Propolis Extract-PVA Nanocomposites of Textile Design : Antimicrobial Effect on Gram Positive and Negative Bacterias

Potential antimicrobial efficiency of propolis extract (bee glue) was experimentally studied on gram positive and negative bacterias by manufacturing propolis extract-based textiles. Pre-samples were prepared by varying percentange concentration of propolis extract in PVA polymer solution and homojenic solutions were electrospun onto polypropylene nonwoven fabric. In-vitro experiments showed that antimicrobial efficiency of extract-containing nanocamposite samples were better than those of not including. According to investigations nanocomposite fabrics with propolis extract sol. were provided antimicrobial effect against to gram positive bacteria (S. aureus) but not to gram negative bacteria (A baumannii and P. aeruginosa). The results indicated that the electrospun PVA/propolis extract nanocomposites provided a good means for healing of wounds or decreasing infection proliferation caused by gram positive bacteria.


INTRODUCTION
Medical textiles contribute to increase on quality of health service.Improving functionality of textiles used in hospitals can limit some problems due to infection spreading.Bed coverings, bedspreadings and some products for patients' personel care are commonly used for supplying sublevel hygenic demands.In order to prevent from hospital infections, these products can be manufactured with pharmasotical materials by considering controversal effects of bacterias causing these problems.By means of bacteria types, it is seen that commonlyknown hospital infections are results of gram positive (Staphylococcus aureus) and gram negative (Klebsiella pneumoniae, Enterobacter spp.and E. Coli from Enterobacteriaceae; Pseudomonas aeruginosa, A. Baumannii and Stenotrophomonas maltophilia from nonpermentative gram-negatives and less frequently Burkholderia cepacia) bacterias.Due to increased drug resistance of these bacterias, preventing of hospital infections take great attention for both patience and personnel health [1].
As considering natural-based pharmasotics, propolis has become a popular material due to its abundant phenolic compounds, mainly with potential antioxidant namely "flavonoids" and aromatic acids.It is a strongly adhesive resinous substance used in traditional medicine and been reported to have a broad spectrum of biological activities, such as anticancer, antioxidant, antiinflammatory, antibiotic, and antifungal activities [2][3][4][5][6][7].
It has recently become popular as a health drink and it has been claimed to prevent diseases such as dermatological wounds, inflammation, heart disease, diabetes, cancer, etc.These facts encourage researchers to draw interest in the extraction of flavonoids from propolis esters and adapt these flavonoids to care products [2,[8][9][10] By means of adapting pharmasotics or drug delivery systems onto textile materials, electrospinning is the most preferable method due to producing compatible nanofibers from a large variety of bulk starting materials.Electrospun bicomponent nanofibrous fabrics with high specific surface area, aspect ratio and porosity as a result of random deposition of fibers, could have a great potential in biomedical applications such as tissue engineering scaffolds, drug delivery carriers, wound dressings etc. [11,12].By handling of electrospun pharmasotic material, healing period of wounds can be limited and their high surface area of electrospun surfaces can provide optimum healing dosage by using low pharmasotic material extense.
In this study, aqueous poly (vinyl alcohol) (PVA) and propolis extract solutions were blended in proper volume ratio and were electrospun under constant conditions.Electrospun PVA/Propolis extract nanofibrous structure were manufacturedand their wound healing performances and bacterial proliferation rates were macroscopically compared by counting colony occured on both subject and control agars.

Electrospinnability of Materials
PVA polymer (Mw:80.000g/mol) was dissolved in distilled water at 60 o C during 8 hours.Propolis extract was diluted with ethanol by concentration of 3% in weight.3% propolis/ethanol solutions were added into 5%, 7%, 9% and 11%PVA/water solutions drop by drop and mixtures were stirred during 15 hours at room temperature.Homogenized solutions were electropun onto PP nonwoven surface (17 g/m 2 ).Potential differences between the tip and the counter electrode (collector) used to electrospin the polymer /propolis solutions were 37 kV.Stationary collector covered with aluminum foil, placed 10 cm above the capillary tip, was used to collect the electrospun fiber material.Feeding rate was undercontrolled during manufacturing and it was adjusted to 2.5 ml/h.This study was carried out by following two stages: (a) electrospinnability of propolis by keeping constant propolis amount in polymer solutions to observe fiber formation; (b) efficiency of propolis/PVA nanostructures electrospun from solutions containing different amounts of propolis extract.Samples were characterised by SEM method and their antimicrobial efficiency were tested against to gram positive bacteria (S. aureus) and gram negative bacteria (A baumannii and P. aeruginosa) either in serum physiologic and human blood.

SEM Micrographs of Electrospun Samples
Figure 3 (a) shows that there is no fiber formation on electrospinning of 3% propolis extract solution and electrospun surface of propolis extract is composed of nearly particular substances.Electrospinning of PVA polymer solution succesfully provides nanoweb formation in various concentration rates, meanly in Figure 3  Easily electrospinnability of PVA polymer solution contribute to adaptable of propolis by getting further away from electrospraying.Electrospinnability of propolis by help of polymer solutions has been investigated with other types of polymers in recent studies [13,14].
By means of fiber fineness, it is manufactured finer nanofibers from 100% PVA polymer solution than those from propolis-containing polymer solutions.Addition of propolis into PVA polymer solution cause nano/micro-sized fiber manufacturing with thicker diameter and homogeneous structural properties.Figure 4 shows the compatibility of propolis extract on PVA polymer.At decreasing concentration of PVA polymer in polymer/propolis mixture, beading formation and clustering are seen on electrospun structure.There is no seen any beading formation on higher concentrations of PVA polymer.From Figure 4, it is observed that network clusters are occured in propolis containing nanofibers.But electrospun fibers from 100% PVA polymer solution or high PVA-containing mixtures exhibits more regular, uniform fiber formation and less network clustering.

Efficiency of Samples on Gram-Negative/-Positive Bacterias in Serum Physiologic Solutions
Subculturation of samples of 0.5 McFarland bacteria, serum physiologic solution and with/without propolis nanostructure is illustrated in Figure 5 and Figure 6. Figure 5 shows subculturation of bacteria and serum physiologic mixture on blood agar and colony proliferation are available for each bacteria types.100 colonies are proliferated in sample shown in Figure 5 (a).However, there are4 colonies in Figure 6 (a).It is seen that propolis nanostructure containing solutions prevents proliferation of Staphylococcus aureus colonies effectively but not of Acinetobacter baumannii and Pseudomonas aeruginosa [15].This proves that propolis extract containing nanostructure is effective on gram positive bacteria, Staphylococcus aureus [16][17][18][19].

Efficiency of Samples on Gram-Negative/-Positive Bacterias in Human Blood
Subculturation of controls (solution in which propolis containing electrospun is not penetrated) result to occurence of 100 colonies profileration.The least proliferation among control solution subculturation is observed in Figure 8c as 60 colonies.But all subculturations from propolis containing solutions, namely illustrated as"sample", lead to decrease on proliferation of S. Aureus bacterias although different concentration amount in polymer solutions.This case declares the effectiveness of propolis extract on preventing proliferation of S. Aureus bacterias in human blood [20].

CONCLUSION
Electrospinnability of propolis is possible by addition of this material into easily electrospun biocompatible polimeric solution.Micro/nanocomposite structure is obtained by mixing propolis extract into PVA polymer solution.According to experiments electropun fabrics with propolis sol., especially 11%, were provided antimicrobial effect against to gram positive bacteria (S. aureus) and not provided antimicrobial effect against to gram negative bacteria (A baumannii and P. aeruginosa).According to studies, S.aureus bacteria is the most initiative and encountered bacteria type among hospital bacterias.By adapting propolis onto hospital textiles or patients'personal care products, hospital infections caused by S.aureus bacteria can be limited by these resistive natural pharmasotic, propolis.

Figure 3 .
Figure3(a) shows that there is no fiber formation on electrospinning of 3% propolis extract solution and electrospun surface of propolis extract is composed of nearly particular substances.Electrospinning of PVA polymer solution succesfully provides nanoweb formation in various concentration rates, meanly in Figure3 (b).According to SEM micrographs, it is seen that propolis extract can be electrospun by adding into PVA polymer solution.