-Cadinen - - 0.879 - - 0.625
γ-Cadinol 5.23 - 18.807 - 7.834 -
tau Cadinol - - 2.563 - 2.261 -
tau Cadinen 0.772 2.394 - - 1.624 -
γ-Cadinen 1.05 0.968 - 0.449 1.017 -
Elemol 0.85 0.388 0.875 - 0.362 -
trans Nerolidol 0.367 3.081 0.304 0.28 0.456 0.317
Spatulenol - - 9.079 - 9.572 0.32
Cariophyllene oxid 2.334 10.531 2.525 0.511 3.171 0.883
Leden - - 0.845 - - -
Cubenol - 1.107 - - - -
tau Murolol - 9.698 5.456 - 3.845 -
Aromadendren epoxy - - 2.453 - 1.118 0.285
TOTAL % 84.228 87.879 87.281 99.128 93.346 97.145
Analyzing the results it is observed that there are a number of qualitative and quantitative differences
depending on phenophase and harvest year. We could mention as the main chemical components: geraniol,
linalool, terpineol, geranyl acetate, γ-cadinol, caryophyllene oxide and tau murololul. An important aspect to
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note, is the presence, in large amounts, in some cases or in small amounts in other cases, of the two
monoterpenes phenol (thymol and carvacrol) specific Thymus genus. For 2013, thymol and carvacrol is
noted those are missing in anthesis and fruiting stage and are present in small quantities in the vegetative
stage. For 2014, the two phenolic monoterpenes are present in all 3 phenophases analyzed in rather large
quantities.
To highlight the cumulative effect of altitude and the stage of development of plants on the chemical
composition of volatile oils Thymus praecox sp polytrichus was chosen, a species that grows in Parang at
different altitudes. For this purpose, the plant material was collected from four different areas with different
altitudes (950m, 1600m, 2069m, 2145m), in 2 consecutive years (2013, 2014).
After analyzing the essential oils of Thymus praecox sp. polytrichus, collected at different altitudes,
during 2013, we have identified a number of 65 chemical compounds. The highest number of chemicals (46
compounds) was identified in populations collected from 2149 m altitude in populations at the stage of
fructification. The lowest number of chemical compounds (22 compounds) was identified in populations
collected from 2069 m altitude, in anthesis stage.
For taxa collected in 2014, the largest number of chemical compounds (41 compounds) was identified
in populations collected from 950 m, at the stage of fructification. The lowest number of chemical
compounds (20 compounds) was identified in populations collected from 1600 m altitude, in the fruiting
stage.
The main chemical compound of the essential oil of Thymus praecox sp polytrichus is δ- Cadinol, the
compound of high percentages in both 2013 and 2014 (except for taxa collected from 950 m at the stage of
fruiting). This compound possesses repellent activities (He et al., 1997; Yatagai et al., 2007). In large
quantities were obtained the following compounds: cis-sabinene hydrate, β caryophyllene, Germacren D-4-
ol and linalool. It should be noted that Germacren D-4-ol was found in large quantities in 2013, being no
longer identified in volatile oils obtained in 2014. Also, Linalool was obtained in large quantities in 2014,
compared with the year 2013.
For taxa collected from 2069 m altitude is distinguished, for 2013, the lack of many monoterpenes
(first terpenes that appear on the chromatogram) compared with other samples studied. The Thymus specific
phenolic monoterpenes (thymol and carvacrol) were identified in small amounts or were not identified, for
both years of the study.
All these differences may be due to altitudinal gradient associated with changes in environmental
factors such as temperature, precipitation, etc. On the other hand, the seasonal increase gradient is associated
with the seasonal changes of the parameters, such as photoperiod, temperature of the air and water
availability. Perhaps combining these factors are putting pressure on the plant, pressure is expressed through
changes in physiology and productivity. In conclusion, our studies reveal that geographical area and altitude
can be factors that influence the quality and quantity of volatile oil produced by aromatic plants.
3. Study of the volatile oils obtained from species of the genus Thymus - microbiological researches
3.1. Documentation using existing literature about the importance of testing essential oils on
microorganisms (Staphyloccocus aureus, Escherichia coli, Candida albicans)
3.2. Testing the antibacterial activity of essential oils used on Staphylococcus aureus
15
3.3. Testing the antibacterial activity of essential oils used on Escherichia coli
3.4. Testing the antifungal activity of essential oils used on Candida albicans
Volatile oils have been used over time in food and cosmetics, but also in medical and pharmaceutical
industry. Environmental factors such as temperature, radiation and photoperiod plays an extremely
important role in the quantity and quality of essential oils. The purpose of this objective is to establish
antibacterial and antifungal activity of essential oils studied, taking into account the altitude at which it was
collected plants, the gradient of the season and their ontogenetic stage, highlighting the importance
therapeutic compounds.
Medicinal and aromatic plants have been used for a long time in the fight against infectious diseases,
but the discovery of antibiotics has led to a decline of medicine based on plant. No doubt antibiotics are, at
present, the group of drugs most used in medicine. However, research alternative methods are necessary
because right from the start antibiotics showed some inconveniences and limitations of use (Siegenthaler W.
and R. Luthy, 1978):
• presence of side effects;
• tolerance and increasing resistance of germs to antibiotics;
• decreased immunity.
The species of Thymus genus exhibit tonic, carminative, digestive, antitussive, expectorant activity
(Mojab F.et al., 2008), for which essential oils have been extensively studied and tested on various
microorganisms. Thus, the essential oil of Thymus vulgaris presents antifungal properties, being tested on
Aspergillus, Candida, Penicillium, Mucor, Cladosporium, Trichoderma, Chaetomium (Segvic Klaric M. et
al., 2006; Giordani R. et al., 2004; Faleiro M.L. et al., 2003).
Currently there are numerous studies on antibacterial activity of essential oils of Thymus genus
(Kowal and Kuprinska, 1979; Marino, 1999; Nelson, 1997; Pina-Vaz et al., 2004; Smithpalmer et al., 1998).
Thymol and carvacrol seems to play an important role in this regard. These phenolic compounds bind to the
amino- and hidroamino groups of proteins across the bacterial membrane, altering the permeability, leading
to the death of bacteria (Juven et al., 1994).
According to the studies made by Pina-Vaz C. et al (2004), the volatile oil derived from Th. vulgaris,
Th. zygioides ssp. zygioides and Th. mastichina can be used for medicinal purposes. It was studied the
antibacterial activity of the main components of the essential oil (carvacrol, thymol, p-cymene and 1.8
cineole) and also the possible interactions between these components. Oils derived from Th. vulgaris and Th.
zygioides showed a similar antibacterial activity, and higher than the oil from Th. mastichina. Also, volatile
oil Th. vulgaris was tested and E. coli (M. Marino, 1999), showing that E. coli cells are destroyed at a
relatively small concentration of the oil.
Potential antimicrobial activity of essential oils of Thymus was investigated by Faleiro et al (2003).
The authors analyze the chemical composition and test the antimicrobial activity of oils obtained from Th.
mastichina ssp. mastichina, Th. camphoratus and Th. lotocephalus species harvested from different areas of
Portugal. The antimicrobial activity of these oils was tested on Candida albicans, Escherichia coli, Listeria
monocytogenes, Proteus mirabilis, Salmonella sp. and Staphylococcus aureus. Thymus species studied have
demonstrated antimicrobial activity, but the microorganisms tested showed different sensitivities. Also, this
antimicrobial activity is due to several components of essential oils. The antimicrobial properties of essential
oil of Th. pubescens and Th. serpyllum species collected before and during flowering, were studied by
16
Rasooli and Mirmostafa (2002). Also, volatile oil extracted from Th. revolutus, a species that grows in
Turkey, present significant antibacterial and antifungal activities.
*To test the antimicrobial activity of essential oils two methods of work were used: Kirby-Bauer disc
diffusion method and microplate method. Sensitivity to essential oils microorganisms studied was tested "in
vitro", putting them in optimal and standardized cultivation (culture medium, inoculation, incubation, etc.).
Diffusion method Kirby-Bauer adopted by CLSI (Clinical Laboratory Standards International, 2009) in the
US is the usual method, widely used in laboratories to test a relatively small number of microbial strains
with rapid growth. By submitting cylinders containing 50μl quantities of samples tested, on the surface of a
solid medium inoculated with a microbial culture, active antimicrobial substance will diffuse into the
environment, with a steady decline of the concentration gradient from the edge of the cylinder toward the
periphery. After the incubation time may define two separate zones: one in which microbial growth is
inhibited by concentrations of the antimicrobial substance, and a zone where the concentration is too low to
inhibit the growth.
The culture medium used is Mueller Hinton medium (for bacteria) and Sabouraud medium (for
yeast), distributed in Petri dishes in a uniform layer thickness of 4 mm, a pH of 7.2 to 7.4 (for bacteria ) and
pH 6.5 (for yeast) measured before pouring into plates. These medium have nutritional value which allows
optimum development of a wide variety of germs and contains no inhibitors of bacterial substances.
The second method used is microplate method (Sarker et al., 2007). We used 96-well microplate,
each containing 80 ml culture medium, 10 ml of diluted bacterial culture, 100 ml essential oil to be tested in
different concentrations (1/10, 1/100 and 1/1000) and 10 ml resazurine, resulting in a total volume of 200 ml
per well. Microplates were incubated at 370 C for 24 hours. Of course, each plate contained wells and
representing control (represented by DMSO). The color changes were then evaluated visually. Thus, growth
and development of microorganisms was indicated by changing color from dark blue to purple. MIC
(minimum inhibitory concentration) is the lowest concentration at which the color changes.
The essential oils studies were tested on Staphylococcus aureus ATCC 25923, Escherichia coli
ATCC 25922 and Candida albicans.
Fig. 8. Testing the antomicrobial activity of essential oils through microplate method
17
Testing the antibacterial and antifungal activity was made for the essential oils of Thymus alternans
(vegetative, anthesis and fruiting stage), Thymus dacicus (vegetative, anthesis and fruiting stage) and
Thymus praecox sp. polytrichus (vegetative, anthesis and fruiting stage, colected from different altitude:
950m, 1600m, 2069m and 2145m from Parîng Mountains) in 2014.
In the case of essential oils of Thymus alternans, our studies show that these oils inhibit the growth
and development of the 3 microorganisms tested, in all stage of development, differing only CMI. Thymus
alternans Klokov is a relatively new species for Romania, being first mentioned in 2002 in the Romanian
flora (Ciocârlan, 2002). Studies regarding the antimicrobial activity of essential oils of Thymus alternans are
very few. We mention here the study conducted by Vitali and collaborators (2016). According with their
studies, the essential oil of Thymus alternans (collected from Slovakia, 2014, only flowering aerial parts)
inhibit the growth and development of the S. aureus, E. faecalis, E. coli and C. albicans. Pseudomonas
aeruginosa was the bacterial species not affected by the Thymus alternans essential oil.
Another species studied by us is Thymus dacicus. According to our results, the essential oils from
this species show antimicrobial activity in all stages of development, differing only CMI. Regarding the
existing studies in the literature, we mention that in this species they are missing, there is only a few data on
the chemical composition of volatile oil (main components: carvacrol - 30%; thymol - 16.8%, nerol, α-
terpineol, linalil ethyl) (Kisgyörgy et al., 1984). These compounds have been identified in our oils in
different proportions. On individuals collected by us, the main chemical compounds are geraniol, linalool,
terpineol, geranyl acetate, γ-cadinolul, caryophyllene oxide and tau murolol, in different percentages,
depending on the developmental stage of the plant.
Another species included in the study is Thymus praecox sp. polytrichus, collected in 3 different
phenophases (vegetative, anthesis and fruiting), from different altitude (950m, 1600m, 2069m and 2145m).
The essential oils of this species inhibit the growth and development of Staphylococcus aureus and
Escherichia coli, in all vegetative phases, except the oil obtained from plants in fruiting stage (1600m).
These oils present also antifungal activity on Candida albicans, except plants collected from 1600m and
2145 m, in anthesis stage. Thymus praecox sp. polytrichus is a rare species and the studies on the
antimicrobial activity are relatively few. This species presents the antimicrobial activity as tested on
Aspergillus fumigatus, A. versicolor, A. ochraceus, A. niger, Trichoderma viride, Penicillium funiculosum,
P. ochrochloron, P. verrucosum var. cyclopium (Petrovic et al., 2016). No data were found on the antifungal
activity of these oils on Candida albicans.
4. Specializing training - information and documentation on the domain of the project
4.1. Traveling within the borders of the country: in view of obtaining biography information in
university centers (Bucharest, Cluj, Arad, Oradea)
During the project displacements for information were made in the following universities and research
centers:
Obergurgl University Centre/Tyrol, Austria
Academy of Sciences of Moldova
Faculty of Biology, University Babes-Bolyai University, Cluj
18
Research Center for the Study of Quality of Horticultural Products, Faculty of Horticulture,
University of Agricultural Sciences and Veterinary Medicine Bucharest
Center for Biological Research "Stejarul" Piatra Neamt
4.2. Traveling abroad: specializing, informing and documentation training
Research internship at the Center for Biological Research "Stejarul" Piatra Neamt, coordinator:
CS I Dr. Elvira Gille, 20-24 May 2013
Research internship at the Center for Biological Research "Stejarul" Piatra Neamt, coordinator:
CS I Dr. Elvira Gille, 25-30 November, 2013
Research internship at the Research Center for the Study of Quality of Horticultural Products,
Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine Bucharest,
coordonator Prof. Dr. Ioan Burzo, 5-12 September 2014
Research internship at the Center for Biological Research "Stejarul" Piatra Neamt, coordinator:
CS I Dr. Elvira Gille, 25-30 November, 2014
5. Dissemination of research results
5.1. Participation in a number of national and international scientific manifestations
Annual Scientific Session of the Faculty of Biology, 24-26 october 2013, Iași, paper: ESSENTIAL
OILS OF THYMUS COMOSUS HEUFF. EX GRISEB. (LAMIACEAE) COLLECTED FROM
DIFFERENT AREAS, authors: Irina Boz, Ioan Burzo, Maria-Magdalena Zamfirache, Rodica
Efrose;
Trends in natural products research: a young scientists meeting of PSE and ÖPhG, University
Centre Obergurgl/Tyrol, Austria, 21–25 July 2013, papers: INFLUENCE OF ALTITUDE ON
THE CHEMICAL COMPOSITION OF ESSENTIAL OILS OF THYMUS BALCANUS BORBÁS,
authors: Irina Boz, Elvira Gille, Irina Mihalache, Maria-Magdalena Zamfirache, Rodica Efrose
and ECOLOGICAL CULTURES OF MEDICINAL AND AROMATIC PLANTS
COMMERCIALIZED IN FOOD SUPPLEMENTS, authors: Elvira Gille, Dana Bobit, Georgiana
Gavril, Irina Boz, Monica Hancianu;
„The Third National Symposium with international participation "Advanced Biotechnologies -
progress and prospects”, Chișinău, Republica Moldova, 24-25 october 2013, paper: DATA ON
THE CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF VOLATILE OILS
OF THYMUS PULEGIOIDES L., authors: Irina Boz, Elvira Gille, Simona Dunca, Maria-
Magdalena Zamfirache
8th CMAPSEEC Conference on Medicinal and Aromatic Plants of Southeast European Countries,
May 2014, Durrës, Albania – paper: MICROMORPHOLOGICAL RESEARCH REGARDING
THE GLANDULAR HAIRS OF THYMUS PRAECOX OPIZ SSP. POLYTRICHUS (A. KERN.
EX BORBAS) JALAS, authors: Boz Irina, Toma Constantin, Zamfirache Maria Magdalena, Gille
Elvira
Phytochemicals in Medicine and Pharmacognosy, organizat de Phytochemical Society of Europe,
April 2014, Piatra Neamt, Romania, paper: MICROMORPHOLOGICAL AND BIOCHEMICAL
19
STUDIES REGARDING THYMUS TAXA FROM ROMANIA FLORA, authors: Irina Boz,
Constantin Toma, Zenovia Olteanu, Ioan Burzo, Elvira Gille, Maria Magdalena Zamfirache
Phytochemicals in Medicine and Pharmacognosy, organizat de Phytochemical Society of Europe,
April 2014, Piatra Neamt, Romania, paper: CONTRIBUTIONS TO THE PHYTOCHEMICAL
STUDY OF THE POLYPHENOLIC FRACTIONS SEPARATED FROM THYMUS
PULEGIOIDES L. NATURAL POPULATIONS HARVESTED IN NORTHERN ROMANIA,
authors: Radu Necula, Irina Boz, Valentin Grigoras, Georgiana Luminita Gavril, Ursula Stanescu
“Young Researchers in Biosciences” International Symposium, Cluj Napoca, Romania, July
2014, paper: CONTRIBUTIONS TO THE KNOWLEDGE REGARDING THE STRUCTURE
OF VEGETATIVE ORGANS OF THYMUS DACICUS BORB., authors: Irina Boz, Constantin
Toma
Annual Scientific Session of the National Institute of Research and Development for Biological
Sciences, December 2014, București, paper: ANATOMICAL AND BIOCHEMICAL
INVESTIGATIONS ON THYMUS ALTERNANS KLOKOV, authors: Irina Boz, Ioan Burzo,
Constantin Crăciun, Andrei Lobiuc
12th edition of the National Symposium with International Participation „MEDICINAL PLANTS
– PRESENT AND PERSPECTIVES”, 06-09 September 2016, Piatra Neamț, Romania, paper:
CONTRIBUTIONS TO THE KNOWLEDGE REGARDING THE STRUCTURE OF
VEGETATIVE ORGANS OF THYMUS DACICUS BORB., authors: Irina Boz, Constantin
Craciun, Andrei Lobiuc
12th edition of the National Symposium with International Participation „MEDICINAL PLANTS
– PRESENT AND PERSPECTIVES”, 06-09 September 2016, Piatra Neamț, Romania, paper: THE
EFFECT OF HARVESTING TIME ON ESSENTIAL OILS COMPOSITION OF THYMUS
PANNONICUS L., authors: Irina Boz, Ioan Burzo, Corneliu Tanase
5.2 Publication of scientific papers in national and international journals recognized CNCS
indexed/top rated ISI.
Irina Boz, Elvira Gille, Radu Necula, Simona Dunca, Maria-Magdalena Zamfirache -
CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF ESSENTIAL OILS
FROM FIVE POPULATIONS OF THYMUS PULEGIOIDES, Cellulose Chemistry and
Technology, 2015, 49 (2): 169-174 (IF- 0.825)
Irina Boz, Ioan Burzo, Maria-Magdalena Zamfirache, Rodica Efrose - ESSENTIAL OILS OF
THYMUS COMOSUS HEUFF. EX GRISEB. (LAMIACEAE) COLLECTED FROM DIFFERENT
AREAS OF ROMANIA, Analele Științifice ale Universității „Alexandru Ioan Cuza“ din Iași,
serie nouă, secțiunea II a. Biologie Vegetală, 2014, 60, 1: 40-45
Irina Boz, Andrei Lobiuc, Corneliu Tănase - CHEMICAL COMPOSITION OF ESSENTIAL
OILS AND SECRETORY HAIRS OF THYMUS DACICUS BORBÁS RELATED TO
HARVESTING TIME, Cellulose Chemistry and Technology, (2016) article submitted for
publication (IF- 0.825)
Radu Necula, Elvira Gille, Valentin Grigoraș, Irina Boz, Ursula Stănescu - CONTRIBUTIONS
TO THE PHYTOCHEMICAL STUDY OF THE POLYPHENOLIC FRACTIONS SEPARATED
20
FROM THYMUS PULEGIOIDES L. NATURAL POPULATIONS HARVESTED IN
NORTHERN ROMANIA, Analele Științifice ale Universității „Alexandru Ioan Cuza“ din Iași,
Sec. II a. Genetică și Biologie Moleculară, article submitted for publication
Irina Boz, Ioan Burzo, Constantin Crăciun, Andrei Lobiuc, ANATOMICAL AND
BIOCHEMICAL INVESTIGATIONS ON THYMUS ALTERNANS KLOKOV COLLECTED
FROM ROMANIAN FLORA, Indian Journal of Experimental Biology (Factor de impact: 1.165),
article submitted for publication
5.3. Achieving and maintaining a Web page
http://www.icbiasi.ro/pd/
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Director proiect,
Dr. Boz Irina