Balaure PC Revista de Chimie 5_2012

8/12/2019 Balaure PC Revista de Chimie 5_2012

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REV. CHIM. (Bucharest) 63 No. 5 2012 http://www.revistadechimie.ro 531

Biocompatible Magnetic MWCNTs Based on PhytocomponentsfromEugenia carryophyllata

CRINA SAVIUC1, ALEXANDRU MIHAI GRUMEZESCU2, OTILIA BANU3, CARMEN CHIFIRIUC1*, DAN MIHAIESCU2,

PAUL BALAURE2, VERONICA LAZAR1

1University of Bucharest, Faculty of Biology, 1-3 Portocalelor Alley, Bucharest, Romania2 Politehnica University of Bucharest Faculty of Applied Chemistry and Materials Science, 17 Polizu Str., 011061 Bucharest,

Romania3 Emergency Institute for Cardiovascular Diseases Prof. Dr.C.C. Iliescu, 258 Fundeni Road, 022328, Bucharest, Romania

The aim of the present study was the phytocomponents extraction from the aromatic waters of Eugeniacarryophyllata by magnetic MWCNT encapusaltion, in order to obtain biocompatible nanoparticles withclinical applications. Eugenia carryophyllata dried buds were hydrodistilled, in microwave conditions, usinga Neo-Clevenger device. The aqueous residue was separated in two equal parts, one for the chloroformextraction and the other for the extraction with magnetic MWCNT. The chemical composition of the aqueousresidue extracts was established by GC-MS analyses. The high content of eugenol and -cariophyllen wasproven in both magnetic MWCNT assisted and chloroform extraction. The MWCNT antimicrobial activitywas tested against 20 Staphylococcus aureus strains. The MIC value for all tested strains was 50mg/mL,smaller concentrations being promoters for bacterial growth probably due to the large surface offered byMWCNT for microbial adherence and to the subinhibitory concentration of encapuslated phytocomponents

with antimicrobial activity.

Keywords: magnetic nanoparticles, Eugenia carryophyllata, encapsulated phytocomponents, MWCNT

Nanoparticles are generally defined as having carbonnanotubes (CNTs) have a lot of applications since theywere discovered. CNTs are crystalline graphiticsheets rolledup into a seamless cylindrical shape [1]. Since theirdiscovery diverse and unique physical properties ofCNTs

have been revealed. Therefore, many novel applicationssuch as nanotube-based magnets, solar cells,superconductors, displays, air pollution filter, hydrogenstorage, medicine and clinical microbiology have beenconceived. However, due to their macromolecular structureand their affinity to agglomerate, carbon nanotubes areinsoluble[2]. This is a serious problem that obstructs theiruniform incorporation into matrices for the fabrication ofadvanced materials. This deficiency can be attenuatedthrough functionalization of carbon nanotube surfaces[3].Therefore, in many cases, carbon nanotubes of either multiwalled (MWCNT) or single-walled (SWCNT) structure areoxidized along their sidewalls in a non destructive way bya HCl/HNO3treatment which functionalizes the surfaces

through hydroxyl, carboxyl, and carbonyl groups [4]. Themagnetic properties of MWCNTs make them possessunique potential applications as magnetic data storage[5],magnetic force microscopes [6], microwave absorbingmaterials [7], or sau magnetic nanomaterials for drugdelivery[8-10].

Essential oils are complex mixtures of phyto-components with synergic or concerted action. Thefitochemistry studies of different plant extracts are orientedto the component isolation and identification from complexmixtures in order to establish correlations between thestructure and the biological and pharmacological activity,respectively. The determination of the essential oil

pharmacological activity is relatively difficult due to theirintra and interspecific variability, and to the workingconditions (low solubility in aqueous media, high volatility).

* email: [email protected]

The simplification of the phytocomponents complexmixture from the essential oils considerably reduces thesynergic/concerted action implications, thus allowing thedetermination of the role of the isolate component. Forthat purpose, aromatic waters represent a remarkablealternative due to the fact that they contain principalcomponents of aqueous medium solubilized oils.

The antimicrobial activity of some volatile oils and theircomponents was proven towards a large variety ofmicroorganisms, fungi, Gram-positive and Gram-negativebacteria. It seems like Gram-negative bacteria are moreresistant to the activity of biocides obtained from plants,due to lipopolisacharides that are found in the externalmembrane; but there are also exceptions [11,12]. Bacterialresistance at drug substances together with the declineregistered in the formulation of new antibiotics representa threat for human health [13].

In this context, our study focused on the developmentof a new, easy and rapid method of obtaining encapsulated

magnetic MWCNT for biomedical applications, based onthe antimicrobial activity of phytocomponents extractedfrom aromatic waters ofE. carryophyllata.

Experimental partAromatic water preparation from Eugenia carryophyllatasamples

E. carryophyllatadried buds were purchased from a localsupplier and used for the essential oil extraction [14]. Threeextractions were performed in microwave conditions usinga Neo Clevenger type apparatus. The essential oils and thearomatic water were collected separately [15]. In scheme1 the experimental steps are presented.

Synthesis and characterization of magnetic nanoparticlesMagnetic MWCNTs were synthesized by plasma processing

and purified as follows: solvent extraction (with benzene,


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dichloromethane and o-dichlorobenzene, successively),elimination of inorganic impurities by nitric acid/concentratedhydrochloric acid treatment, washing with ultra-pure water,thermal treatment at 300 oC. The morphology was determined

by Transmission Electron Microscopy (TEM). A TEM image ofthe obtained magnetic nanoparticles is presented in figure 1.

Phytocomponents extraction from aromatic watersAqueous residues from the hydrodistillationofEugenia

carryophyllatasamples were combined and filtered, 500mL of aromatic water being obtained. This was extractedby two procedures: simple solvent extraction (withchloroform) and magnetic MWCNT extraction (scheme2) using 800 mg of nanoparticles. For the phytocomponentsencapsulation, the nanoparticle-aromatic water mixturewas stirred for 30 min in order to suspend the nanotubesand then felt in a magnetic field for 5 min. The resulted

nanoparticles were isolated by placing a strong magnetNdFeB, (100 kgf) on the bottom of the suspension beaker,the aqueous residue being thus eliminated [16].

The isolated encapsulated MWCNTs were washed withultra-pure water (3200 mL) to remove the hydrosolublecompounds. Encapsulated MWCNTs were divided in twohalfs, one being used in the GC-MS analysis and the otherone for the biological activity testing.

GC-MS analysisGas chromatographic analysis was performed using an

Agilent 6890 Series GC System. Detection was carried outwith a 5973 mass-selective single quadrupole detector(Agilent technologies). Operation control and data processwere carried out by Agilent Technologies ChemStationsoftware (Santa Clara, CA, USA). The mass spectrometerwas calibrated before use with perfluorotributy lamine(PFTBA) as a calibration standard.

Scheme 1. Schematic diagram of theexperimental part

Scheme 2. Phytocomponentsextraction procedure from aromatic

water, using magnetic MWCNTs


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The working conditions were: H2-carrier gas, flow: 1,2mL/min, temperature program 50/300oC with a ramp rateof 5oC/min; the temperature of the injector and of thedetector was 250oC, and a DB5-MS (30m; 0.25 mm id;0.25 m) column.

Antimicrobial activity determination of magnetic MWCNTsMicrobial strains

The antimicrobial activity of MWCNTs was testedagainst 20Staphylococcus aureusstrains, recently isolatedfrom clinical specimens, and a reference strain. The strainswere identified with a Vitek automatic system II [17]. The

bacterial suspensions used were obtained from bacterialcultures of 15-18 h grown in solid media (Chapmanmedium), that were adjusted at an optic density appropriatewith the McFarland 0.5 standard (containing 1.5x108colonyforming units (UFC)/mL) [18].

Table 1

Quantitative determination of minimal inhibitoryconcentration (MIC) by serial microdilution technique

The MIC value [19] for MWCNT was determined bytwofold microdilution technique, in 96 multi-well plates,starting from 50 to 0.39mg/mL, for each tested bacterialstrain. Positive and negative controll (serial dillutions forMWCNT) was used for microbial growth. The plates wereincubated for 24 h at 37C, and MIC values were determinedby macroscopic examination [20] (as the lowestconcentration of compound which inhibited the microbialgrowth) and spectrofotometric (A620 for the obtainedmicrobial cultures) [21].

Results and discussionsHR-TEM characterisation

Two representative HR-TEM images of MWCNTs areshown in figure 1. The multiple graphitic walls of the CNTsare clearly visible in this TEM image that also shows a 10-11 nm diameter tube.

Fig. 1. Transmission ElectronMicroscopy images of the

MWCNTs


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GC-MS analysisTotal ion chromatograms (TIC) for the two extraction

methods are presented in figure 2. The identifiedphytocomponents were the same in both methods:eugenol and -cariophyllene. The principal componentspercentage was 92.42% and 5.44% from the total area, forthe MWCNT extraction.

Minimal Inhibitory ConcentrationThe MIC value for all tested strains was the same (50mg/

mL). The subinhibitory concentrations are bacterial growthpromoters, fact that can be attributed to the large surfaceoffered by the MWCNTs for microbial adherence (thisfavours the metabolic process acceleration) as well as tothe low concentrations of antimicrobial compounds fromcharged MWCNT (fig. 3).

ConclusionsEncapsulation of carbon nanotubes was proven to be

an efficient method of extraction of aromatic waterscompounds, in comparison with the control extraction withorganic solvent. The TIC analysis for the two extractionmethods chloroform and MWCNTs has demonstrated

the presence of principal components from the E.carriophyllata volatile oil, eugenol and -cariophyllene, inthe aromatic water. Also, it was proven that thephytocomponents extracted from the E. carriophyllataaromatic water exhibit antibacterial activity on all of theS.

Fig. 2. Aromatic water chromatogram after extraction with magnetic MWCNTs (a) and chloroform extraction (b)

Fig. 3. Graphical representation of microbial cultures optical densities at 24h of incubation (A490 nm), at differentconcentrations of the coated magnetic MWCNTs

aureus tested strains, at 50 mg/mL, while lowerconcentrations stimulated the bacterial growth. This wasprobably due to the specific nature of MWCNTs that offer alarge contact surface for microbial adherence.

In conclusion, magnetic MWCNTs can become efficienttarget systems for natural compounds with antimicrobialactivity, having applications in the anti-infectious therapyfield.

Acknowledgment: The present study was financed by the POSDRU107/1.5/S/80765 European Program and Human Resources no. 135/2010 (Contract nr. 76/2010).

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Manuscript received: 14.11.2011

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