Fabrication, characterization and bioevaluation of novel ... · as antimicrobials agents. The...

Romanian Biotechnological Letters Vol. 20, No. 3, 2015 Copyright © 2015 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 20, No. 3, 2015 10521

Fabrication, characterization and bioevaluation of novel antimicrobial composites based on polycaprolactone,

chitosan and essential oils

Received for publication, June 14, 2014 Accepted, April 04, 2015

PETRUŢA STOICA1,2, MARIANA CARMEN CHIFIRIUC2,*, MARIA RÂPĂ1, CORALIA BLEOTU3, LILIANA LUNGU4, GRIGORE VLAD1, RALUCA GRIGORE5, 6, ȘERBAN BERTEȘTEANU5,6, ELISAVET STAVROPOULOU7, VERONICA LAZĂR2

1S.C. I.C.P.E. Bistrița S.A., Bistriţa, Parcului Street, No.7, County Bistrița-Năsăud, Romania

2Department of Microbiology and Immunology, Faculty of Biology, University of Bucharest, Romania

3Cellular and Molecular Pathology Department, "Stefan S. Nicolau" Institute of Virology, Bucharest, Romania

4Institute of Organic Chemistry "C.D. Nenitzescu”, Spl. Independentei 202B, 060023, Bucharest, Romania

5Head & Neck Surgery Clinic, Colţea Clinical Hospital, Bucharest, Romania 6 University of Medicine and Pharmacy Carol Davila, Bucharest, Romania 7Democritus University of Thrace,Medical School,68100-Alexandroupolis,Greece *Corresponding author : [email protected]

Abstract The continuously spreading use of implantable medical devices (IMDs) in all fields of

medicine increased the risk of microbial colonization leading to difficult to treat infections. The objective of this study was to obtain and characterize by physico-chemical and biological methods, new biopolymeric composites with anti-adherence properties based on polycaprolactone (PCL) dopped with chitosan (CS) and essential oils (EOs) as antimicrobial agents, for further use in the manufacture or coating of IMDs.

The EOs were extracted from cinnamon (CIN), coriander (COR) and tea tree (TEA), and were characterized by GC-MS. Biopolymeric composite films (PCL/CS, PCL/CS/EOs) were obtained by solvent evaporation in two variants and further characterized by DSC and FTIR techniques.

The in vitro antimicrobial activity of EOs was determined by the minimum inhibitory concentration (MIC) assay in liquid medium, towards Gram-positive and Gram-negative bacterial strains. The anti-adherence activity of films was analyzed against Staphylococcus aureus ATCC 29213 by viable cell counts assay. The cytotoxicity of the obtained films was assessed using the HT29 cell line. The results of this study suggest that CIN EO strongly inhibited the growth of all tested bacterial species, followed by TEA and COR EOs. From all tested composites, only PCL/CS/CIN1 exhibited a significant anti-adherence effect.

Keywords: biopolymeric composite, antimicrobial activity, cytotoxicity, anti-adherence

1. IntroductionAdhesion of microorganisms on the surface of medical devices is considered an essential

step in the pathogenicity of infectious diseases [1]. When polymeric medical devices are implanted into the human body, they become easily colonized by microbial cells [2-4], causing devices-related infection, responsible of high rates of morbidity and mortality, and thereby significantly increasing health care costs [2, 5-9]. Prevention of such infections remains a major challenge to deliver quality medical care. In this regard, biomaterials industry

PETRUŢA STOICA, MARIANA CARMEN CHIFIRIUC, MARIA RÂPĂ, CORALIA BLEOTU, LILIANA LUNGU, GRIGORE VLAD, RALUCA GRIGORE, ȘERBAN BERTEȘTEANU, ELISAVET STAVROPOULOU, VERONICA LAZĂR


has focused on the design of anti-infective polymers, which are usually impregnated or mixed with different antimicrobial agents [2, 8, 10-13]. Composite systems are becoming more and more popular in biomedical applications because of the desired properties obtained by the combination of their constituents. From this perspective biocompatible polymeric matrices are one of the most frequently used for the development of implantable medical devices (IMDs).

Polycaprolactone (PCL), one of the earliest polymers synthesized by the Carother’s group in the early 1930s, is a hydrophobic, semi-crystalline substance with low melting point (59–64oC [14, 15]. PCL is soluble in chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclohexanone and 2-nitropropane at room temperature. It has a low solubility in acetone, 2-butanone, ethyl acetate, dimethylformamide and acetonitrile and is insoluble in alcohol, petroleum ether and diethyl ether [14, 16]. Due to their appropriate physico-chemical properties, PCL biomaterials have been proposed for multiple applications over the last two decades (sutures, wound dressings, fixation devices, contraceptive devices, dentistry), drug delivery and tissue engineering (scaffolds, bone, cartilage, tendon and ligament, cardiovascular, blood vessel, skin, nerve) [14, 17, 18].

Enzyme-embedded PCL-based coating, co impregnated with antibiotic (gentamicin sulfate) exhibited antibacterial properties against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus [19].

Biodegradation of PCL is slow in comparison to other polymers, so it is suitable for long-term delivery approaches, extending over a period of more than 1 year. PCL also has the ability to form compatible blends with other polymers, which can affect the degradation kinetics, facilitating tailoring to achieve the desired release profiles [14, 20-22].

Chitosan (CS) is a copolymer of N-acetyl glucosamine and glucosamine units. CS is a unique basic polysaccharide with high molecular weight that occurs in nature in the cell walls of some fungi, insects, etc. It is a positively charged polysaccharide prepared by the N-deacetylation of chitin with 40-50% aqueous alkali at 120-160oC [23]. CS is hydrophilic and soluble in weak aqueous acids (pH<6.3) [24, 25]. Chitosan is biodegradable, biocompatible and non-toxic, properties which are appropriate for many applications in the pharmaceutical and biomedical field, such as the controlled release of drugs, space filling implants and wound management [23].

Chitosan and its derivatives have attracted a great interest due to their antimicrobial properties. Allan and Hardwiger (1979) reported for the first time the antimicrobial activity of chitosan [26, 27]. A recent study, in which PCL / CS polymers blends were investigated for their antimicrobial properties has shown that blends based on low molecular weight chitosan exhibited a pronounced inhibitory effect [28].

Chitosan and polycaprolactone are biodegradable biomaterials approved by Food and Drug Administration (FDA) [29].

In order to overcome some of the PCL properties drawbacks, such as high price and long biodegradability cycles, it is blended with a cheaper biodegradable natural polymer, such as starch, cellulose, or chitin and chitosan [30].

Also, the bioactivity of PCL can be enhanced when combined with natural polymers. The hydrophilic nature of CS will enhance its wettability and permeability, with a consequent accele-ration of the PCL hydrolytic degradation [31]. The PCL / CS polymer blends reported in different studies were obtained by polymers bending in solution as well as by the melting technique.

The combination of these two polymers leads to high porosity, hydrophilicity and high mechanical strength [32, 33]. Also, by combining the synthetic polymers with natural polymers the biocompatibility can be increased [34].

Fabrication, characterization and bioevaluation of novel antimicrobial composites based on polycaprolactone, chitosan and essential oils


The cytocompatibility of PCL was improved by the immobilization of chitosan rich in NH2 groups onto the PCL membranes surface [35].

Essential oils (EOs) are composed of a very complex mixture of volatile molecules that are produced in the secondary metabolism of aromatic and medicinal plants. The main group of EOs is composed of terpenes and terpenoids and other aromatic and aliphatic constituents, all characterized by a low molecular weight [36]. The chemical composition, the quality and the quantity of EOs are influenced by many factors, such as: the soil composition, plant organ, vegetative cycle phase and climate [37-40]. The hydrophobicity is a very important characteristic of EOs and of their components, enabling them to partition the lipids of the bacterial cell and mitochondrial membranes, disturbing the cell structures, rendering them more permeable and causing metabolic disturbances, leading to microbial cells death [41, 42]. This indicates that minor or even trace EOs elements may be critical for the antimicrobial activity. The complexity of EOs prevents bacteria to develop tolerance and resistance [42, 43].

The usage of EOs to combat hospital-acquired infections and to control epidemic multi-resistant bacteria, such as methicillin-resistant Staphylococcus aureus shows promising results. The EOs of eucalyptus, tea tree, thyme white, lavender, lemon, lemongrass, cinnamon, grapefruit, clove bud, sandal wood, peppermint, kunzea and sage, proved to be active against problematic microorganisms, like methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus and Candida strains [44].

The antimicrobial activity and applications of nanocomposite coatings based on PCL and thymol have been reported [45].

Taking into account the consecrated antimicrobial properties of EOs, we have focused our attention to obtain biomaterials incorporating cinnamon EO, coriander EO and tea tree EO as antimicrobials agents.

The objective of this study was to obtain new antimicrobial composites based on polycaprolactone (PCL), chitosan (CS) and essential oils (EOs) and to characterize them by physico-chemical and biological methods. The chemical composition of the essential oils (CIN, COR and TEA) was investigated by GC-MS. The antimicrobial activity of CIN, COR and TEA essential oils was tested against Staphylococcus aureus ATCC 29213, Enterococcus fecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8730 and Klebsiella pneumoniae IC 134202 bacterial strains. The chemical structure of antimicrobial composites was evidenced by fourier transform infrared spectroscopy (FTIR). Thermal analysis was performed by differential scanning calorimetry (DSC). Antibacterial adherence and cytotoxicity tests were used to prove the use of antimicrobial composites for the biomedical field.

2. Materials and Methods

2.1. Materials PCL was obtained from Sigma-Aldrich (Mw = 45,000, Milwaukee, Wisconsin USA).

Chitosan was obtained from Sigma-Aldrich (Iceland); Cyclohexanone (extra pure), was purchased from Scharlab S.L (Barcelona, Spain). The cinnamon essential oil (CIN), coriander essential oil (COR) and tea tree essential oil (TEA) were supplied by Cozac Plant SRL (Bucharest, Romania). Tryptic Soy Broth (TSB) medium was acquired from Biomedics (Madrid, Spain); Plate Count Agar (PCA) medium was obtained from Oxoid (England); Dimethylsulphoxide (DMSO) solvent was purchased from Sigma-Aldrich; Phosphate Buffered Saline (PBS) was obtained from Biochrom. The bacterial strains were provided by



the collection of the Department of Microbiology of the Faculty of Biology (University of Bucharest, Romania).

The HT-29 ATCC HTB-38TM cells were used in the evaluation of cytotoxicity. The cells were maintained as an adherent culture in Dulbecco’s Modified Essential Medium (DMEM) (Sigma, USA) supplemented with 10% heat-inactivated (FBS) fetal bovine serum (Sigma, USA) at 37oC, 5% CO2, in a humid atmosphere.

2.2. Preparation of antimicrobial composites films Biopolymeric composites films (PCL/CS, PCL/CS/EOs) were prepared by solvent

evaporation at room temperature conditions. PCL film was used as control. EOs were added to the PCL solution (10% w/v) prepared in cyclohexanone and stirred magnetically for 5 min at 40oC and 400 rpm for obtaining of homogeneous dispersions. Then the CS solution (1% w/v prepared in acetic acid aqueous solution1% v/v) was added, followed by magnetic stirring for 5 min at 40oC and 400 rpm. The mixtures were molded in Petri glass dishes and kept at room temperature conditions for seven days for the solvent evaporation. The proportion of the compounds PCL/CS/EOs was 4: 1: 0.2 and respectively 4: 1: 0.04. The proportion for the PCL/ CS was 4: 1, as shown in Table 1.

Table 1.The composition of biopolymeric composites films

Code Sample Composition, volume ratio

PC PCL/CS = 4:1

PCCI1 PCL/CS/CIN = 4:1: 0.2

PCT1 PCL/CS/TEA = 4:1: 0.2

PCC1 PCL/CS/COR = 4:1: 0.2

PCCI2 PCL/CS/CIN = 4:1: 0.04

PCT2 PCL/CS/TEA = 4:1: 0.04

PCC2 PCL/CS/COR = 4:1: 0.04

2.3. Gas chromatography/mass spectrometry (GC-MS) analysis of essential oils (EOs) The GC–MS analysis was carried out using an Agilent 6890 N gas chromatograph

interfaced with an Agilent mass selective detector 5975B (Agilent Technologies, USA). Oven temperature program: 100–3200C, at 10oC/min (39 min analysis times); the injector parameters were: initial temperature 250 °C, pressure 6.89 psi, split ratio 2:1, carrier gas helium. A HP-5MS 5% phenylmethylsiloxane capillary column was used for the separation of the sample components with the following characteristics: max. temperature 325°C, nominal length 30.0 m, nominal diameter 250.0 μm, nominal film thickness 0.25 μm, initial flow 1.0 mL/min. The mode of operating was constant flow. Interface temperature was 2800C; standard electronic impact (EI), MS source temperature: 2300C; MS quadrupole temperature: 1500C; mass scan range: 29 – 850 amu at 70 eV, 1 µl of sample was injected into the system.

The constituents of the EOs were identified by comparing their mass spectra obtained with those in the mass spectra library (NIST, Wiley). For quantification purposes, area percentages were obtained by integration of chromatograms [46].

2.4. Differential Scanning Calorimetry (DSC) measurements The DSC tests were performed using a METTLER-TOLEDO apparatus DSC-823e

model, using STARe software, version 9.10, and previously calibrating with indium standard.



The weighted samples were sealed in 40 µl aluminum crucibles with a small hole in the lids, and the first heating curves were measured from room temperature to 100°C at a heating rate of 10°C/min, as first heating. The melting temperature and the enthalpies of melting (ΔH) of the samples were determined. The melting region in this experiment was defined by onset temperature (Ton) and maxim melting temperatures (Tm).

2.5. Fourier Transform Infrared Spectroscopy (FTIR) measurements The functional groups present in the prepared films were identified by FTIR using a

Spectrum BX Spectrometer. The spectra were recorded in the range of 400 to 4000 cm-1 with a resolution of 4 cm-1.

2.6. EOs Minimum Inhibitory Concentration (MIC) assay MIC tests were carried out according to [47] by using 96 multi-well plates. The stock

solutions of EOs were transferred into the first well, and serial microdilutions were performed by using TSB medium. Microbial suspensions of 0.5 McFarland density (1:5 volume/well) were added to the wells and the plates were incubated at 37ºC for 24 h. The experiments were carried out using DMSO as solvent. MIC was defined as the lowest concentration of EOs at which the microorganisms did not demonstrate visible growth in the liquid medium.

2.7. Anti-adherence test The bacterial anti-adherence effect was demonstrated using 24 multi-well plates.

Standard pieces of 1cm x 1cm of PCL, PCL/CS and PCL/CS/EOs were sterilized by exposure to direct UV light for 30 min on both sides and distributed in 24 multi-well plates (one per well). The Staphylococcus aureus ATCC 29213 suspension of 0.5 McFarland density was diluted 1:100, for reaching an inoculums with density of 105–106 (CFU/ml). S. aureus inoculums prepared in TSB medium were added in each well, to completely cover films pieces. Samples were incubated for 24h at 37oC. The inhibitory effect of staphylococcal colonization on the surface of films was analyzed by viable cell count assay. After the incubation period, biopolymeric films pieces were gently washed with sterile PBS for not removing the bacterial cells adhered on films surface and placed in 2 ml Eppendorf tubes containing 1000 µl PBS. Samples were centrifuged for 5 min at 10000 rpm, followed by 30s vortexing in order to disperse the bacterial cells on films surfaces, and ten-folds microdilutions were performed from the obtained suspensions. Three replicates of each dilution were plated on PCA medium for viable cell counts assay and the Colony Forming Units (CFUs) were calculated [48-50].

2.8. Cytotoxicity test and cell cycle analysis

5x105 HT29 cells were seeded in 3.5-cm diameter wells, in RPMI medium supplemented with 10% FBS, 400 IU/ml penicillin and 200 µg/ml streptomycin. HT29 were incubated for 24 h at 37oC in humidified and 5%CO2 atmosphere. The sterile film pieces of 0.7cm x 0.7cm were added over the monolayer, and then incubated for another 24 h and 48h, in the same conditions. The effect of samples on HT29 cell was observed using inverted microscope Zeiss Observer D1 and was evaluated by Trypan Blue Exclusion method. The cells were trypsinised, centrifuged for 6 minutes at 1200 rpm and resuspended in 1 mL PBS. 50 µL of cell suspension was stained with 50 µL Trypan blue solution (0.4%), for 1 minute, and the mixture was placed in a haemocytometer and viable and non-viable cells were counted. Each experiment was repeated three times. The rest of harvested cells were fixed in cold ethanol (70%) and stored at – 20oC overnight. The samples were then centrifuged, washed with PBS, re-suspended in 100 µl PBS, treated with 1 mg/mL RNase A at 37oC for 15 minutes, and stained with 100 μg/mL propidium iodide for 1 h at 370C. DNA content of cells was



quantified on a Beckman Coulter EPICS XL flow cytometer and analyzed using FlowJo 8.8.6 software (Ashland, Oregon, USA).

3. Results and discussion 3.1. Chemical composition of EOs The GC-MS technique revealed the presence of 12 substances in the composition of

cinnamon EO, of which 10 could be identified. The chromatogram revealed the cinnamaldehyde to be the major compound, with a relative abundance of 86.27 % followed, in smaller amounts, by o-methoxycinnamic aldehyde (2.57%), and cinnamic acid (1.93%), as well as by other compounds, in percents under 1%.

Several reported studies have shown that CIN EO are very complex mixtures of compounds and many variations have been found in their chemical composition [51], but of these, cinnamaldehyde is the main component, with percentages varying from 79.38% [51-55] to 41.62% [41].

For coriander EOs, the chromatogram showed 24 components from which 22 compounds were identified. The main compounds identified in tested COR EO was linalool (61.30%), and in smaller percentages, α-pinene (7.38%), DL-limonene (5.80%), camphor (4.82%), α-terpinenyl acetate (2.15%), geranyl acetate (1.78%), linalool oxide (1.45%), and others. Our results are in good agreement with the literature data, stating that coriander seeds oil is rich in linalool (60-70%) [56]. Zorca et al. (2006) identified 45.31% linalool as major component of coriander oil obtained by hydrodistillation and 72.10 % linalool for the oil obtained by supercritical CO2

extraction [57]. Nazrul et al., (2009) reported linalool (37.65%) as major component from seeds oil of coriander from Bangladesh [58], while other authors report 69.60% linalool [59].

In case of the tested tea tree EO, 29 components were detected, from which 27 were identified.Terpinen-4-ol was found in the highest amount (28.87%) followed by γ-terpinene (16.00%), α-terpinene (9.83%), DL-limonene (8.99%), p-cymene (6.51%), α-pinene (5.88%), α-terpinolene (4.23%), trans-caryophyllene (3.12%) and other compounds in smaller percentages. Other GC-MS performed tests have been shown that terpinen-4-ol represents the majoritary chemical component of TEA, with a percentage of 39.8% [60] and respectively, 41.6% [61].

3.2. DSC measurements of the biopolymeric films Melting temperature and heat of fusion (ΔH) determined from the DSC termograms of

the samples (Figure1 and Figure 2) are summarized in (Table 2).

Figure 1. DSC termograms of biopolymeric

composites films: a) PCL(film);

b) PCL(granules); c) PC; d) PCC1 ;

e) PCCI1 ; f) PCT1 .



Figure 2. DSC termograms of biopolymeric composites films: a)

PCL(film); b) PCL (granules); c) PC; d) PCC2 ; e) PCCI2 ; f) PCT2

Table 2. DSC characteristics

Characteristics Code sample

PCL (granules)

PCL (film)

PC

PCCI1 PCT1 PCC1 PCCI2 PCT2 PCC2

ΔH (J/g) 67.07 71.20 88.89 84.53 83.85 98.10 64.14 63.83 73.13 Ton (0C) 56.69 60.42 56.42 53.46 59.36 59.40 57.33 58.91 59.31 Tm (

0C) 63.46 67.02 63.80 62.32 65.94 65.80 69.26 68.78 68.78

Data from Table 2 and Figures 1 and 2 reveal that PCL film recorded the melting temperature (Tm) at 67.02 0C and the enthalpy of melting (∆H) at 71.2 J/g, respectively. PCL/CS composite shows lower Tm than PCL pure, due to the hydrophilic character of CS, that likely leads to poor adhesion with the hydrophobic PCL. The lower Tm recorded for PCL/CS/EOs in ratio 4:1:0.2 v/v % in comparison with PCL film (by about 2–5 0C) is due to the lower melt viscosity of chitosan and EOs while the higher ∆H (by about 12 - 27 J/g) is caused by the higher crystallinity of the composites. We can suppose here that EOs can act like plasticizers. It is proposed that this is also a result of less space available for molecular motion within this composite. Instead, the values of Tm in the composites PCL/CS/EOs in ratio 4:1:0.04 v/v % increased (by about 2 0C) while the ∆H decreased (by about 7 J/g) than PCL film. This implies a decreased in crystallinity with decrease of EOs content, probably caused by chitosan movement of the polymer segments and thereby making them more easily to arrange the polymer chain. This melting behavior shows that PCL/CS/EOs composites in the ratio 4:1:0.2 are easier to process than those in ratio 4:1:0.0.4.

3.3. FTIR measurements of the biopolymeric films IR absorbance spectra of PCL, PC, PCC1, PCCI1, PCT1, PCC2, PCCI2 and PCT2

biopolymeric films are presented in Figures 3 and 4. The spectra clearly suggest the presence of the various functional groups corresponding

to the raw materials used to obtain the biopolymeric films by solvent evaporation. The PC spectra show the characteristic absorption bands at 1148 and 841cm-1 attributed

to the glycoside bond specific to chitosan. These bands are also present in all of the biopolymeric investigated composites films. In other studies the bands assigned to the glycoside bond of chitosan were reported at 890 and 1147 cm-1 [62], 905 and 1157 cm-1[63], as well as 897 and 1154 cm-1 [64].

Also, the amine N-H symmetrical vibration band reported in the IR spectrum for chitosan at 1650 cm-1[65] can be attributed to the bands 1685, 1698, 1689, 1675, 1679, 1669, 1681cm -1 , confirming the presence of chitosan in all the investigated samples.



The characteristic absorbance band of ester in PCL film was shown at 1739 cm-1. All the investigated biopolymeric composites films presented bands at 1755 cm-1 (PC), 1764 and 1742 cm-1 (PCT1 and PCT2), 1765 and 1757 cm-1 ( PCCI1 and PCCI2), 1756 and 1764 cm-1 (PCC1 and PCC2). In the case of PCL, the ester bands were identified in other studies at 1750 cm-1[66], 1734 cm-1[62] and 1724 cm-1[67].

Figure 3. FTIR spectra of PCL/CS/EOs composites in the ratio 4:1:0.2 in comparison to PCL and PC

Figure 4. FTIR spectra of PCL/CS/EOs composites in the ratio 4:1:0.04 in comparison to PCL and PC In the case of the samples with incorporated EOs, which have chemical groups similar to

those of PCL and CS, it can be observed a difference between the bands in the regions 2800-3000 cm-1 and 1650-1800 cm-1. The modifications in those regions are attributed to the essential oil incorporation into the samples.

3.4. Quantitative assay of the Minimum Inhibitory Concentration (MIC) of EOs Antimicrobial activity of essential oils was confirmed by the MICs data. Microbial

susceptibility to the tested oils is shown in Table 3. CIN EO showed the highest antimicrobial activity against both Gram positive and Gram negative bacteria, followed by TEA and COR EOs. The MICs values confirm the strong inhibitory effect of CIN EO on a broad spectrum of bacterial strains. Similar studies have also reported the strong inhibitory effect of CIN EO against Staphylococcus aureus, Escherichia coli, including E. coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Salmonella typhimurium, MRSA, Streptococcus pyogenes, Bacillus cereus, Bacillus subtilis and Klebsiella pneumoniae [41, 51, 68-70].



Table 3. Minimum Inhibitory Concentration (MIC) of cinnamon, coriander and tea tree EOs.

Bacteria MIC (µl/ml)

Cinnamon EO (CIN)

Coriander EO (COR)

Tea tree EO (TEA)

Staphylococcus aureus ATCC 29213 0.39 6.25 3.13 Pseudomonas aeruginosa ATCC 27853 0.19 25 25

Escherichia coli ATCC 8730 0.39 25 6.25 Klebsiella pneumoniae IC 134202 0.78 12.5 12.5 Enterococcus fecalis ATCC 29212 1.56 25 25

The antimicrobial activity of the tested COR EO was better against the S. aureus ATCC

29213. Another study also reported that COR EO exhibited a broad spectrum inhibitory effect, S. aureus ATCC 25923 being the most sensitive, while P. aeruginosa ATCC 27853 more resistant [71]. In case of TEA EO, the results revealed that S. aureus and E. coli were more susceptible to the EO action. Similar MIC values were obtained for COR and TEA EOs on P. aeruginosa, K. pneumoniae and E. fecalis (Table 3).

Numerous studies have shown that the CIN EO broad spectrum antimicrobial activity is attributed to cinnamaldehyde. The lipophilicity or hydrophobicity and the chemical structure of the main compounds, such as the presence of functional polar groups and aromaticity have an important role in antimicrobial activity [51, 72, 73], these characteristics enabling the partition among the lipids of the bacterial or fungal cell membrane and mitochondria, disturbing the cellular structures and rendering them more permeable, which will lead to cell death [51, 74]. So, the antibacterial activity of cinnamaldehyde can be due to the lipophilicity of phenylpropanoids, which can penetrate the microbial membrane, react with the membrane enzymes and proteins as well as with the phospholipids bilayer, causing the impairment of microbial enzymatic systems and/or of the genetic material functionality [51, 68, 70]. Singh et al., (1995), cited by Sheng-Yang Wang et al., (2005) have shown that cinnamaldehyde is a strong antifungal agent [75, 76].

The essential oil of Melaleuca alternifolia (tea tree oil) consists largely of cyclic monoterpenes of which about 50% are oxygenated and about 50% are hydrocarbons. It exhibits a broad-spectrum antimicrobial activity that can be principally attributed to terpinen-4-ol [77-79].

The strong antimicrobial activity of cinnamon essential oil tested in this work could thus be attributed to the major product cinnamaldehyde (86.27%) and the antimicrobial activity of tea tree essential oil could be associated with the terpinen-4-ol (28, 87%). The presence of these chemical compounds was confirmed by GC-MS analysis.

However, it must be taken into account that the antimicrobial activity of the EOs is due to complex interactions between individual components that lead to an overall higher activity, as also demonstrated for COR EO as compared to its main constituent (linalool) [71, 80].

3.5. Anti-adherence properties of the obtained biopolymeric films In this study, we have also tested the anti-adherence activity of antimicrobial composites

based on polycaprolactone, chitosan and essential oils, against Staphylococcus aureus ATCC 29123 using viable cell counting assay. It was observed that the antimicrobial composite PCCI1 totally reduced (CFUs/ml = 0) the viable staphylococcal cells adhering to the tested surfaces, when comparing with other composites, and with control (PCL film). The other composites did not exhibit any anti-adherence activity (Table 4).

Table 4. CFUs of Staphylococcus aureus ATCC 29213, calculated after viable cell counts

assay by removing cells adhered on films surfaces at 24h.

Code sample PCL (film)

PC PCCI1 PCT1 PCC1 PCCI2 PCT2 PCC2

CFU/ml 1,93x107 2,83x107 0 2,73x107 1,36x107 2,8x107 3x107 2,1x107



These observations are consistent with the results of MIC, in other words, the anti-adherence activity against Staphylococcus aureus ATCC 29123, is due to the influence of EOs (CIN, COR and TEA). The quantitative assay (MIC) showed that CIN EO strongly inhibited S. aureus growth at very low MICs values, while TEA and COR EOs exercised inhibitory effect against S. aureus at higher MICs. Therefore, increasing staphylococcal adhesion observed in case of the PCCI2 composite, as compared to PCCI1 could be explained by the low amount of EOs incorporated into the polymeric material, unable provide an inhibitory effect and anti-adherence effect respectively, probable hypothesis in the case of composites based on COR and TEA EOs.

3.6. Cytotoxicity assay results Originally biocompatibility referred to the ability of a material to induce an appropriate

host response in a specific application. In vitro biocompatibility, or cytotoxicity, is generally evaluated through cell cultures systems. Hence, biocompatibility is a factor that must be considered before the selection of polymers to be used in the design of medical devices [14].

The obtained results showed that after 24h, although the morphology of HT29 cells developed under the films pieces was altered compared with control cells and the rest of the cells from the well (Figure 5), a > 90% cellular viability was registered for the majority of film pieces. However, in case of PCCI1 and PCCI2 the area with affected morphology was higher, and the viability decreased to nearly 80%. PCCI1 and PCCI2 pieces films increased cell mortality when were maintained in HT29 cells for 48h reaching 50% and respectively 60%.

Figure 5. Morphology of HT29 cells after 24 h, analyzed by Optical Inverted Microscope (OIM): A) control

HT29 cells; B) cells treated with PCL; C) cells treated with PC; D) cells treated with PCC1; E) cells treated with PCC2; F) cells treated with PCCI1; G) cells treated with PCCI2; H) cells treated with PCT1; I) cells treated with

PCT2 (x200).



These effects were confirmed by the appearance of a subG0 peak in cell cycle analysis, correlated with apoptosis (Figure 6). The peak was strongly increased in the case of PCCI1 and PCCI2. In adition, the PCCI1 and PCCI2 films modified the percent of cells in diferent cell cycle phases. In case of PCCI2 we noticed a slight increase in the S and G2 phases; these values were double in case of PCCI1 comparing to HT29 control cells.

Therefore, the alteration of cells morphology after 24 h of contact of HT29 cells with the films pieces, the decreasion of cell viability by 50-60% after 48h for HT29 cells maintained in the presence of PCCI1 and PCCI2 films pieces, the presence in both cases of a subG0 peak in cell cycle compared with control cells as well as modification the percent of cells in diferent cell cycle phases exercised by the PCCI1 and PCCI2 films suggest that the biopolymeric films exhibit cytotoxic effect.

Figure 6. Flow cytometry diagrams of cell cycle analysis of the HT29 cells treated with biopolymeric films

pieces: A) PCL film; B) PC film ; C) PCC1 film; D) PCC2 film; E) PCCI1 film; F) PCCI2 film; G) PCT1 film; H) PCT2 film; I) control HT29 cells.

Conclusions We report the fabrication of new antimicrobial and biocompatible composites based on

polycaprolactone (PCL), chitosan (CS) and essential oils (EOs) and their characterization by physico-chemical and biological methods. The obtained results suggest that the composites with the formulation PCL/CS/CIN (4:1:0.2) were resistant to staphylococcal cells adhesion.



The cytotoxic nature of these composites eliminating their applicability as biomaterials, but they could be represent an interesting approach for the food packaging and surfaces industry.

Acknowledgements

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI, project number 164/2012 and POSDRU/159/1.5/S/133391.

References

1. KATSIKOGIANNI M, MISSIRLISY F. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria material interactions. European Cells and Materials, l8, 37-57 (2004).

2. ZHANG W, CHU PK., JI J, ZHANG Y, LIU X, FU RKY, HA PCT, YAN Q. Plasma surface modification of poly vinyl chloride for improvement of antibacterial properties. Biomaterials, 27(1), 44-51 (2006).

3. JONES DS, DJOKIC J, GORMAN SP. The resistance of polyvinylpyrolidone iodine-poly(ε-caprolactone) blends to adherence of Escherichia coli. Biomaterials, 26(14), 2013-20 (2005).

4. BOWER CK, PARKER JE, HIGGIN AZ, OEST ME, WILSON JT, VALENTINE BA, BOTHWELL MK, McGUIRE J. Protein antimicrobials barriers to bacterial adhesion: in vitro and in vivo evaluation of nisin-treated implantable materials. Colloids and Surfaces B: Biointerfaces, 25(1), 81-90 (2002).

5. NICHOLAS RL, RAAD II. Management of bacterial complications in critically ill patients: surgical wound and catheter-related infections. Diagnose microbiology infectious disease, 33(2), 121–30 (1999).

6. VUONG C, OTTO M. Staphylococcus epidermidis infections. Microbes and infection, 4(4), 481–9, (2002). 7. THRELKELD MG, COBBS CG, MANDELL GL, DOUGLAS RG, BENNETT MJ. Principles and

practice of infectious disease. New York: Churchill Livingstone, pp. 783 (1995). 8. CHEN KS, KU YA, LEE CH, LIN HR, LIN FH, CHEN TM. Immobilization of chitosan gel with

cross-linking reagent on PNIPAAm gel/PP nonwoven composites surface. Materials Science and Engineerind: C, 26(2), 1–7 (2005).

9. ICONARU SL., CHAPON P, COUSTUMER P L, PREDOI D. Antimicrobial Activity of Thin Solid Films of Silver Doped Hydroxyapatite Prepared by Sol-Gel Method. The Scientific World Journal, doi:10.1155/2014/1653518.

10. KALYON BD, OLGUN U. Antimicrobial efficacy of triclosan-incorporated polymers. American Journal of Infection Control, 29 (20), 124–5 (2001).

11. CIOBANU CS, MASSUYEAU F, CONSTANTIN LV, PREDOI D. Structural and physical properties of antibacterial Ag-doped nano-hydroxyapatite synthesized at 100°C. Nanoscale Research Letters, 6 (2011).

12. ICONARU SL, CIOBANU CS, COUSTUMER PL, PREDOI D. Structural characterization and magnetic properties of iron oxides biological polymers. Journal of Superconductivity and Novel Magnetism, 25(8), 2547-2828 (2012).

13. CIOBANU CS, ICONARU SL, CHIFIRIUC MC, COSTESCU A, COUSTUMER PL, PREDOI D. Synthesis and antimicrobial activity of silver doped hydroxyapatite nanoparticles. Journal of Biomedicine and Biotechnology, http://dx.doi.org/10.1155/2013/916218, (2013).

14. WOODRUFF MA, HUTMACHER DW. The return of a forgotten polymer—Polycaprolactone in the 21st century. Progress in Polymer Science, 1-40 (2010).

15. VAN NATTA FJ, HILL JW, CARRUTHERS WH. Polymerization and ring formation ε-caprolactone and its polymers. Journal of the American Chemical Society, 56, 455-459 (1934).

16. COULEMBIER O, DEGEE P, HEDRICK JL, DUBOIS P. From controlled ring-opening polymerization to biodegradable aliphatic polyester: especially poly(beta-malic acid) derivatives. Progress in Polymer Sciences, 31, 723-747 (2006).

17. SMITH R. Biodegradable polymers for industrial applications. Woodhead Publishing Limited and CRC Press LLC, pp.4 (2005).

18. PATHIRAJA AG & ADHIKARI R. Biodegradable synthetic polymers for tissue engineering. Eurpean Cells and Materials, 5, 1-16 (2003).



19. RACHNA ND, HIREN JM., VENUGOPALAN PV. Novel Biocatalytic Polymer-Based Antimicrobial Coatings as Potential Ureteral Biomaterial: Preparation and In Vitro Performance Evaluation. Antimicrobial Agents and Chemotherapy, 55(2), 845–853 (2011).

20. MERKLI A, TABATABAY CR. GURNY, HELLER J. Biodegradable polymers for the controlled release oculars drugs. Progress in Polymers Sciences, 23, 563-580 (1998).

21. FREIBERG S & ZHU X. Polymer microspheres for controlled drug release. International Journal of Pharmaceutics, 282, 1-18 (2004).

22. SINHA VR, BANSAL K, KAUSHIK R, KUMRIA R, TREHAN A. Poly-epsilon-caprolactone microspheres and nanospheres: an overview. International Journal of Pharmaceutics, 278, 1-23 (2004).

23. RADHAKUMARY C, PRABHA D NAIR, MATHEW S, REGHUNADHAN CP NAIR. Biopolymer composite of chitosan and methyl methacrylate for medical applications. Trends in Biomaterials and Artificial Organs, 18(2), 117-124 (2005).

24. SARASAM A., DMYTRYK J, KHAJOTIA S, S MADIHALLY. Effect of blending with polycaprolactone on the anti-bacterial properties of chitosan for periodontal tissue engineering .http://madihally.okstate.edu/proceedings/OUpaper.pdf

25. KHOR E & LIM LY. Implantable applications of chitin and chitosan. Biomaterials, 24(13), 2339-49 (2003). 26. ALLAN CR, HARDWIGER LA. The fungicidal effect of chitosan on fungi of varying cell wall

composition. Experimental Mycology, 3, 285–287 (1979). 27. KONG M, CHEN XG, XING K, PARK HJ. Antimicrobial properties of chitosan and mode of action: A

state of the art review. International Journal of Food Microbiology, 144, 51–63 (2010). 28. SAHOO R, JAYSHREE SJP., MANIRAO R, SAHOO S. Blend effect of PCL on antimicrobial activity

of different molecular weight chitosan. European Scientific Journal, 10(21), 36-47 (2014). 29. WANG TJ, WANG IJ, LU JN, YOUNG TH. Novel chitosan-polycaprolactone blends as potential

scaffold and carrier for corneal endothelial transplantation. Molecular Vision, 18, 255–264 (2012). 30. ABDOLMOHAMMADI S, SIYAMAK S, IBRAHIM NA, YUNUS WMZW, RAHMAN MZA,

AZIZI S, FATEHI A. Enhancement of Mechanical and Thermal Properties of Polycaprolactone/ Chitosan Blend by Calcium Carbonate Nanoparticles. International Journal of Molecular Sciences, 13, 4508-4522 (2012).

31. NEVES SC, MOREIRA LST, MORONI L, RUI L, REIS CA VAN BLITTERSWIJK, ALVES NM, KARPERIEN M, MANO JF. Chitosan/Poly(ε-caprolactone) blend scaffolds for cartilage repair. Biomaterials, 32, 1068-1079 (2011).

32. ROOZBAHANI F, SULTANA N, ISMAIL AF, NOUPARVAR H. Effects of Chitosan Alkali Pretreatment on the Preparation of Electrospun PCL/Chitosan Blend Nanofibrous Scaffolds for Tissue Engineering Application. Journal of Nanomaterials,. doi:10.1155/2013/641502.

33. JIN RM, SULTANA N, BABA S, HAMDAN S, ISMAIL AF. Porous PCL/Chitosan and nHA/PCL/Chitosan Scaffolds for Tissue Engineering Applications: Fabrication and Evaluation. Journal of Nanomaterials, article ID 357372, 1-8 (2014).

34. SARASAM A, MADIHALLY SV. Characterization of chitosan–polycaprolactone blends for tissue engineering applications. Biomaterials, 26, 5500–5508 (2005).

35. ZHU Y, GAO C, LIU X, SHEN J. Surface Modification of Polycaprolactone Membrane via Aminolysis and Biomacromolecule Immobilization for Promoting Cytocompatibility of Human Endothelial Cells. Biomacromolecules, 3, 1312-1319 (2002).

36. BAKKALI F, AVERBECK S, AVERBECK D, IDAOMAR M. Biological effects of essential oils – A review .Food and Chemical Toxicology, 46, 446–475 (2008).

37. FALEIRO ML. The made of antimicrobial action of essential oils. Science against microbial pathogens: communicating current research and technological advances, 1143-1156 (2011).

38. MIGUEL MG, DUARTE J, FIGUEIREDO AC, BARROSO JG, PEDRO LG. Thymuscarnosus Boiss: effect of harvesting period, collection site and type of plant material on essential oil composition. Journal of Essential Oil Research, 17, 422-426 (2005).

39. ANGIONI A, BORRA A, CORONEO V, DESSI S, CABRAS P. Chimical composition seasonal variability, and antifungal activity of Lavandastoechas L. ssp. stoechas essential oils from stem/leaves and flowers. Journal of Agriculture and Food Chemistry, 54, 4364-4370 (2006).

40. FIGUEIREDO AC, BARROSO JG, PEDRO LG., SALGUEIRO L, MIGUEL MB, FALEIRO ML. Portuguese Thymbra and Thymus species volatiles: chemical composition and biological activities. Current Pharmaceutical Design, 14, 3120-3140 (2008).

41. SHAREEF AA. Evaluation of antibacterial activity of essential oils of Cinamomum sp. and Boswellia sp. Journal of Basrah Research, 37(5A), 60-71 (2011).



42. NIMBARTE S, KULKARNI A. Comparative phytochemical analysis and resilience pattern exhibited by thyme and tea tree oil against selected poultry isolates. Journal of Agriculture and Veterinary Science, 4(4), 113-117 (2013).

43. BURT S. Essential oils: their antimicrobial proprieties and potential application in foods-a review. International Journal of Food Microbiology, 94, 223-253 (2004).

44. AMR E. EDRIS. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: A Review. Phytotherapy Research, doi10.1002/ptr 2072 (2007).

45. SANCHEZ-GARCIA MD, OCIO MJ, GIMENEZ E, LAGARON JM. Novel polycaprolactone nanocomposites containing thymol of interest in antimicrobial film and coating applications. Journal of plastic film & sheeting, 24, 239-251 (2008).

46. RAILEANU M, TODAN L, VOICESCU M, CIUCULESCU C, MAGANU M. A way for improving the stability of the essential oils in an environmental friendly formulation. Materials Science and Engineering:C, 33(6), 3281-3288 (2013).

47. CHIFIRIUC MC, MIHAESCU G, LAZAR V. Medical microbiology and virology. Pub.by University of Bucharest, pp. 426-428 (2011).

48. HACHEM R, REITZEL R, BORNE A, JIANG Y, TINKEY P, UTHAMANTHIL R, CHANDRA J, GHANNOUM M, RAAD I. Novel antiseptic urinary catheters for prevention of urinary tract infections: correlation of in vivo and in vitro test results. Antimicrobial agents and chemotherapy, 53(12), 5145-5149 (2009).

49. CHAIBAN G, HANNA H, DVORAK T, RAAD I. A rapid method of impregnating endotracheal tubes and urinary catheters with gendine: a novel antiseptic agent. Journal of Antimicrobial Chemotherapy, 55, 51-56 (2005).

50. BURTON E, GAWANDE PV, YAKANDAWALA N., LoVETRI K., ZHANEL GG, ROMEO T, FRIESEN A. D., MADHYASTHA S. Antibiofilm Activity of GlmU Enzyme Inhibitors against Catheter-Associated Uropathogens. Antimicrobial agents and chemotherapy, 50(5), 1835-1840 (2006).

51. EL-BAROTY GS, ABD EL-BAKY HH, FARAG RS., SALEH MA. Caracterisation of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils. African Journal of Biochemistry Research, 4(6), 167-174 (2010).

52. JHAM GN., ONKAR DD, VANIA MMV. Identification of the major fungitoxic component of cinnamon bark oil. Fitopatolgia Brasileira, 30( 4), 404-407 (2005).

53. KOROCH A, RANARIVELO L, BEHRA O, JULIANI RH, SIMON JE. Quality attributes of ginger and cinnamon essential oils from Madagascar. Issue in new crops and new uses. Eds by J. Janick and A. Whipkey, 338-341 (2007).

54. SENANAYAKE UM, LEE TH., WILLS RBH. Volatile constituents of cinnamon (Cinamomum zeylancium) oils. Journal of Agricultural and Food Chemistry, 26, 822-824 (1978).

55. GUDDADARANGAVVANAHALLY KJ, LINGUMALLU JR, KUNNUMPURATH KS. Chemical composition of volatile oil from Cinnamomum zeylancium buds. Zeitschriftfür Naturforschung, 57c, 990-993 (2002).

56. MOMIN AH., ACHARYA SS., GAJJAR AV. Coriandrum stativum – review of advances in phytophar-macology. International Journal of Pharmaceutical Sciences and Research, 3(5), 1233-1239 (2012).

57. ZORCA M, GAINAR I, BALA D. Supercritical CO2 extraction of essential oil from coriander fruit. University of Bucharest annals, 2, 79-83 (2006).

58. NAZRUL IB, JARIPA B, MAHBUBA S. Chemical composition of leaf and seed essential oil of Coriandrum stativum L. from Bangladesh. Bangladesh Journal of Pharmacology, 4, 150-153 (2009).

59. FAROOQ A, MUHAMMAD S, ABDULLAH IH, NAZAMID S, SHAHID I, UMER R. Physicochemical composition of hydro-distilled essential oil from coriander (Coriandrum sativum L.) seeds cultivated in Pakistan. Journal of Medicinal Plant Research, 5(15), 3537-3544 (2011).

60. COX SD, MANN CM, MARKHAM JL, GUSTAFSON JE, WARMINGTON JR., GRANTB WS. Determining the antimicrobial actions of Tea Tree oil. Molecules, 6, 87-91 (2001).

61. HART PH, BRAND C, CARSON CF, RILEY TV, PRAGER RH, FINLAY-JONES JJ. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes . Inflammatory Research, 49, 619–626 (2000).

62. DUAN K., CHEN H, HUANG J, YU J, LIUN S., WANG D, LI Y. One-step synthesis of amino-reserved chitosan-graft-polycaprolactone as a promising substance of biomaterial. Carbohydrate Polymers, 80 (2), 498-503 (2010).



63. BURGNEROTTO J, LIZARDI J, GOYCOOLEA FM, ARGUELLES-MONAL W, DESBRIERES J, RINAUDO M. An infrared investigation in relation with chitin and chitosan characterization. Polymer, 42, 3569-3580 (2001).

64. FERNANDES LL., RESENDE CX., TAVARES DS, SOARES GA. Cytocompatibility of chitosan and collagen-chitosan scaffolds for tissue engineering. Polymer, 21(1), 1-6 (2011).

65. WANG T, TURHAN M, GUNASEKARAN S. Selected propriettes of pH-sensitive biodegradable chitosan- poly(vinyl alcohol) hydrogel. Polymer International, 53, 911-918 (2004).

66. SOUSA I, MENDES A, BARTOLO PJ. PCL scaffolds with collagen bioactivator for application in tissue engineering. Procedia Engineering, 59, 279-284 (2013).

67. TRIPATHY AR., MacKNIGHT WJ., KUKUREKA SN. In-situ copolymerization of cyclic poly(butylene terephthalate) oligomers and ε-caprolactone. Macromolecules, 37, 6793-6800 (2004).

68. HUELANDER IM, ALAKOMI HL, LATVAKALA K., MATTILA SANDHOLM T, POL L, SMID EJ., GORRIS LGM, VON WRIGHT A. Caracterisation of the action of selected essential oil component on Gram- negative bacteria. Journal of Agriculture and Food Chemistry,46, 3590-3595 (1998).

69. FRIEDMAN M, P HENIKA R, MANDRELL RE. Bactericidal activities of plant essential oils and some of their isolated constituents against campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. Journal of Food Protection, 65(10), 1545-1560 (2002).

70. BABU AJ, SUNDARI AR, INDUMATHI J, SURJAN RVN, SRAVANTHI M. Study on the antimicrobial activity and Minimum Inhibitory Concentration of essential oils of spices. Veterinary World, 4, (7), 311-316 (2011).

71. SILVA F, FERREIRA S, QUEIROS JA. Coriander (Coriandrum stativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry. Jurnal of Medical Microbiology, 60, 1479-1486 (2011).

72. FARAG RS, DAW ZY, ABO-RAYA SH. Influence of some essential oils on Aspergillus parasiticus growth and aflatoxins production in a synthetic medium. J. food Sci., 54, 74-6 (1989b).

73. DAW ZY, EL-BAROTY GS, MAHMOUD AE. Inhibition of Aspergillus parasiticus growth and aflatoxin production by some essential oils. Chem. Mikrobiol. Technol. Lebensm., 16(5/6), 129-135 (1994).

74. SIKKEMA J, DE BONT JAM, POOLMAN B. Interaction of cyclic hydrocarbons with biological membranes. J. Biol. Chem., 269, 8022-8028 (1994).

75. SINGH HB., SRIVASTAVA M, SINGH AB, SRIVASTAVA AK. Cinnamon bark oil, a potent fungitoxicant against fungi causing respiratory tract mycoses. Allergy, 50, 995–999 (1995).

76. WANG SY, CHEN PF, CHANG ST. Antifungal activities of essential oils and their constituents from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood decay fungi. Bioresource Technology, 96, 813–818 (2005).

77. ANGELINI P, PAGIOTTI R, GRANETTI B. Effect of antimicrobial activity of Melaleuca alternifolia essential oil on antagonistic potential of Pleurotus species against Trichoderma harzianum in dual culture. World J. Microbiol. Biotechnol., 24, 197–202 (2008).

78. RICCIONIAND L & ORZALI L. Activity of Tea Tree (Melaleuca alternifolia, Cheel) and thyme (Thymus vulgaris, Linnaeus). Essential Oils against Some Pathogenic Seed Borne Fungi. Journal of Essential Oil Research, 23, 43-47 (2011).

79. RADULOVI NS, BLAGOJEVI PD, STOJANOVI-RADI ZZ, STOJANOVI NM. Antimicrobial Plant Metabolites: Structural Diversity and Mechanism of Action. Current Medicinal Chemistry, 20, 932-952 (2013).

80. DUMAN AD, TELCI I, DAYISOYLU KS, DIGRAK M, DEMIRTAS I, ALMA MH. Evaluation of bioactivity of linalool-rich essential oils from Ocimum basilucum and Coriandrum sativum varieties. Nat. Prod.Commun., 5, 969–974 (2010).

Fabrication, characterization and bioevaluation of novel ... · as antimicrobials agents. The...

Documents

Transcript of Fabrication, characterization and bioevaluation of novel ... · as antimicrobials agents. The...