1

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)

2

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

3

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

4

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.

5

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

6

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

7

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

8

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.

9

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.

10

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.

11

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 - -

12

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 - - - -

13

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

14

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/

Bibliography:

1. Anwar F., Hussain A. I., Sherazi S. T. H., Bhanger M. I. - Changes in composition and antioxidant

and antimicrobial activities of essential oil of fennel (Foeniculum vulgare Mill.) fruit at different

stages of maturity. Journal of Herbs, Spices and Medicinals Plants. 2009, 15: 1-16;

2. Bakkali F., Averbeck S., Averbeck D., Idaomar M. – Biological effects of essential oils – A review,

Food and Chemical Toxicology, 2008, 46: 446-475;

3. Bosabalidis A.M. – Structural features of Origanum sp., In Oregano: the genra Origanum and Lippia,

Taylor and Francis, 2002, 11-27;

4. Bosabalidis A.M., Skoula M. - A comparative study of the glandular trichomes on the upper and

lower leaf surfaces of Origanum x intercedens, Rech. J. Essential Oil Res., 1998, 10: 277–286;

5. Bruni A., Modenesi P. - Development, oil storage and dehiscence of peltate trichomes in Thymus

vulgaris (Lamiaceae). Nord J Bot., 1983, 3: 245–251;

6. Burt S. – Essential oil: their antibacterial properties and potential applications in food - a review, Int.

J. Food Microbiol., 2004, 94: 223-253;

7. Celiktas O. Y., Kocabas E. E. H., Bedir E., Sukan F. V., Ozek T. , Baser K. H. C. - Antimicrobial

activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and

seasonal variations. Food Chemistry. 2006, 100: 553-559;

8. Chalchat J. C., Garry R. P., Muhayimana A. - Essential oil of Tagetes minuta from Rwanda and

France: chemical composition according to harvesting location, growth stage and part of plant

extracted. Journal of Essential Oil Research. 1995, 7:375-386;

9. Ciocârlan V. - Specii noi în flora României, Buletinul Grădinii Botanice Iași, Tomul II, 2002, p. 97-

98;

10. Ciocarlan V. - Flora ilustrată a Romaniei. Pteridophyta et Spermatophyta, Ed. Ceres, București,

2009;

11. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility

testing; nineteenth informational supplement. CLSI document M100-S19, Wayne, Pa: Clinical and

Laboratory Standards Institute, 2009;

12. Esau K. – Anatomy of seeds plants, J. Willey, New York, 1977;

13. Faleiro M.L., Miguel M.G., Ladeiro F., Venancio F., Tavares R., Brito J.C., Figueiredo A.C.,

Barroso J.G., Pedro L.G. - Antimicrobial activity of essential oils isolated from Portuguese endemic

species of Thymus, Soc. Applied Microbiol., 2003, 36: 35–40;

14. Giordani R., Regli P., Kaloustian J., Mikaïl C., Abou L., Portugal H. - Antifungal Effect of Various

Essential Oils against Candida albicans. Potentiation of Antifungal Action of Amphotericin B by

Essential Oil from Thymus vulgaris, Phytother. Res., 2004, 18: 990–995;

21

15. Gustafson J.E., Liew Y. C., Chew S., Markham J.L., Bell H.C., Wyllie S.G., Warmington J.R. –

Effects of tea tree oil on Escherichia coli, Lett. Appl. Microbiol., 1998, 26: 194-198;

16. Hammer K.A., Carson C.F., Riley T.V. – In vitro activity of Malaleuca alternifolia (tea tree) oil

against dermatophytes and other filamentous fungi, J.Antimicrob.Chemother., 2002, 50: 195-199;

17. Hayasshi K., Kamiya M., Hayasshi T. – Virucidal effects of the stem distillate from Houttuynia

cordata and its components on HSV-1, influenza virus and HIV, Planta Med., 1995, 61:237-241;

18. He K, Zeng L, Shi G, Zhao GX, Kozlowski JF, McLaughlin JL -Bioactive Compounds from

Taiwania cryptomerioides. J. Nat. Prod., 1997, 60: 38-40;

19. Helander I.M., Alakomi H.L., Latva-Kala K., Mattila-Sandholm T., Pol I., Smid E.J., Gorris L.G.M.,

Von Wright A. - Characterization of the action of selected essencial oil components on Gram-

negative bacteria, J. Agric. Food Chem., 1998, 46: 3590-3595;

20. Hussain A. I., Anwar F., Sherazi S. T. H., Przybylski R. - Chemical composition. Antioxidant and

antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations.

Food Chemistry. 2008, 108: 986-995;

21. Jalas, J., Flora Europaea. Cambrige University Press. 1972, 3: 172-182.

22. Juven, B.J., Kanner, J., Schued, F., Weisslowicz, H., 1994. Factors that interact with the antibacterial

action of thyme essential oil and its active constituents. J. Appl. Bacteriol. 76, 626-63;

23. Kisgyörgy Z., K. Csedő, H. Hörster, I. Gergely and G. Rácz – The volatile oil of more important

indigenous Thymus species occurring in the composition of Serpylli herba. Rev. Medic. Tg. Mureş,

1984, 29, (1-2): 124-130;

24. Kosalec I., Pepeljnjak S., Kustrak D. – Antifungal activity of fluid extract and essential oil from

anise fruits (Pimpinella anisum L., Apiaceae), Acta Pharm., 2005, 55:377-385;

25. Kowal, T., Kuprinska, A., 1979. Antibacterial activity of the essential oils from Thymus pulegioides.

Herba Pol. 25, 303-310;

26. Knobloch K., Pauli A., Iberl B., Weis N., Weigand H. – Mode of action of essential oil components

on whole cell of bacteria and fungi in plate tests, In P. Schreier, Bioflavour, 1988, 87: 287-299;

27. Knobloch K., Pauli A., Iberl B., Weigand H., Weis N. – Antibacterial and antifungal properties of

essential oil components, J. Essen. Oil Res., 1989, 1:119-129;

28. Maissoneuve, S.A. - European Pharmacopoeia. vol 1, Sainte Ruffine, France, 1983;

29. Mojab F., Poursaeed M., Mehrgan H., Pakdaman S. – Antibacterial activity of Thymus daenensis

methanolic extract, Pak. Pharm. Sci., 2008, 21(3):210-213;

30. Marino, M., Bersani, C., Comi, G., 1999. Antimicrobial activity of the essential oils of Thymus

vulgaris L. measured using a bioimpedometric method. J. Food Prot. 62(9), 1017-23;

31. Marotti M., Piccaglia R., Giovanelli E. - Effects of variety and ontogenic stag on the essential oil

composition and biological activity of fennel (Foeniculum vulgare Mill.). Journal of Essential Oil

Research. 1994, 6: 57-62;

32. Morales R. – The history, botany and taxonomy of the genus Thymus, The genus Thymus, Ed. Taylor

and Francis, 2002, 1-44;

33. Nelson, R.R., 1997. In vitro activities of five plant essential oils against methicillin-resistant

Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. J. Antimicrob. Chemother.

40, 305-306;

34. Novgorodov S.A., Gudz T.I. – Permeability transition pore of the inner mitochondrial membrane can

operate in two open states with different selectivities, J. Bioenerg. Biomembr., 1996, 28: 139-146;

35. Nada V. Petrović, Slobodan S. Petrović, Mihailo S. Ristić, Ana M. Džamić, Ana D. Ćirić - Chemical

Composition and Antimicrobial Activity of Thymus praecox Opiz ssp. polytrichus Essential Oil from

Serbia, Rec. Nat. Prod., 2016, 10 (5), 633-638;

36. Petrovic´ N. V., Slobodan S. Petrovic´, Ana M. Dzami, Ana D. Ciri, Mihailo S. Ristic, Stoja L.

Milovanovic, Slobodan D. Petrovic - Chemical composition, antioxidant and antimicrobial activity of

22

Thymus praecox supercritical extracts, J. of Supercritical Fluids 2016, 110:117–125;

37. Pina-Vaz, C., Goncalves, R.A., Pinto, E., Costa-de-Oliveira, S., Tavares, C., Salgueiro, L., Cavaleiro,

C., Goncalves, M.J., Martinez-de-Oliveira J., Antifungal activity of Thymus oils and their major

compounds. J. Eur. Dermatol. Venereol. 2004, 18 (1), 73-78;

38. Rasooli I., Mirmostafa S.A. - Antibacterial proprieties of Thymus pubescens and Thymus serpyllum

essential oils, Fitoterapia, 2002, 73(3): 244-50;

39. Richardson P. M. - The chemistry of the Labiate: An introduction and overview. In-Advances in

Labiate Science, ed. Royal Botanic Gardens KEW, Whitstable, 1992, 291-297;

40. Rota C., Carraminana J.J., Burillo J., herrera A. – In vitro antimicrobial activity of essential oils from

aromatic plants against selected foodborne pathogeni, J. Food Prot, 2004, 67:1252-1256;

41. Sarker SD, Nahar L, Kumarasamy Y - Microtitre plate-based antibacterial assay incorporating

resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of

phytochemicals, Methods, 42(4):321-324, 2007;

42. Segvic Klaric M., Kosalec I., Mastelic J., Pieckova E., Pepeljnak S. - Antifungal activity of thyme

(Thymus vulgaris L.) essential oil and thymol against moulds from damp dwellings, Soc. Applied

Microbiol., 2007, 44: 36–42;

43. Sikkema J., De Bont J.A., Poolman B. – Interactions of cyclic hydrocarbons with biological

membrans, J. Biol. Chem., 1994, 269: 8022:8028;

44. Siegenthaler W., Luthy R. - Current chemotherapy, Proceeding 10th international congress of

chemotherapy, Washington DC, Am. Soc. Microbiol, 1978;

45. Skocibusic M., Bezic N., Dunkic V. - Phytochemical composition and antimicrobial activity of the

essential oils from Satureja subspicata Vis. Growing in Croatia. Food Chemistry. 2006, 96: 20-28;

46. Smithpalmer. A., Stewart. J., Fyfe. L., 1998. Antimicrobial properties of plant essential oils and

essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26, 118-122;

47. Soylu E. M., Soylu S., Kurt S. – Antimicrobial activity of the essential oil of varios plants against

tomato late blight disease agent Phytophthora infestans, Mycopathologia, 2006, 161: 119-129;

48. Souto-Bachiller F. A., De-Jesus-Echevarria M., Cardenas-Gonzalez O. E., Acuna-Rodriguez M. F.,

Melendez P. A., Romero-Ramsey L. - Terpenoid composition of Lippia dulcis. Phytochemistry,

1997; 44: 1077-1086;

49. Şerbănescu-Jitariu, G., Andrei, M., Mitroiu-Rădulescu, N.E. - Practicum de biologie vegetală. Edit.

Ceres, Bucureşti, 1983;

50. Terblanche F. C. - The characterization, utilization and manufacture of products recovered from

Lippia scaberrima Sond. PhD. thesis, Pretoria, University of Pretoria, 2000;

51. Turina A.V., Nolan M.V., Zygadlo J.A., Perillo M.A. – Natural terpenes: self-assembly and

membrane partitioning, Biophys. Chem., 2006, 122: 101-113;

52. Uribe-Hernandez C, Hurtado-Ramos J, Olmedo-Arcega E R, Martinez-Sosa MA - The essential oil

of Lippia graveolens H.B.K. from Jalisco, Mexico.Journal of Essential Oil Research. 1992, 4: 647-

649;

53. Van Vuuren S. F., Viljoen A. M., Ozek T., Demirici B., Baser K. H. C. - Seasonal and geographical

variation of Heteropyxis natalensis essential oil and the effect thereof on the antimicrobial activity.

South African Journal of Botany. 2007, 73, 3: 441-448;

54. Viljoen A. M., Petkar S., Van-Vuuren S. F., Cristina Figueiredo A., Pedro L. G., Barroso J. G. -

Chemo-Geographical Variation in Essential Oil Composition and the Antimicrobial Properties of

"Wild Mint" - Mentha longifolia subsp. polyadena (Lamiaceae) in Southern Africa. Journal of

Essential Oil Research. 2006, 18: 60-65;

55. Luca A. Vitali, Stefano Dall’Acqua, Filippo Maggi, Pavol Martonfi, Fabrizio Papa, Dezemona

Petrelli, Stefania Sut, Giulio Lupidi - Antimicrobial and antioxidant activity of the essential oil from

23

the Carpathian Thymus alternans Klokov, Natural Product Research,

http://dx.doi.org/10.1080/14786419.2016.1224874, 2016: 1-10;

56. Yatagai M, Makihara H, Oba K - Volatile compenents of Japanese cedar cultivars as repellents

related to resistance to Cryptomeria bark borer. J. Wood. Sci., 2002, 48: 51-55.

Director proiect,

Dr. Boz Irina