*3.6. Reprocessing Performance*

After being placed in a constant temperature and humidity box for 24 h, the cut dumbbell-shaped spline was used for the tensile test. Figure 8 provides the stress-strain curve of WPU2 film and WPU2 film after reprocessing. The tensile strength of WPU2 film after reprocessing was 3.39 MPa, compared with the original spline, its tensile strength increased and the elongation at break reduced, which indicated that the film had a good reprocessing performance.

**Figure 8.** Tensile properties of the reprocessed WPU2 film.


**3. Genetic resources of cotton — Content and location**

The details pertaining to all cotton genetic resource holdings and their locations, evaluations and conservation are presented below:

#### **3.1. Wild species and related stocks**

**2.2. Germplasm needs for India's cotton improvement**

21st century are as follows:

92 World Cotton Germplasm Resources

per hectare).

effects on fibre quality.

production of cotton at present.

Germplasm demand depends on various considerations and criteria. Primarily, it depends on the predominant species grown, genetic constitution of the cultivar / hybrid developed for large scale cultivation, the fibre quality requirements of the textile industry and export needs and incidentally various byproduct utility potential. The future attention of the breeders may be more towards the improvement of the *G. hirsutum* cotton because of the changed species composition involving more than 90 per cent area under cultivation of *G. hirsutum* cotton. Some special interest will be shown to the utilization of *G. barbadense* germplasm in cotton improve‐ ment because of the need to develop superior cultivars of *G. barbadense* with extra-long staple coupled with better adaptation to Indian conditions and also in the exploitation of the potential of the *G. hirsutum* x *G. barbadense* F1 commercial hybrids to develop ELS cotton hybrids with high yield potential. Further, the private seed companies holding a major stake in seed business have great role to play in meeting the needs for high quality seeds of superior varieties of the predominant cultivated species preferred by the farmers. The major breeding goals in cotton are based on conventional and changing cropping practices and industry requirements of the

Wide adaptation: agronomic and ecological stability including-Areas of cultivation and their agro-climatic conditions, soil fertility, temperature both day and night temperatures, rainfall characteristics (onset, duration, intensity and occurrence characteristics), adaptation and other factors (like adaptation to various abiotic stresses such as late sowings, droughts, excess rainfall

Response to advanced production technologies: this will include higher fertilizer use, higher planting densities, mechanization of cultivation practices with insect resistant cotton, chemical herbicide use for weed management and other practices like constraints management, manual and machine picking of cotton, double cropping patterns, continuous improvement in yield

Resistance to biotic and abiotic stresses: this includes resistance/tolerance to various kinds of pest and disease reactions and abiotic factors causing serious recurring yield losses and adverse

Cotton requirements for the textile industry: the total quantitative demands of national textile consumption of cotton, extra-factory consumption and export needs (including qualitative needs in terms of share of various staple types like short staple, medium staple, superior medium, long staple and extra-long staple and fibre quality parameters consistent with the desired levels for each of above staple categories besides for various spinning systems).

Improving the utility of cottonseeds: important aspects include elimination of gossypol, increased oil content and improved fatty acid profiles of the oil and protein in seed etc., to

Specialty cotton requirements: these are based on considerations for production of organic cotton and naturally colour-linted cottons with appreciable fibre quality parameters for spinning and weaving, although the market requirement is low at less than 1% of the global

improve the utility value of seed and its nutritional qualities for food and feed.

at critical phases of crop growth and major weather aberrations).

Cotton plant belongs to the family *Malvaceae* and genus *Gossypium* L., which comprises about 50 species, 45 of which are diploid (2n=2x=26) and the remaining 5 being allotetraploid (2n=4x=52) and whose geographical distribution spans the tropical and sub-tropical regions of the world [17]. The two 'A' genome diploid and two 'AD' genome allotetraploid species are cultivated for their lint fibre. The four species in cultivation have their botanical and geo‐ graphical races such as (*africanum, acerifolium, wightianum, persicum, and kuljianum under G. herbaceum; races indicum, bengalense, burmanicum, cernuum, sinense, and soudanense under G. arboreum; races latifolium, punctatum, morilli, yucatanense, marie-galante, palmeri and richmondii under G. hirsutum and races barbadense and brasiliense under G. barbadense* as described by Hutchinson, 1951 [3, 17] are available in distinct accessions of germplasm of cotton maintained in gene banks of CICR Nagpur and its regional station Coimbatore and certain other State Agricultural University Centres like UAS Dharwad, GAU Surat, TNAU etc. Intermediary germplasm-genetic stocks and breeding lines developed out of crosses between wild and cultivated species and between cultivated species have been integrated into the maintenance under germplasm for gene banks at CICR and State Agricultural University (SAU) centers and used for crop improvement programmes. The list of *Gossypium* species and those available in India is given in Table 2.

**3.2. Genetic resources of cultivated cottons**

maintained in India are presented in Table 3.

**Primary gene pool**

**Secondary gene pool**

The collections-accessions reportedly available mainly in the CICR Nagpur gene bank [or in its regional stations at Coimbatore especially for *G*. *barbadense* / Sirsa (limited to some diploid cotton accessions] have been described [23, 14, 24, 15]. The total cotton accessions available in India was reported as 7484 in *G. hirsutum*, 263 in *G. barbadense*, 1877 in *G. arboreum* and 530 in *G. herbaceum,* besides wild species and perennials [25]. Additional accessions of *G. hirsutum* and *G. barbadense* acquired after 2011 are to be documented after evaluation and added to the figures; similarly the germplasm additionally available after 2011 for the two diploid species of Asiatic cottons, *G. herbaceum* and *G. arboreum* also have to be incorporated in the total holdings after assessment and characterization. The details of the wild primary, secondary, and tertiary cotton (*Gossypium*) species and the cultivated tetraploid species maintained in several countries has been previously reported [26]. Similar materials of major gene pools including germplasm of the two cultivated tetraploid and the two cultivated diploid species

**Species Genome Collection maintained at CICR**

*G. tomentosum* Nuttal ex Seaman AD3 1 *G. darwinii* Watt AD5 1

*G. anomalum* Wawra B1 1 *G. triphyllum* Hochreutiner B2 1 *G. capitis viridis* Mauer B3 1 *G. barbosanum* Phillips & Clement B3? 1 *G. longicalyx* Hutchinson & Lee F1 1 *G. thurberi* Todaro D1 1 *G. trilobum* (DC) Skovsted D8 1 *G. davidsonii* kellogg D2-d 1 *G. klotschianum* Andersson D2-k 1 *G. armourianum* Kearney D2-1 2 *G. harknessii* Brandigee D2-2 1 *G. aridum* Skovsted D4 1 *G. lobatum* H Gentry D7 1

*G. herbaceum* L. A1 given below *G. arboreum* L. A2 given below

**Nagpur**

Cotton Germplasm in India — New Trends http://dx.doi.org/10.5772/58622 95

**Table 2.** Species of *Gossypium* and their availability in India (in cultivation or in Species Gardens):-Taxonomic classification of *Gossypium* L. species [18-22].

#### **3.2. Genetic resources of cultivated cottons**

**Genome Species of** *Gossypium* **(if available**

94 World Cotton Germplasm Resources

D3-k *klotzschianum* Andersson Yes D4 *aridum* Skovsted Yes D5 *raimondii* Ulbrecht Yes

E1 *stocksii* Masters Yes Note:

E2 *somalense* Hutchinson Yes

F1 *longicalyx* Hutchinson & Lee Yes G *australe* Mueller Yes

G1 *bickii* Prokhanov Yes

classification of *Gossypium* L. species [18-22].

E3 *areysianum* Deflers E4 *incanum* Hillcoat E? *ellenbickii* Mauer E? *bricchettii* Vollesen E? *benadirense* Mattei

G *nelsonii* Fryxell

**'Yes')**

C2 *robinsonii* Mueller *rotundifolium* Fryxell D1 *thurbei* Todaro Yes *enthyle* Fryxell

D2-1 *armourianum* Kearney Yes *londonderriense* Fryxell D2-2 *harknessii* Brandigee Yes *marchantii* Fryxell D3-d *davidsonii* Kellog Yes *anapoides* Stewart

D6 *gossypioides* Standley Yes **Allotetraploids 2n = 4x = 52**

D7 *lobatum* Gentry Yes (AD)1 *hirsutum* Linn. Cultivation D8 *trilobum* Skovsted Yes (AD)2 *barbadense* Linn. cultivation

D9 *laxum* Phillips (AD)3 *tomentosum* Nuttal Yes

D11 *schwendimanii* Fryxell (AD)5 *darwinii* Watt. yes

**Table 2.** Species of *Gossypium* and their availability in India (in cultivation or in Species Gardens):-Taxonomic

D10 *turneri* Fryxell (AD)4 *mustelinum* Masters

**Diploid Species 2n = 2x = 26 Diploid Species 2n = 2x = 26** ? *trifurcatum* Vollesen *pilosum* Fryxell C1 *sturtianum* Willis Yes *exiguum* Fryxell C1-n *nandewarense* Dereda Yes *nobile* Fryxell

**Genome Species of** *Gossypium* **(if available**

In addition, 82 perennials, 141 land races *(G. arboreum and G. barbadense), 20* distinct *cernuum* race collections with big boll weight , up to 6.4g and 44.4% GOT are available; also 40 synthetic polyploids, 20 male sterile lines from Israel etc., GMS, TGMS and EGMS lines are also maintained at CICR.

**'Yes')**

The collections-accessions reportedly available mainly in the CICR Nagpur gene bank [or in its regional stations at Coimbatore especially for *G*. *barbadense* / Sirsa (limited to some diploid cotton accessions] have been described [23, 14, 24, 15]. The total cotton accessions available in India was reported as 7484 in *G. hirsutum*, 263 in *G. barbadense*, 1877 in *G. arboreum* and 530 in *G. herbaceum,* besides wild species and perennials [25]. Additional accessions of *G. hirsutum* and *G. barbadense* acquired after 2011 are to be documented after evaluation and added to the figures; similarly the germplasm additionally available after 2011 for the two diploid species of Asiatic cottons, *G. herbaceum* and *G. arboreum* also have to be incorporated in the total holdings after assessment and characterization. The details of the wild primary, secondary, and tertiary cotton (*Gossypium*) species and the cultivated tetraploid species maintained in several countries has been previously reported [26]. Similar materials of major gene pools including germplasm of the two cultivated tetraploid and the two cultivated diploid species maintained in India are presented in Table 3.

the Malwa plateau in Central India representing distinct agro-climate, apart from perennial cottons grown as backyard cottons for house-hold use. Attributes of breeders' interest in the collections included closed boll types in *G. herbaceum* with storm proof nature, big bolls with >6g boll weight, high ginning and high seed number per boll in *G. arboreum* and cold resistance

in *G. hirsutum.* The details of the surveys and collections made are given in Table 4.

1977 CICR/NBPGR ICAR Assam and Meghalaya *Cernuum* types of *G.*

1978 CICR/GPAC ICAR Tinnies tract of Tamil Nadu Diploid cultivated

Pradesh

1984 CICR/NBPGR ICAR Assam and Manipur *G. arboreum* race

1987 NBPGR/CICR ICAR Assam regions *Cernuum* types of *G.*

**Table 4.** Surveys and collections made by the CICR/NBPGR and their description

**4. Funding sources for cotton germplasm activities**

1981 CICR ICAR Malwa plateau of Madhya

ICAR Saurashtra region in Gujarat

and Raichur area in Karnataka

1986 NBPGR ICAR Gujarat western tract *Desi* cotton material Resistance to moisture stress,

The Indian Council of Agricultural Research (ICAR), New Delhi is the main funding agency for all the germplasm related activities in cotton and the ICAR is an autonomous body under the Ministry of Agriculture and Department of Agricultural Research and Education (DARE) of the Government of India. The Central Institute for Cotton Research (CICR) Nagpur, the National Bureau of Plant Genetic Resources (NBPGR) New Delhi and also Central Institute for Research on Cotton Technology (CIRCOT), Mumbai are national level Institutes under the ICAR to take care of all cotton research related activities including germplasm related matters. The ICAR may seek collaboration with the FAO in germplasm exploration and collection activities as and when such activities are planned. The responsibility for cotton germplasm is mainly vested with the CICR. The CICR has played a pivotal role in germplasm acquisition, maintenance, evaluation and promoting utilization in breeding and other related researches. India is probably the third richest country in general and in certain respects probably the richest in the world in its total holdings and diversity of cotton genetic resources including wild species, cultivated species and even certain perennial genotypes of cotton [15]. The National

1979 CICR/

GPAC/UAS/GAU

**Year Organization Funded by Regions of expedition Material collected Characters observed**

*arboreum*

variants

*cernuum*

*herbaceum*

*G. herbaceum* types and variants

High ginning, big long bolls, loculi retention after

Introgressed types of early

Closed boll types, round boll,

High seed number per boll, long big bolls, high ginning

bursting

*G. hirsutum* Local variants, cold

introductions

Cotton Germplasm in India — New Trends http://dx.doi.org/10.5772/58622 97

resistance

types

storm proof types

high yield potential

Big boll, higher boll numbers, abiotic resistance

**Table 3.** Description of the wild primary, secondary, and tertiary cotton (*Gossypium*) species and the germplasm of cultivated cottons of the two cultivated tetraploid and two cultivated diploid cotton species maintained in India

#### **3.3. Germplasm exploration and collection**

The exploration of certain regions in India was carried out by the Germplasm Advisory Committee (GPAC) involving the CICR and the NBPGR in seven expeditions. The areas represented formerly predominant areas of Asiatic cotton cultivation in Southern and Central India, NE Hill regions with *cernuum* types of *G. arboreum* and some *G. hirsutum* cotton under the Malwa plateau in Central India representing distinct agro-climate, apart from perennial cottons grown as backyard cottons for house-hold use. Attributes of breeders' interest in the collections included closed boll types in *G. herbaceum* with storm proof nature, big bolls with >6g boll weight, high ginning and high seed number per boll in *G. arboreum* and cold resistance in *G. hirsutum.* The details of the surveys and collections made are given in Table 4.

**Table 4.** Surveys and collections made by the CICR/NBPGR and their description

**Species Genome Collection maintained at CICR**

*G. gossypioides* (Ulbrich) Standley D5 1 *G. raimondii* Ulbrich D6 1

*G. sturtianum* J.H Wills C1 1 *G. stocksii* Master E1 1 *G. somalense* Hutchinson E2 1 *G. australe* F. Mueller G 2 *G. bickii* Prokanov G1 1

Germplasm 7484 Cultivars 110 Land races/wild stocks 7 Cytogenetic stocks/mutants 32/1 Total 7633

Germplasm 530 Cultivars 3 Land races / wild stocks 1/0 Total 534

Germplasm and all other accessions 1877

Germplasm and all other accessions 530

**Table 3.** Description of the wild primary, secondary, and tertiary cotton (*Gossypium*) species and the germplasm of cultivated cottons of the two cultivated tetraploid and two cultivated diploid cotton species maintained in India

The exploration of certain regions in India was carried out by the Germplasm Advisory Committee (GPAC) involving the CICR and the NBPGR in seven expeditions. The areas represented formerly predominant areas of Asiatic cotton cultivation in Southern and Central India, NE Hill regions with *cernuum* types of *G. arboreum* and some *G. hirsutum* cotton under

**Tertiary gene pool**

96 World Cotton Germplasm Resources

**Cultivated tetraploid species**

**Cultivated diploid species**

*G. arboreum* L.

*G. herbaceum* L.

*G. hirsutum* L. AD1

*G. barbadense* L. AD2

**3.3. Germplasm exploration and collection**

**Nagpur**

## **4. Funding sources for cotton germplasm activities**

The Indian Council of Agricultural Research (ICAR), New Delhi is the main funding agency for all the germplasm related activities in cotton and the ICAR is an autonomous body under the Ministry of Agriculture and Department of Agricultural Research and Education (DARE) of the Government of India. The Central Institute for Cotton Research (CICR) Nagpur, the National Bureau of Plant Genetic Resources (NBPGR) New Delhi and also Central Institute for Research on Cotton Technology (CIRCOT), Mumbai are national level Institutes under the ICAR to take care of all cotton research related activities including germplasm related matters. The ICAR may seek collaboration with the FAO in germplasm exploration and collection activities as and when such activities are planned. The responsibility for cotton germplasm is mainly vested with the CICR. The CICR has played a pivotal role in germplasm acquisition, maintenance, evaluation and promoting utilization in breeding and other related researches.

India is probably the third richest country in general and in certain respects probably the richest in the world in its total holdings and diversity of cotton genetic resources including wild species, cultivated species and even certain perennial genotypes of cotton [15]. The National Gene Bank of Cotton in India is located at the Central Institute for Cotton Research, Nagpur including its regional station at Coimbatore. The total collection represents almost entirely cultivated accessions of *G. hirsutum, G. barbadense, G. arboreum,* and *G. herbaceum*. In addition, the collection also consists of race stock accessions of each cultivated species, 26 wild species, and 32 synthetic introgressed derivatives.

Common and standard procedures were also provided for recording observations with comparable uniformity over locations. Provision was made for recording data on all quality attributes of fibre (technological characters) as well as cottonseed and biomass, harvest index

Cotton Germplasm in India — New Trends http://dx.doi.org/10.5772/58622 99

**Figure 2.** Germplasm Index Card (GIC) designed by CICR Nagpur for recording data on cotton germplasm

Nuclear Magnetic Resonance (NMR) and other instruments [23].

The Germplasm Index Card designed by the CICR was used as the basis for germplasm cataloguing. The Catalogue of Cotton Genetic Resources was compiled based on germplasm data gathered all over the country, particularly at CICR Nagpur, Maharashtra state (Central zone) and its regional centres at Coimbatore in Tamil Nadu (Southern zone) and Sirsa in Haryana state (Northern zone) representing all the three major cotton growing zones of the country. Data for special applications were also recorded by the departments of Pathology, Entomology, Soil Science Agronomy, Physiology, Biochemistry etc. Fibre quality data were generated by the Central Institute for Research on Cotton Technology (CIRCOT), Mumbai and its regional units and that for oil content and seed oil index were obtained at the CICR by using

Close collaboration for data recording and evaluation was ensured between the CICR, the CIRCOT, Mumbai, the National Bureau of Plant Genetic Resources (NBPGR) New Delhi, Indian Agricultural Research Institute (IARI), New Delhi and all the State Agricultural Universities (SAUs) in cotton growing states of the country as partners with the CICR as

**6.2. Cotton genetic resources catalogue**

etc. (Figure 2).

High level of coordination is being achieved in germplasm exchange and utilization with the world level International Institute for Plant Genetic Resources and national level institutions (NBPGR, CICR, CIRCOT and SAUs), The availability of equipment and techniques for mass screening for several parameters in the laboratory, in the fields and glass houses, has made possible to generate precise data in various environments and enabled to have a new-look and re-look at the germplasm for various breeding and gene deployment strategies. It also helped to develop varieties and hybrids for meeting the demands of the farmers, the textiles industry for various end-uses and to overcome inter-fibre competition based on consumer preferences and the cottonseed utilization and biomass utilization industries. Now more information has been accumulated to improve cottonseed yield and quality profile useful for agro-based industrial exploitation. This is to meet human needs for edible oil, protein and cellulose to contribute to enhanced food and nutritional security and also develop various by-products for use as animal feed and further processing in ancillary industries [27, 10].

#### **5. Sharing of germplasm resources**

Sharing of germplasm may take place as per Government of India policies and based on intercountry government protocols and exchange programmes on mutual basis of agreements, if any. Within the country, the germplasm is readily available for public sector research institu‐ tions carrying out research on cotton and related activities.


*Article* **Antifungal Activity of** *Lavandula angustifolia* **Essential Oil against** *Candida albicans***: Time-Kill Study on Pediatric Sputum Isolates**

**Stefan Mijatovic 1 , Jelena Antic Stankovic 2,\*, Ivana Colovic Calovski 1 , Eleonora Dubljanin 1 , Dejan Pljevljakusic 3 , Dubravka Bigovic 3 and Aleksandar Dzamic 1**

**Abstract:** The aim of our study was to determine the susceptibility of 15 *Candida albicans* sputum isolates on fluconazole and caspofungin, as well as the antifungal potential of *Lavandula angustifolia* essential oil (LAEO). The commercial LAEO was analyzed using gas chromatography-mass spectrometry. The antifungal activity was evaluated using EUCAST protocol. A killing assay was performed to evaluate kinetics of 2% LAEO within 30 min treatment. LAEO with major constituents' linalool (33.4%) and linalyl acetate (30.5%) effective inhibited grows of *C*. *albicans* in concentration range 0.5–2%. Fluconazole activity was noted in 67% of the isolates with MICs in range 0.06–1 µg/mL. Surprisingly, 40% of isolates were non-wild-type (non-WT), while MICs for WT ranged between 0.125–0.25 µg/mL. There were no significant differences in the LAEO MICs among fluconazoleresistant and fluconazole-susceptible sputum strains (*p* = 0.31) and neither among caspofungin non-WT and WT isolates (*p* = 0.79). The 2% LAEO rapidly achieved 50% growth reduction in all tested strains between 0.2 and 3.5 min. Within 30 min, the same LAEO concentration exhibited a 99.9% reduction in 27% isolates. This study demonstrated that 2% solution of LAEO showed a significant antifungal activity which is equally effective against fluconazole and caspofungin susceptible and less-susceptible strains.

**Keywords:** *Candida albicans*; *Lavandula angustifolia*; essential oil; fluconazole; caspofungin; time-kill curve

### **1. Introduction**

Yeasts of the genus *Candida*, as members of the skin and mucosal microbiota, can cause opportunistic fungal infections, both superficial and invasive. For hospitalized pediatric patients, *Candida* is a significant pathogen identified in the setting of bloodstream infections. Children with acute myeloid leukemia (AML), hematopoietic stem cell transplant (HSCT) recipients and patients admitted to intensive care units (ICU) have the highest risk of invasive candidiasis development [1]. With the risky patients, abnormal *Candida* colonization correlates with a higher probability of its invasive infection [2]. According to the origin, the mentioned mycoses are endogenous, so the damage and the increased permeability of the mucous membranes as consequences of the underlying disease, cytostatic or radiation therapy are the entry points for these yeasts. Studies of the pediatric candidiasis showed that *Candida albicans* was the most common isolated species, but collectively *Candida parapsilosis*, *Candida glabrata*, *Candida lusitaniae* and *Candida krusei* predominated [3,4].

Prophylactic and pre-emptive therapies with high-risk patients are very important in prevention of invasive candidiasis. As drugs with low toxicity, azoles and echinocandins are widely used in prevention and treatment of the fore mentioned mycoses [5]. The

**Citation:** Mijatovic, S.; Stankovic, J.A.; Calovski, I.C.; Dubljanin, E.; Pljevljakusic, D.; Bigovic, D.; Dzamic, A. Antifungal Activity of *Lavandula angustifolia* Essential Oil against *Candida albicans*: Time-Kill Study on Pediatric Sputum Isolates. *Molecules* **2022**, *27*, 6300. https://doi.org/ 10.3390/molecules27196300

Academic Editors: Raffaele Pezzani and Sara Vitalini

Received: 24 August 2022 Accepted: 20 September 2022 Published: 24 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

introduction of echinocandins into standard protocols alone or in combination with other antifungals has greatly improved the clinical outcomes. Although with lower incidence of adverse events than other antifungals, echinocandins are only available as intravenous formulations [6]. *Candida* resistance to echinocandins has emerged and it is most commonly associated with *C. glabrata* [7]. On the other side, azoles have good tolerability, but a narrow activity spectrum. *C. krusei* is intrinsically resistant to fluconazole whereas *C. glabrata* is highly resistant to the mentioned antifungal [8]. However, the increasing resistance of *C. albicans* and non-albicans species to these antifungals, recurrent infections and persistent colonization require a different strategy in prophylaxis and in treatment of the described disease.

Traditionally, essential oils (EOs) and extracts of aromatic plants have been used in the treatments of wound infections and burns [9,10]. Some EOs in certain cultures are used in aromatherapy as a relaxant and as a carminative and a sedative agent [11]. In addition, dried parts of plants are used as repellents for various arthropods [12]. However, in vitro and clinical studies show different and often conflicting results regarding the efficacy of EOs in human medicine. So far, the antibacterial and antifungal effects of aromatic plants' extracts and EOs have been investigated. EOs of oregano, thyme and lavender, cloves, cinnamon and tea tree showed broad and potent anti *Candida* activity in in vitro studies [13–15]. It is assumed that EOs interfere with fungal membranes, but are distinct from currently available polyenes [16]. Furthermore, inhibition of cell wall polysaccharide synthesis could be another method for EOs antifungal activity [17]. EOs show in vitro activity against *Candida* azole-resistant strains, implicating a different mode of action [18]. It is certain that EOs have an antifungal effect, but there are some limitations. In a laboratory, determinations of effective antimicrobial concentration are difficult to compare because their outcome is influenced primarily by the chemical composition of EOs and methodologies of research. Lipophilic character makes them potentially toxic, especially if used at high concentration and should not be administrated orally and parenterally. Due to their broad-spectrum antimicrobial activity, EOs and their components would be very interesting solution for improving prophylactic and therapeutic failures related to the emergence of resistant strains. Aromatherapy could be an additional method for the prevention of massive upper respiratory tract colonization and the consequent bloodstream, but also some systemic *Candida* infections.

The aim of our study was to determine the susceptibility of *Candida albicans* sputum isolates to *Lavandula angustifolia* essential oil (LAEO) as well as to determine its fungicidal potential on tested cultures in a liquid phase. In addition, we compared the antifungal activity of LAEO to a selected azole (fluconazole) and a representative echinocandin (caspofungin).

### **2. Results**

### *2.1. Microbial Identification*

Out of the 15 analyzed *Candida* isolates, 10 strains were recognized as *C. albicans* using chromogenic media. Additional five isolates were identified by matrix-assisted laser desorption in ionization-time of flight mass spectrometry (MALDI-TOF MS). Peptide mass fingerprinting of these isolates as well as *C. albicans* ATCC 10259 performed by MALDI-TOF MS are shown in Figure 1.

**Figure 1.** MALDI-TOF mass spectra (*m*/*z* 3000 to 15,000) of: (**a**) *C. albicans* PED02; (**b**) *C. albicans* PED06; (**c**) *C. albicans* PED12; (**d**) *C. albicans* PED13; (**e**) *C. albicans* PED15; (**f**) *C. albicans* ATCC 10259. **Figure 1.** MALDI-TOF mass spectra (*m*/*z* 3000 to 15,000) of: (**a**) *C. albicans* PED02; (**b**) *C. albicans* PED06; (**c**) *C. albicans* PED12; (**d**) *C. albicans* PED13; (**e**) *C. albicans* PED15; (**f**) *C. albicans* ATCC 10259.

### *2.2. Chemical Composition of Essential Oil 2.2. Chemical Composition of Essential Oil*

A total of 50 compounds were detected in LAEO (Figure 2), of which 30 were identified (97.4% of whole composition) (Table 1). These constituents could be classified in six different groups: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpene hydrocarbons and oxygenated diterpenes. In Table 1, the 10 main components determined by European A total of 50 compounds were detected in LAEO (Figure 2), of which 30 were identified (97.4% of whole composition) (Table 1). These constituents could be classified in six different groups: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpene hydrocarbons and oxygenated diterpenes. In Table 1, the 10 main components determined by European Pharmacopoeia

representing 84.6% of the whole LAEO composition are bolded [19]. The main constituents of the LAEO were linalool and linalyl acetate, constituting 33.4% and 30.5%, respectively. The contribution of 1,8-cineole (5.0%) and camphor (4.6%) deviated from the ranges determined by European Pharmacopoeia and the ISO 3515 standard, therefore this LAEO can be considered an essential oil of non-standard quality.

**Figure 2.** Chromatogram of *L. angustifolia* essential oil showing the separation of 50 chemical components.

**Table 1.** Composition of the *L. angustifolia* essential oil used in this study.

1 European Pharmacopoeia. 2 *L. angustifolia* essential oil.

### *2.3. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)*

The MICs of LAEO that effectively inhibited grows of *C*. *albicans* sputum strains ranged from 0.5 to 2% (mean MIC 1.17%; MIC50 1%; MIC90 2%) (Table 2). LAEO MFCs for the tested isolates ranged between 1–4% (mean MFC 1.93%; MFC50 2%; MFC90 2%). The MIC and MFC of LAEO for *C. albicans* ATCC 10259 were in the range observed among clinical isolates (MIC 2%; MFC 2%).

1 LAEO = *L. angustifolia* essential oil; 2 MIC = minimum inhibitory concentration (values expressed in %vol./vol.); MIC for fluconazole and caspofunin are expressed in µg/mL; 3 MFC = minimum fungicidal concentration (values expressed in %vol./vol.); MFC for fluconazole and caspofunin are expressed in µg/mL.

Five clinical strains (*C. albicans* PED01, PED03, PED07, PED08 and PED09) were resistant to fluconazole with MICs 64 µg/mL, whereas MICs for susceptible isolates ranged from 0.06 to 1 µg/mL (mean MIC 21.57 µg/mL; MIC50 0.5 µg/mL; MIC90 64 µg/mL). Fluconazole MFC50 and MFC90 were >64 µg/mL. Six sputum isolates (*C*. *albicans* PED03, PED07, PED08, PED09, PED12 and PED15) were categorized as caspofungin non-WT strains with MICs > 0.25 µg/mL, while MIC for WT isolates ranged between 0.125–0.25 µg/mL (mean MIC 0.57 µg/mL; MIC50 0.25 µg/mL; MIC90 0.5 µg/mL). Caspofungin concentration 2 µg/mL completely kills 50% and 90% of tested isolates. In addition, there were no significant differences in the LAEO MICs among fluconazole-resistant (median 1%) and fluconazole-susceptible (median 1%) sputum strains (Mann–Whitney U test; *p* = 0.31). In addition, no LAEO MICs differences were observed among caspofungin non-WT (median 1%) and WT (median 1%) isolates (Mann–Whitney U test; *p* = 0.79).

### *2.4. Time-Kill Assay*

The time-kill curves of the 2% LAEO against the 16 *Candida* strains are illustrated in Figure 3. Time intervals to reach 50%, 90% and 99.9% reduction in the number of yeast cells from the starting inoculums for each strain are summarized in Table 3. After 30 min exposure to LAEO in 4 strains, the cell count was reduced ≥3.0 log10 CFU/mL in range from 3.0 to 3.2 log10 CFU/mL, whereas in the remaining 12 strains, the reduction ranged only 0.47 to 2.47 log10 CFU/mL. This implicates that the mentioned concentration of LAEO showed more fungistatic than fungicidal activity during 30 min exposure.

*2.4. Time-Kill Assay*

The time-kill curves of the 2% LAEO against the 16 *Candida* strains are illustrated in Figure 3. Time intervals to reach 50%, 90% and 99.9% reduction in the number of yeast cells from the starting inoculums for each strain are summarized in Table 3. After 30 min exposure to LAEO in 4 strains, the cell count was reduced ≥ 3.0 log10 CFU/mL in range from 3.0 to 3.2 log10 CFU/mL, whereas in the remaining 12 strains, the reduction ranged

showed more fungistatic than fungicidal activity during 30 min exposure.

**Figure 3.** Time-kill of *C. albicans* sputum strains on different times (0–30 min): (**a**) untreated (grow controls) of *C. albicans* (PED01–PED15) and *C. albicans* ATCC 10259; (**b**) *C. albicans* isolates (PED01– PED15) and *C. albicans* ATCC 10259 treated with 2% concentration of lavender essential oil (LAEO); Within 30 min exposure in 4 strains (PED01, PED03, PED05, PED06) cell count was reduced ≥ 3.0 log10 CFU/mL, where in rest 12 strains reduction ranged from 0.47 log10 CFU/mL (PED04) to 2.47 log10 CFU/mL (PED08). **Figure 3.** Time-kill of *C. albicans* sputum strains on different times (0–30 min): (**a**) untreated (grow controls) of *C. albicans* (PED01–PED15) and *C. albicans* ATCC 10259; (**b**) *C. albicans* isolates (PED01–PED15) and *C. albicans* ATCC 10259 treated with 2% concentration of lavender essential oil (LAEO); Within 30 min exposure in 4 strains (PED01, PED03, PED05, PED06) cell count was reduced ≥ 3.0 log10 CFU/mL, where in rest 12 strains reduction ranged from 0.47 log10 CFU/mL (PED04) to 2.47 log10 CFU/mL (PED08).

> A 50% growth reduction for all sputum strains was rapidly achieved between 0.2 and 3.5 min (approximately 0.83 min). In addition, the time points required to reach 90% and 99.9% were more variable among the tested strains. In fact, within 30 min of exposure to LAEO, 90% reduction was observed with nine strains (*C*. *albicans* PED01, PED03, PED05, PED06, PED07, PED08, PED09, PED13 and PED14) in time range 30 s–18 min. LAEO exhibited 99.9% reduction in four strains (*C*. *albicans* PED01, PED03, PED05 and PED06) between 1.8 and 29.7 min. For other isolates, together with *C. albicans* ATCC 10259 no reduction was achieved in the CFU of 90% and 99.9% during 30 min of LAEO exposure.

> The number of *C*. *albicans* cells significantly declined over time in all LAEO treated sputum strains (Friedman test; *p* < 0.05) (Figure 4). Pairwise comparisons using Wilcoxon signed rank test for related samples showed significant differences in all measurement times except 5 min–15 min (*p* = 0.14) and 15 min–30 min (*p* = 0.06) (Figure 4).

**Table 3.** Times for 2% *L. angustifolia* essential oil to achieve 50%, 90% and 99.9% reductions in growth of starting inoculum during 30 min treatment. *C. albicans* PED15 0.5 > 30 > 30 *C. albicans* ATCC 10259 0.8 > 30 > 30

*Molecules* **2022**, *27*, x FOR PEER REVIEW 7 of 14

**Strains 2% Solution of LAEO 1**

*C. albicans* PED01 0.2 0.7 5 *C. albicans* PED02 1.4 > 30 > 30 *C. albicans* PED03 1.1 2 29.7 *C. albicans* PED04 3.5 > 30 > 30 *C. albicans* PED05 0.2 0.6 2 *C. albicans* PED06 0.2 0.5 1.8 *C. albicans* PED07 0.4 3.3 > 30 *C. albicans* PED08 0.2 0.7 > 30 *C. albicans* PED09 1.1 15.2 > 30 *C. albicans* PED10 1.3 > 30 > 30 *C. albicans* PED11 0.6 > 30 > 30 *C. albicans* PED12 0.6 > 30 > 30 *C. albicans* PED13 0.7 18 > 30 *C. albicans* PED14 0.4 3.1 > 30

of starting inoculum during 30 min treatment.

**Table 3.** Times for 2% *L. angustifolia* essential oil to achieve 50%, 90% and 99.9% reductions in growth

**50% Reduction 2 90% Reduction 99.9% Reduction**

1 LAEO = *L. angustifolia* essential oil; 2 Values are expressed in minutes.

**Figure 4.** Box plot of cell counts (log10 CFU/mL) of 15 *C. albicans* sputum strains noted from preexposure (0 min) and within 30 min exposure to 2% lavender essential oil (LAEO). Friedman test **Figure 4.** Box plot of cell counts (log10 CFU/mL) of 15 *C. albicans* sputum strains noted from preexposure (0 min) and within 30 min exposure to 2% lavender essential oil (LAEO). Friedman test with post hoc pairwise comparison with applied Bonferroni correction showed significant differences among all pairs (\* *p* < 0.05) except 5 min–15 min and 15 min–30 min.

Among sputum strains 2% LAEO was reducing the cell counts rapidly after 1 min by approximately 0.49 log10 CFU/mL (68%) (Figure 5). This activity remained, reducing cell viability by approximately 0.73 log10 CFU/mL (81%), 0.85 log10 CFU/mL (86%), 0.91 log10 CFU/mL (87%) and 1.03 log10 CFU/mL (91%) within 2, 5, 15 and 30 min, respectively (Figure 5). In *C. albicans* ATCC 10259 cells reduction calculated at 2, 5, 15 and 30 min were 0.41 log10 CFU/mL (60%), 0.48 log10 CFU/mL (67%), 0.77 log10 CFU/mL (83%), 0.77 log10 CFU/mL (83%) and 0.91 log10 CFU/mL (88%), respectively (Figure 5).

**Figure 5.** Bar chart of mean (±SD) reduction in log10 CFU/mL levels of 15 *C. albicans* sputum isolates as well as *C. albicans* ATCC 10259 from 1 to 30 min exposure to 2% of *L. angustifolia* essential oil. Within 1 min treatment mean cell reduction in sputum strains was 0.49 log10 CFU/mL (68%), while in 30 min their cell viability mean reduced 1.03 log10 CFU/mL (91%). In *C. albicans* ATCC 10259 cell count reduction ranged from 0.41 log10 CFU/mL (61%) to 0.91 log10 CFU/mL (88%) calculated in 1 min and 30 min, respectively. **Figure 5.** Bar chart of mean (±SD) reduction in log10 CFU/mL levels of 15 *C. albicans* sputum isolates as well as *C. albicans* ATCC 10259 from 1 to 30 min exposure to 2% of *L. angustifolia* essential oil. Within 1 min treatment mean cell reduction in sputum strains was 0.49 log10 CFU/mL (68%), while in 30 min their cell viability mean reduced 1.03 log10 CFU/mL (91%). In *C. albicans* ATCC 10259 cell count reduction ranged from 0.41 log10 CFU/mL (61%) to 0.91 log10 CFU/mL (88%) calculated in 1 min and 30 min, respectively.

with post hoc pairwise comparison with applied Bonferroni correction showed significant differ-

Among sputum strains 2% LAEO was reducing the cell counts rapidly after 1 min by approximately 0.49 log10 CFU/mL (68%) (Figure 5). This activity remained, reducing cell viability by approximately 0.73 log10 CFU/mL (81%), 0.85 log10 CFU/mL (86%), 0.91 log10 CFU/mL (87%) and 1.03 log10 CFU/mL (91%) within 2, 5, 15 and 30 min, respectively (Figure 5). In *C. albicans* ATCC 10259 cells reduction calculated at 2, 5, 15 and 30 min were 0.41 log10 CFU/mL (60%), 0.48 log10 CFU/mL (67%), 0.77 log10 CFU/mL (83%), 0.77 log10

ences among all pairs (\* *p* < 0.05) except 5 min–15 min and 15 min–30 min.

CFU/mL (83%) and 0.91 log10 CFU/mL (88%), respectively (Figure 5).

### **3. Discussion**

**3. Discussion**  As natural aromatic plant products, EOs are mainly composed of terpens and terpenoids. LAEO is present with approximately 1.3% in the flowering aerial part of the plant and contains more than 100 compounds [20]. It was shown that the LAEO and its main constituents can kill or inhibit the growth of fungi [21]. The main components of this LAEO that exhibit fungistatic and fungicidal effects are monoterpene alcohols (60–65%), such as linalool (20–50% per fraction) and linalyl acetate (25–46% per fraction) [20]. The other components are cis-ocimene, terpinen4-ol, limonene, cineol, camphor, lavandulyl acetate, lavandulol and α-terpineol, β-caryophyllene, geraniol, α-pinene. Non-terpenic aliphatic components such as 3-octanon, 1-octen-3-ol, 1-octen-3-y-lacetate, 3-octanol are present as well [22]. They are determined based on the chemical compositions LAEO, but EOs components could differ among other *Lavandula* spp. [23]. A chemical analysis of *L. binaludensis* EO showed that γ-terpinen with participation of 71.6% is its major content [24]. Zuzarte et al. showed that the main component of *L. viridis* EO was 1,8-cineole with participation of 34.3–42.2% [25]. Out of 10 lavender EO components shown in Table 1, 8 As natural aromatic plant products, EOs are mainly composed of terpens and terpenoids. LAEO is present with approximately 1.3% in the flowering aerial part of the plant and contains more than 100 compounds [20]. It was shown that the LAEO and its main constituents can kill or inhibit the growth of fungi [21]. The main components of this LAEO that exhibit fungistatic and fungicidal effects are monoterpene alcohols (60–65%), such as linalool (20–50% per fraction) and linalyl acetate (25–46% per fraction) [20]. The other components are cis-ocimene, terpinen4-ol, limonene, cineol, camphor, lavandulyl acetate, lavandulol and α-terpineol, β-caryophyllene, geraniol, α-pinene. Non-terpenic aliphatic components such as 3-octanon, 1-octen-3-ol, 1-octen-3-y-lacetate, 3-octanol are present as well [22]. They are determined based on the chemical compositions LAEO, but EOs components could differ among other *Lavandula* spp. [23]. A chemical analysis of *L. binaludensis* EO showed that γ-terpinen with participation of 71.6% is its major content [24]. Zuzarte et al. showed that the main component of *L. viridis* EO was 1,8-cineole with participation of 34.3–42.2% [25]. Out of 10 lavender EO components shown in Table 1, 8 are in range classified by the European Pharmacopoeia [19]. Two components of 1,8-cineole and camphor were in higher percentage (5.0% and 4.6%, respectively). These deviations in EOs' composition sometimes reflect non-uniformity in the bioactivity results and they could be consequences of climatic, geographical, seasonal and geological factors, physiological characteristics of plants and extraction procedures.

are in range classified by the European Pharmacopoeia [19]. Two components of 1,8-cineole and camphor were in higher percentage (5.0% and 4.6%, respectively). These deviations in EOs' composition sometimes reflect non-uniformity in the bioactivity results and they could be consequences of climatic, geographical, seasonal and geological factors, physiological characteristics of plants and extraction procedures. The action mechanism of essential oils remains somewhat controversial. An exceptional antifungal activity of EOs is probably based on their lypophilic nature which typically integrates into membrane structures thus increasing the cell permeability and leaching of intracellular membranes [26]. It has been noticed that among lavender EOs, the component α-pinene shows the best antifungal effect [25]. Other components such as The action mechanism of essential oils remains somewhat controversial. An exceptional antifungal activity of EOs is probably based on their lypophilic nature which typically integrates into membrane structures thus increasing the cell permeability and leaching of intracellular membranes [26]. It has been noticed that among lavender EOs, the component α-pinene shows the best antifungal effect [25]. Other components such as linalool 1,8-cineol and linalyl acetate are also potent, contributing to the overall antifungal activity of [27,28].  As an acyclic monoterpene alcohol, linalool differs from most of the monoterpenes that exhibit significant antifungal activity. It is reasonable to speculate that the presence of alcohol components primarily determines the antifungal activity and then whether the constituent has a cyclic or acyclic structure. Probably these terpene alcohol moieties disrupt the microbial cell membrane thus increasing the permeability to protons and larger ions [29].

In this investigation, LAEO exhibited considerable antifungal effects against *C. albicans* sputum isolates. Our results correlate with those of several previous studies in which MIC values for LAEO range between 0.5–2% [30,31]. However, LAEO MIC50 and MIC90 values were twice higher than the values obtained in the previous experiments. This may be a consequence of the denser inocula that we used, as well as the origin of the tested strains. D'Auria et al. showed that higher concentrations of LAEO are necessary for growth inhibition of *C. albicans* oropharingeal isolates [21]. Moreover, a small number of studies included the mentioned isolates in their research, meaning that we could thoroughly compare our results with them. Nevertheless, the authors who compared *Candida* susceptibility to EOs by vapor diffusion and microdilution reported that MIC values in the vapor phase were lower than in the liquid phase [32,33]. Mandras et al. obtained an LAEO MIC90 of 1% in the liquid phase; whereas in the vapor phase, the growth of all *C. albicans* strains tested at a 0.06% concentration was inhibited [34]. High volatility, poor water solubility and dispersibility of EOs and their components could be the main reasons for unequal inter-laboratory results.

The experiments performed in our study showed that LAEO MICs of the all-tested fluconazole-resistant *C. albicans* strains were not different from the MICs of fluconazolesusceptible strains implicating that *C. albicans* does not exhibit cross-resistance to the mentioned EO and fluconazole. This could be a consequence of different antifungal actions in which azoles interfere with cytochrome P-450-dependent enzyme lanosterol 14α-demethylase, which is responsible for the production of ergosterol and where several mechanisms of fluconazole resistance are possible: a decrease in the affinity of lanosterol 14α-demethylase, hyperproduction of the same enzyme, as well as a loss of sterol desaturase, leading to alteration in the ergosterol synthesis [8]. Moreover, EOs can disrupt the function of efflux pumps which, through an active transfer, scavenge the fluconazole across the plasma membrane allowing its easier entry inside the yeast cell [35]. Some authors implicate that EOs alter the synthesis of many cell wall polysaccharide components, such as β-glucan, chitin and mannan [17]. Surprisingly, our data have shown that strains in which the caspofungin MIC values were higher than 0.25 µg/mL did not require higher LAEO concentrations for their growth inhibition.

Grounded on the fact that MFC values were once or twice higher than MICs (Table 1), it is suggested that LAEO has more fungicidal than fungistatic activity during 24 h exposure [36]. Based on the MFC found for each *Candida* species, time-kill curves were constructed in order to evaluate the effect of MFC90 LAEO concentration during short time exposure. The kill curves obtained with 2% LAEO indicate that this concentration is primary fungistatic (< 3 log10 CFU/mL) against most *C. albicans* isolates for 30 min. Previous studies have shown that during a short time exposure, 2% concentration of EOs exhibit a predominantly fungistatic activity against *Candida*, but fungicidal activity may also be observed, depending on the strain, type of EO and the test conditions [21,37]. However, in our investigation, the aforementioned LAEO concentration led to 0.301 log10 CFU/mL (50%) yeast cells reduction within 5 min for all the tested strains. The fact that 99.9% of growth reduction was not achieved for most isolates during 30 min implies that the decreasing of the significant reduction effect observed in the first minutes of the exposure is probably in relation to fungal cell stress adaptation. This characteristic was also observed during exposure of EOs and their components to yeast cells where many stress responses and signaling pathways were induced, such as oxygen and free radical detoxification, heat shock proteins and chaperones, autophagy and vacuolar degradation mechanisms [38]. The same study showed that these responses are arising from early 15 min to late 90 min of exposure to certain EOs components.

Regarding the antifungal efficiency of the 2% LAEO against *C. albicans* strains, our EO was able to significantly reduce the number of yeast cells during a short time exposure. Unfortunately, only one study monitored the effect of LAEO against *Candida* during 30 min of exposure [21]. In the same study, 2% LAEO concentration killed 99% of the cells within 5 min and 100% of the cells were killed within 15 min. This study was performed on

one strain only, so it is difficult to compare the results obtained then with the ones in our investigation. However, in our investigation the mean cell reduction calculated in 5 min and 15 min for sputum strains was approximately 86% and 88%, respectively. Similarly, cell reduction in *C. albicans* ATCC 10259 calculated within 5 min and 15 min was 83%.

### **4. Materials and Methods**

### *4.1. Microorganisms*

Fifteen species of *Candida albicans* isolated from sputum of pediatric patients with AML, diagnosed at the Institute for Mother and Child Health Care of Serbia "Dr Vukan Cupic", were included in this study. Initially, isolation was performed on Sabouraud dextrose agar (SDA) (HiMedia, Mumbai, India). Identification of *Candida* strains was performed after subcultivation on HiCromeTM Candida Differential Agar (HiMedia, Mumbai, India) according to the specific appearance of colonies defined by the manufacturer. For isolates that could not be identified by the aforementioned chromogenic medium, protein fingerprint analysis obtained by matrix-assisted laser desorption in ionization-time of flight mass spectrometry (MALDI-TOF MS) was used. After subcultivation on SDA at 37 ◦C for 24 h colony adhering to the toothpick was directly transferred onto a 96-spot polished steel target plate (Bruker Daltonics, Bremen, Germany) for spotting and allowed to air drying. The bacterial test standard was used as a positive control (Bruker Daltonics, Bremen, Germany). Immediately, 1 µL of 70% formic acid and alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics, Bremen, Germany) were spotted onto the target plate. After drying at room temperature samples were analyzed using MALDI-TOF MS Biotyper Sirius one IVD System (Bruker Daltonics, Bremen, Germany) in automatic runs operated by flexControl, ver. <IP_ADDRESS> (Bruker Daltonics, Bremen, Germany). The yeast identification was achieved using MBT Compass software, ver. 4.1.100 (Bruker Daltonics, Bremen, Germany), based on the comparison of generated mass spectra with database (MBT Compass Library, Revision H, 3893 species/entries). The log score values > 1.7 indicated reliable species identification. American Type Culture Collection strain *C. albicans* ATCC 10259 as control strain suggested for EOs assays was included in the investigation as well. Complete mycological examinations were performed at the National Referential Medical Mycology Laboratory (NRMML), Faculty of Medicine, University of Belgrade (Serbia).

### *4.2. Essential Oil*

*L. angustifolia* essential oil was obtained from Herba® (Belgrade, Serbia) lot N◦ 02560115 and stored at −18 ◦C until the chemical and mycological investigations were to be conducted.

### *4.3. Chemical Identification of Essential Oil*

The gas chromatography with flame ionization detection (GC-FID) analyses were carried out with a HP-7890 Series A apparatus (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a split-splitless injector, a flame ionization detector (FID), and a HP-5 capillary column (25 m × 0.32 mm i.d., film thickness 0.52 mm). The oven temperature was programmed rising from 40 to 260 ◦C at 4 ◦C/min; injector temperature was 250 ◦C; detector temperature was 300 ◦C; carrier gas was H2 (1.0 mL/min). The relative contents expressed as percentages were obtained from electronic integration of the peak areas measured using FID.

The gas chromatography-mass spectrometry (GC/MS) analyses were performed under almost the same analytical conditions as the GC-FID analyses, with a HP G1800C Series II GCD analytical system (HewlettPackard, Palo Alto, CA, USA) equipped with a HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 mm). Helium (1.0 mL/min) was used as carrier gas, and the transfer-line temperature (MSD) was heated at 260 ◦C. Mass spectra were acquired in the EI mode (70 eV) over the m/z range 40–450 amu. Aliquot of 1 µL of sample diluted in EtOH (10 mL/mL) was injected in split mode (1:30). The identification of the constituents was performed by comparing their mass spectra and retention indices (RIs) with those obtained from authentic samples and/or 4 listed in the NIST/Wiley mass-spectra libraries, using different types of searches (PBM/NIST/AMDIS) and available literature data [39,40].

### *4.4. Antifungal Agents*

Fluconazole and caspofungin (Sigma–Aldrich, Taufkirchen, Germany) powders were dissolved in 100% dimethyl sulfoxide (DMSO) at final concentration 12.8 mg/mL and 0.8 mg/mL, respectively. Prepared stock solutions were stored at −70 ◦C until use.

### *4.5. Antifungal Susceptibility Testing*

### 4.5.1. Preparation of Inoculums

Different colonies of 15 *Candida albicans* isolates and a control strain from overnight culture on SDA at 37 ◦C were suspended in 6 mL distilled water and homogenized with a gyratory vortex to the final density of the 0.5 McFarland standard. Working suspension was made by further 1:10 dilution of the standardized suspension resulting in 1–5 × 105 CFU/mL. Cell counts and viability of yeasts were confirmed in triplicates using a hemocytometer chamber by adding trypan blue.

### 4.5.2. Determination of LAEO, Fluconazole and Caspofungin MIC and MFC Values

LAEO stock solution was prepared in pure ethanol (1:2) and then diluted in RPMI 1640 broth with L-glutamine (Capricorn Scientific, Ebsdorfergrund, Germany), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich, Taufkirchen, Germany), supplemented with 2% glucose to obtain its final concentration of 8% (*vol./vol.*). Tween 80 in final concentration of 0.5% was added to enhance the EO solubility. Initially, 200 µL of freshly prepared LAEO stock solution was poured in the first rows of flat-bottomed 96-well microtiter plates. One hundred µL of RPMI 1640 with MOPS and 2% glucose were added in the remained wells. Nine serial two-fold dilutions were performed by the withdrawal of a 100 µL aliquot from the concentrated well into the succeeding one. Preparations of fluconazole and caspofungin work solutions, together with the susceptibility testing, were performed according to the EUCAST (European Committee on Antimicrobial Susceptibility Testing) document E.DEF 7.3.2. After adding of 100 µL appropriate working yeast suspension (1–5 × 105 CFU/mL), the final concentration of LAEO ranging between 4–0.008%, 64–0.125 µg/mL for fluconazole and 4–0.008 µg/mL for caspofungin were obtained. EO-free and antifungals-free grow control and sterility control wells were also included. The final solvent concentrations did not affect yeast growth as their concentration did not exceed 2% for ethanol and 1% for DMSO. All microtiter plates were incubated at 37 ◦C for 24 h in aerobic atmosphere.

After incubation, the plates were estimated to fungal growth. Minimal inhibitory concentration (MIC) of LAEO was defined as the lowest concentration, showing complete inhibition of visible growth. For fluconazole and caspofungin, MIC determinated the lowest concentration reducing ≥ 50% of the culture growth in comparison with the drug-free growth control reading by the microplate reader on 450 nm (Tecan Sunrise, Mannedorf, Switzerland). EUCAST clinical breakpoints are used to categorize the fluconazole susceptibility. Due to the significant inter-laboratory variation in caspofungin MIC ranges, a recommended epidemiological cut-off (ECV) value of 0.25 µg/mL was used to categorize the isolates into wild-type (WT) and non-WT [41]. MIC50 and MIC90 were defined as the lowest concentrations of each substance capable of inhibiting 50% and 90% of the tested isolates, respectively.

The minimal fungicidal concentration (MFC) of EO and tested drugs were estimated after MIC determination as the lowest concentration that kills 99.9% yeast cells after subculturing 10 µL of broth taken from all the wells without turbidity on SDA.  Each experiment was performed in duplicates on three different dates. The results were reported as modal values.

### *4.6. Time-Kill Essay*

In order to evaluate the further fungicidal effect of LAEO MFC90, a time-kill assay on 15 *C. albicans* sputum isolates and *C. albicans* ATCC 10259 was performed in a liquid phase according to the method proposed by Klepser et al. with some modifications [42]. One hundred microliters of a working suspension 1–5 × 105 CFU/mL was poured onto flat-bottomed 96-well micro titration plates and 100 µL of 4% EO, previously prepared by dissolving in pure ethanol (1:2) and further diluting in RPMI 1640 broth with MOPS, 2% glucose and 0.5% Tween 80, were added (the final EO concentration was 2%). Comparatively, nontreated growth controls were prepared as well. After incubation at 0, 1, 2, 5, 15 and 30 min, 10 µL from each test samples were removed for three serial 1:10 dilutions (10 µL samples and 90 µL distilled water). Colonies were counted after 100 µL inoculation of the second and the third dilution on SDA and incubation at 37 ◦C for 48 h in aerobic atmosphere. The limit of detection was ≥ 102 CFU/mL. The experiment was performed in duplicates and average values are included for further observations. Time-kill curves were constructed by plotting logarithmic values of colony counts (log10 CFU/mL) as a function of time (in minutes). In order to determine fungicidal or fungistatic character of LAEO, a reduction in the growth ≥ 3 log10 CFU/mL of the starting inoculums was defined as a fungicidal activity of EO [43]. Approximate time intervals to achieve 50% (≥0.301 log10 CFU/mL), 90% (≥1 log10 CFU/mL) and 99.9% (≥3 log10 CFU/mL) of growth reduction were calculated with non-linear interpolation function using Excel (version 16; Microsoft Corp., Redmond, WA, USA) for Windows 10 (Microsoft Corp., Redmond, WA, USA). Ultimately, roughly counted, the overall reduction in *C. albicans* sputum strains for each time interval expressed as a log10 CFU/mL was calculated using the following Equation (1):

$$mean\,\log\_{10}{\text{CFU/mL}}\\
\text{reduction} = -\log\_{10}{\left(1-\frac{mean\,\%\,\text{reduction}}{100}\right)}\tag{1}$$

### *4.7. Statistical Analysis*

Normality distribution of the variables was verified using the Kolmogorov–Smirnov and Shapiro–Wilk tests. MIC values were compared by using the Mann–Whitney U test. For comparison of the time-kill test results Friedman test with post hoc pairwise comparison (Wilcoxon signed-rang test) with applied Bonferroni correction were used to determine the correlation between log10 CFU/mL values measured at different times. In all the cases, *p* values < 0.05 were considered as statistically significant. All statistical analyses were performed using Excel (version 16; Microsoft Corp., Redmond, WA, USA) for Windows 10 (Microsoft Corp., Redmond, WA, USA) and R Commander (R package ver. 2.7-1; R Core Team, Vienna, Austria).

### **5. Conclusions**

As expected, the major components of LAEO were linalool and linalyl acetate. Our results demonstrated the efficacy of LAEO in growth reduction in *C. albicans* sputum isolates. The 2% solutions of LAEO possess potent and rapid antifungal activity that is primary fungistatic within 30 min. The same concentration is equally effective against fluconazole susceptible and fluconazole resistant, as well as against WT and non-WT caspofungin isolates. Although the prophylactic treatment of invasive candidiasis is based on systemic antifungal agents, further studies are necessary to assess the application of the essential oils as aromatherapy with high-risk patients.

**Author Contributions:** S.M. performed all the mycological experiments, analyzed the data and wrote a draft of the paper; J.A.S. designed all the experiments, reviewed and revised the draft manuscript; D.B. and D.P. performed the experiments and analyzed data related to chemical identification of essential oil. I.C.C., E.D. and A.D. oversaw the laboratory work, analyzed the data, reviewed and revised the draft manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia through the Grant Agreement with University of Belgrade—Faculty of Pharmacy No. 451-03-68/2022-14/200161.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

### **References**


**1. Introduction**

274 Biomaterials – Physics and Chemistry

[37] Daamen WF, van Moerkerk HT, Hafmans T, Buttafoco L, Poot AA, Veerkamp JH, et al.

[39] Semler EJ, Ranucci CS, Moghe PV. Mechanochemical manipulation of hepatocyte

[40] Nagai N, Yunoki S, Satoh Y, Tajima K, Munekata M. A method of cell-sheet preparation

[41] Yao C, Roderfeld M, Rath T, Roeb E, Bernhagen J, Steffens G. The impact of proteinase-

[42] Isenberg BC, Williams C, Tranquillo RT. Small-diameter artificial arteries engineered in

[43] Iwai S, Sawa Y, Ichikawa H, Taketani S, Uchimura E, Chen G, et al. Biodegradable

[44] Lepidi S, Grego F, Vindigni V, Zavan B, Tonello C, Deriu GP, et al. Hyaluronan

[45] Shum-Tim D, Stock U, Hrkach J, Shinoka T, Lien J, Moses MA, et al. Tissue engineering

[46] Wildevuur CR, van der Lei B, Schakenraad JM. Basic aspects of the regeneration of

[47] Eriksson AC, Whiss PA. Measurement of adhesion of human platelets in plasma to protein surfaces in microplates. J Pharmacol Toxicol Methods. 2005;52:356-65. [48] Polanowska-Grabowska R, Simon CG, Jr., Gear AR. Platelet adhesion to collagen type I,

[49] Kulik E, Ikada Y. In vitro platelet adhesion to nonionic and ionic hydrogels with

[50] Lee JH, Khang G, Lee JW, Lee HB. Platelet adhesion onto chargeable functional group

Eur Cell Mater. 2006;11:43-56.

matrices. Biomaterials. 2006;27:1608-16.

prosthesis. J Thorac Cardiovasc Surg. 2004;128:472-9.

animal model. Eur J Vasc Endovasc Surg. 2006;32:411-7.

kinetics under shear. Thromb Haemost. 1999;81:118-23.

gradient surfaces. J Biomed Mater Res. 1998;40:180-6.

different water contents. J Biomed Mater Res. 1996;30:295-304.

vitro. Circ Res. 2006;98:25-35.

1999;68:2298-304; discussion 305.

Bioeng. 2000;69:359-69.

2004;98:493-6.

1987;8:418-22.

Preparation and evaluation of molecularly-defined collagen-elastinglycosaminoglycan scaffolds for tissue engineering. Biomaterials. 2003;24:4001-9. [38] Wahl DA, Czernuszka JT. Collagen-hydroxyapatite composites for hard tissue repair.

aggregation can selectively induce or repress liver-specific function. Biotechnol

using collagenase digestion of salmon atelocollagen fibrillar gel. J Biosci Bioeng.

induced matrix degradation on the release of VEGF from heparinized collagen

polymer with collagen microsponge serves as a new bioengineered cardiovascular

biodegradable scaffold for small-caliber artery grafting: preliminary results in an

of autologous aorta using a new biodegradable polymer. Ann Thorac Surg.

small-calibre neoarteries in biodegradable vascular grafts in rats. Biomaterials.

collagen type IV, von Willebrand factor, fibronectin, laminin and fibrinogen: rapid

Hydroxyapatite and Bioglass® are two well-known biomaterials, belonging to the vast class of ceramic supplies, both highly biocompatible and widely applied in the biomedical field. In spite of a huge research regarding engineering applications of both inorganic materials, still many aspects of their tissue integration mechanism have not been completely cleared at a molecular level. Thus, *in silico* studies play a fundamental role in the prediction and analysis of the main interactions occurring at the surface of these biomaterials in contact with the biological fluid when incorporated in the living tissue (prevalently bones or teeth). Hydroxyapatite [HA, Ca10(PO4)6(OH)2] owes its relevance and use as a biomaterial since it constitutes the majority of the mineral phase of bones and tooth enamel in mammalians (Young & Brown, 1982). For sake of completeness, we mention that hydroxyapatite is also studied as an environmental adsorbent of metals and a catalyst (Matsumura & Moffat, 1996; Toulhat et al., 1996). One of the first applications of HA in biomedicine dates back to 1969, when Levitt *et al.* hot-pressed it in powders for biological experimentations (Levitt et al., 1969). From then on, several commercial forms of HA have appeared on the market. The material has also been utilized for preparing apatitic bioceramic, due to its bioresorbability which can be modulated changing the degree of cristallinity. There are so many examples of applications, from Mg2+-substituted hydroxyapatite (Roveri & Palazzo, 2006) to the synthesis of porous hydroxyapatite materials by colloidal processing (Tadic et al., 2004), starch consolidation (Rodriguez-Lorenzo et al., 2002), gel casting (Padilla et al., 2002) and more. Furthermore, recent applications follow a biologically inspired criterion to combine HA to a collagen matrix aiming at the improvement of mechanical properties and bioactivity (Wahl et al., 2007). However, a complete review of all the practical as well as hypothetical uses of HA in the biomaterial area is outside the scope of this Chapter.

Inside the bone, a highly hierarchical collagen-mineral composite, hydroxyapatite is in the form of nano-sized mineral platelets (Currey, 1998; Fratzl et al., 2004; Weiner & Wagner, 1998) and contains carbonate ions for the 4-8 weight % (Roveri & Palazzo, 2006). In section 2.1 of this Chapter, two aspects of defects which can be encountered in a synthetic or natural HA sample will be presented. The first aspect deals with non-stoichiometric surfaces and

*In Silico* Study of Hydroxyapatite and Bioglass®: How Computational Science Sheds Light on Biomaterials

focusing exclusively on Gaussian type functions.

**2.1 Defects in hydroxyapatite bulk and surfaces**

2009; Corno et al., 2008).

oxygen atom of the OH-

symmetry equivalent.

computational time. Our calculations are performed either with the pure GGA PBE functional (Perdew et al., 1996) or with the hybrid B3LYP (Becke, 1993), both well-known functionals. Two *ab initio* approaches are possible within DFT and they differ for the type of basis set functions. Indeed, a localized Gaussian basis set can be considered, as in the present case, or a plane waves one, also extremely diffuse. An *excursus* of advantages and disadvantages of these approaches is not useful in this context and will be omitted, by

All the calculations mentioned in this Chapter have been performed using the CRYSTAL code in its latest release (Dovesi et al., 2005a; Dovesi et al., 2005b; Dovesi et al., 2009). This periodic quantum-mechanical software has been developed by the Theoretical Chemistry group of the University of Turin (Italy) together with the Daresbury Laboratory (UK) since 1988. CRYSTAL is capable of computing systems with every dimensionality, from molecules to real infinite crystals and it supports massive parallel calculations. This code uses local Gaussian basis sets and can deal with many electronic structure methods, from Hartree-Fock to Kohn-Sham Hamiltonians. Structural, electrostatic and vibrational properties of the studied materials have been characterized with the program. Another crucial aspect in modeling is the graphical visualization and representation of structures. For all the images displayed in this Chapter, MOLDRAW (Ugliengo et al., 1993), J-ICE (Canepa et al., 2011b) and VMD (Humphrey et al., 1996) programs were used. Further more precise computational details can be read in a number of our recent papers on both HA (Corno et al., 2009; Corno et al., 2006; Corno et al., 2007; Corno et al., 2010) and bioactive glasses (Corno & Pedone,

Hydroxyapatite (HA) is a mineral which occurs in nature in two polymorphs, a monoclinic form, thermodynamically stable at low temperatures, and an hexagonal form, which can be easily stabilized by substitution of the OH- ions (Suda et al., 1995). These ions are aligned along the c axis (the [001] direction), as highlighted in Fig. 1. The single crystal structure of the hexagonal form of HA is characterized by the *P63/m* space group. The mirror plane,

an intrinsic static disorder of these ions, which can point, with no preference, in one of the two opposite directions ([001] or [00-1]). The result is a fractional occupation of the sites in the solved crystallographic structure (50% probability for each direction). As *ab initio* simulation cannot take into account the structural disorder, we reduced the symmetry to

configuration found, both the OH- ions point in the same direction, as reported in Fig. 1. The

the *ab* plane. Moreover, there are six phosphate ions inside the crystallographic cell, all

The bulk structure of crystalline HA, fully characterized in the literature (Corno et al., 2006), has been considered as a starting point to model the surfaces which are experimentally found to be the most important: (001) in terms of reactivity, and (010) in terms of exposure in the crystal habit (Wierzbicki & Cheung, 2000). Those surfaces have already been fully characterized at an *ab initio* level, and all the structural, geometrical and electronic properties

ion is close to three Ca ions, which form an equilateral triangle in

perpendicular to the [001] direction, is compatible with the column of OH-

*P63*, removing the mirror plane and fixing the directions of the OH-

**2. Hydroxyapatite and Bioglass® as computational case study**

277

ions because of

ions. In the most stable

their adsorptive behavior towards simple molecules (water and carbon monoxide). The second concerns the inclusion of carbonate ions in the pure HA bulk structure to simulate the apatite bone tissue. These examples of applying sophisticated computational techniques to the investigation of defects in HA represent a very recent progress achieved in our laboratory inside this biomedical research area, which has being carried out since 2003. For the interested reader, a summary of the last years work on simulation of HA in our research group has been recently published (Corno et al., 2010).

As for bioactive glasses, the first synthesis was performed in 1969-71 by Larry Hench in Florida (Hench et al., 1971). He had synthesised a silicate-based material containing calcium and phosphate and had implanted this composition in rats' femurs (Hench, 2006). The result was a complete integration of the inorganic material with the damaged bone. This very first composition was called Bioglass® 45S5 (45SiO2 - 24.5Na2O - 24.5CaO - 6P2O5 in wt. % or 46.1 SiO2, 24.4 Na2O, 26.9 CaO and 2.6 P2O5 in mol %) and has been introduced in clinical use since 1985. The interest has been then to investigate the steps of the bioactivity mechanism leading to the formation of a strong bond between the material and the biological tissue. The most renowned hypothesis is the so-called Hench mechanism and its crucial step resides in the growth of a thin amorphous layer of hydroxy-carbonated apatite (HCA) (Hench, 1998; Hench & Andersson, 1993; Hench et al., 1971). Indeed, on that layer biological growth factors are adsorbed and desorbed to promote the process of stem cells differentiation. Moreover, before the growth of HCA, several other chemical reactions occur, dealing particularly with the exchange of sodium and calcium ions present in the Bioglass® with protons derived from the biological fluid. The influence of the chemical components of the inorganic material on its bioactivity has recently been object of scientific research and discussion. For instance, additives such as fluorine (Christie et al., 2011; Lusvardi et al., 2008a), boron, magnesium (A. Pedone et al., 2008) and zinc (Aina et al., 2011) were considered in a number of systematic studies. In section 2.2 of this Chapter, the role of phosphate concentration inside models with the 45S5 composition will be highlighted, since these changes in content can affect the crucial mechanism of gene activation and modify the local environment of the silicon framework and of Na and Ca sites, as well as the dissolution rate of silica (O'Donnell et al., 2009).

The joint use of experimental and theoretical techniques nowadays has reached a very large diffusion due to the completeness of the derived information. Particularly, in the biological or biochemical field, computational methods are essential to the investigation of interfacial mechanisms at a molecular level. Moreover, very often the interplay between experimental and calculated data allows researchers to improve both methodologies. A huge amount of examples could be reported, but for sake of brevity here we limit to our own experience of collaboration with a number of experimentalists. Indeed, in our research papers, dealing either with HA or with bioactive glasses, there is always a detailed comparison with measured data, for instance by means of NMR (Pedone et al., 2010), IR and Raman spectroscopy and of adsorption microcalorimetry (Corno et al., 2009; Corno et al., 2008). In our computational studies, we refer to quantum-mechanical techniques, which are very accurate but also quite heavy as for the need of resources. Usually, high parallel computing systems are required to run the simulations and we have successfully used the supercomputers of several HPC centers, such as the Barcelona Supercomputing Centre (Spain) or the CINECA Supercomputing Center (Italy).

The most used theoretical framework in the last decade's literature is the Density Functional Theory, which grants a good compromise in terms of accuracy of the representation and

their adsorptive behavior towards simple molecules (water and carbon monoxide). The second concerns the inclusion of carbonate ions in the pure HA bulk structure to simulate the apatite bone tissue. These examples of applying sophisticated computational techniques to the investigation of defects in HA represent a very recent progress achieved in our laboratory inside this biomedical research area, which has being carried out since 2003. For the interested reader, a summary of the last years work on simulation of HA in our research

As for bioactive glasses, the first synthesis was performed in 1969-71 by Larry Hench in Florida (Hench et al., 1971). He had synthesised a silicate-based material containing calcium and phosphate and had implanted this composition in rats' femurs (Hench, 2006). The result was a complete integration of the inorganic material with the damaged bone. This very first composition was called Bioglass® 45S5 (45SiO2 - 24.5Na2O - 24.5CaO - 6P2O5 in wt. % or 46.1 SiO2, 24.4 Na2O, 26.9 CaO and 2.6 P2O5 in mol %) and has been introduced in clinical use since 1985. The interest has been then to investigate the steps of the bioactivity mechanism leading to the formation of a strong bond between the material and the biological tissue. The most renowned hypothesis is the so-called Hench mechanism and its crucial step resides in the growth of a thin amorphous layer of hydroxy-carbonated apatite (HCA) (Hench, 1998; Hench & Andersson, 1993; Hench et al., 1971). Indeed, on that layer biological growth factors are adsorbed and desorbed to promote the process of stem cells differentiation. Moreover, before the growth of HCA, several other chemical reactions occur, dealing particularly with the exchange of sodium and calcium ions present in the Bioglass® with protons derived from the biological fluid. The influence of the chemical components of the inorganic material on its bioactivity has recently been object of scientific research and discussion. For instance, additives such as fluorine (Christie et al., 2011; Lusvardi et al., 2008a), boron, magnesium (A. Pedone et al., 2008) and zinc (Aina et al., 2011) were considered in a number of systematic studies. In section 2.2 of this Chapter, the role of phosphate concentration inside models with the 45S5 composition will be highlighted, since these changes in content can affect the crucial mechanism of gene activation and modify the local environment of the silicon framework and of Na and Ca sites, as well as the dissolution

The joint use of experimental and theoretical techniques nowadays has reached a very large diffusion due to the completeness of the derived information. Particularly, in the biological or biochemical field, computational methods are essential to the investigation of interfacial mechanisms at a molecular level. Moreover, very often the interplay between experimental and calculated data allows researchers to improve both methodologies. A huge amount of examples could be reported, but for sake of brevity here we limit to our own experience of collaboration with a number of experimentalists. Indeed, in our research papers, dealing either with HA or with bioactive glasses, there is always a detailed comparison with measured data, for instance by means of NMR (Pedone et al., 2010), IR and Raman spectroscopy and of adsorption microcalorimetry (Corno et al., 2009; Corno et al., 2008). In our computational studies, we refer to quantum-mechanical techniques, which are very accurate but also quite heavy as for the need of resources. Usually, high parallel computing systems are required to run the simulations and we have successfully used the supercomputers of several HPC centers, such as the Barcelona Supercomputing Centre

The most used theoretical framework in the last decade's literature is the Density Functional Theory, which grants a good compromise in terms of accuracy of the representation and

group has been recently published (Corno et al., 2010).

rate of silica (O'Donnell et al., 2009).

(Spain) or the CINECA Supercomputing Center (Italy).

computational time. Our calculations are performed either with the pure GGA PBE functional (Perdew et al., 1996) or with the hybrid B3LYP (Becke, 1993), both well-known functionals. Two *ab initio* approaches are possible within DFT and they differ for the type of basis set functions. Indeed, a localized Gaussian basis set can be considered, as in the present case, or a plane waves one, also extremely diffuse. An *excursus* of advantages and disadvantages of these approaches is not useful in this context and will be omitted, by focusing exclusively on Gaussian type functions.