Bio-nanofibrous mats as potential delivering systems of natural substances

Abstract

Considering the increasing resistance of numerous bacteria to antibiotics, a novel wound dressing material was developed with naturally acquired olive leaf extract, which shows not only good antimicrobial activity, but also very good antioxidant activity. Besides that, the leaves are treated as waste in agriculture, giving an impact on waste management. An environmentally friendly procedure, electrospinning, was used for the first time to prepare polysaccharide nanofibrous mats with incorporated olive leaf extract, with the unique property of releasing the active phenolic components in a prolonged manner over 24 hours. The developed electrospun mats were characterized using scanning electron microscopy, high-performance liquid chromatography and ultraviolet-visible spectroscopy for determination of free radical scavenging activity by 2,2-diphenyl-1-picrylhydrazyl, antimicrobial testing and release kinetics. Antimicrobial tests have shown that electrospun mats with olive leaf extract achieve reduction towards the tested microorganisms: Staphylococcus aureus (G+), Escherichia coli (G-), Enterococcus faecalis (G+) and Pseudomonas aeruginosa (G-), while the high antioxidant activity of olive leaf extract was preserved during the electrospinning procedure. Release of olive leaf extract from electrospun mats was mathematically modeled, and the release kinetics evaluation indicates the appropriateness of the Korsmeyer–Peppas model for fitting the obtained results of release ability due to erosion of polysaccharide nanofiber mats.

Keywords

electrospinning olive leaf extract release antioxidant activity antimicrobial activity

The treatment of complex wounds on different areas represents a challenging area worldwide.¹ Wound infections are the most expensive complications following surgery and are still a major source of bacteria that drive the nosocomial infections rates in hospitals. In European hospitals the overall rates of surgical site infections range between 3% and 4% of patients undergoing surgery.² Wound infections are also one of more serious complications related particularly to chronic wounds. The burden of treating chronic wounds is growing rapidly due to increasing healthcare costs,³ an aging population⁴ and a sharp rise in the incidence of diabetes⁵ and obesity⁶ worldwide.

In order to prevent wound infections and promote healing, dressings with incorporated antimicrobial active agents have been developed.^7–18 Many of these active components are poorly water soluble, cause skin irritation, lose their functionality when in contact with wound-fluid and might be either very toxic or not safe for patients^19,20 and the environment. Owing to the mentioned possible unwanted side effects of synthetic drugs as used in local therapies, natural extracts have gained particular importance over recent years.^21–23 Bearing this in mind, olive leaves were used in this study, since they contain natural phenolic components with many beneficial properties for health.^24–26 The two main phenolic compounds in olive leaves are oleuropein (OLE) and its hydrolytic derivative hydroxytyrosol (HTS), which are known to possess antibacterial, antifungal, antioxidative and antiviral properties. OLE contributes close to 75% of total phenolics in olive leaves,²⁷ whereas HTS is usually present in smaller amounts (depending on the amount of hydrolyzed OLE). Although HTS concentration in olive leaves is significantly lower than that of OLE, studies suggest that the contribution to the powerful antioxidant and antimicrobial activities of olive leaf extracts comes from HTS, which is currently classified as one of the most potent natural antioxidants discovered to date.²⁸ With the exploitation of rich phenolic compounds in olive leaves, the environmental impact in the research study was introduced, since olive leaves represent a great amount of waste material in olive production. It is known that during the yearly spring pruning, each olive tree produces around 25 kg of olive leaves, that is, agricultural waste.²⁹

Textural properties for the dressing formulation are also of great importance.^30,31 The materials produced by electrospinning possess by their nature properties showing advantages compared to fibers made by other methods.^32–36 The nanofiber morphology enables excellent comfortability³⁷ and significant water absorption capability,³⁸ as well as promoting wound healing by stopping bleeding (i.e. hemostasis) by exchange of gases (i.e. respiration) and by introducing biopolymers directly to the damaged skin for covering of the wound, protection against infection and elimination of scars forming.³⁹ In addition, the ease of incorporation of active components into nanofibers via a spinning process makes them extra attractive as an advanced wound dressing. The incorporated ingredient plays an active role in the wound healing process. Besides the effectiveness of the active ingredient, the release pattern is also of great importance. Controlled delivery dressing is an excellent mean for delivering active substances to the wound site in a consistent and sustained way over a desired period of time. Naturally derived polymeric dressings are potentially useful in the treatment of local infections, where it is beneficial to have increased local concentration avoiding an excess beyond the wound site.

The desired antimicrobial/antioxidative properties are usually obtained by adding synthetic components (such as drugs, nanoparticles, nanocapsules, etc.) into polymer solution or attached onto the formed nanofiber surface. The possible toxic impact or side effects of certain synthetic components has resulted in growing interest in using alternative natural incidences. Components extracted from olive leaves have potential applications promoting health. Therefore, our unique approach was to use the olive leaf extract, which has antimicrobial/antioxidative properties, prepared by environmentally friendly water extraction combined with solvent-free electrospinning. The success of formation of three-dimensional (3D) mats using the polymer solutions was evaluated by scanning electron microscopy (SEM). The presence of phenolic components was determined by high-performance liquid chromatography (HPLC) analysis. The antioxidative effectiveness of the incorporated active components was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging, while the antimicrobial properties were tested on four types of bacteria, mostly present in infected skin wounds. An ultraviolet-visible (UV/VIS) spectrophotometer allowing continuous on-line monitoring was used to obtain an overview of the 24 h release mechanism.

Experimental details

Material and chemicals

Fresh olive leaves were collected as waste from spring cutting (March 2012) of trees (Portorož, Slovenia). The leaves were dried at 40℃ in a tray air dryer, packed tightly and stored in a dry and dark place until their further use. Before each experiment the leaves were grinded on a laboratory scale, in small quantities, so the heating of the raw material was minimal.

Carboxymethyl cellulose (CMC; average molecular weight (Mw): 90.000 g/mol, degree of substitution (DS): 0.70; ACROS Organics, Geel, Belgium), alginic acid sodium salt from brown algae (NaAlg; Mw: 120 000 g/mol; M:G ratio 1.56; Sigma Aldrich Chemie GmbH, Steinheim, Germany), calcium chloride (CaCl₂; Mw: 110.98 g/mol, dehydrated, granulated, purity of 97%, Sigma Aldrich GmbH, Steinheim, Germany) and polyethylene oxide (PEO; Mw: 600 000 g/mol, ACROS Organics, Geel, Belgium) were used to prepare the basic solution to be blended with olive leaf extract. Separate solutions of CMC (7% w/v), NaAlg (2.5% w/v), CaCl₂ (1% w/v) and PEO (5% w/v) were prepared using Milli-Q water.

Pegatex® S non-woven, kindly supplied by PEGAS NONWOVENS s.r.o. (Znojmo, Czech Republic) was used as a mat for nanofiber deposition. The mat is a non-woven fabric manufactured by means of spun bond technology made from 100% polypropylene (PP) fibers.

Methanol for HPLC analysis was purchased from Sigma-Aldrich (Slovenia) and acetic acid was purchased from Merck (Germany). Standards, HTS (≥98%) and OLE (≥80%), were purchased from Extrasynthese (France).

The stable radical DPPH• for evaluation of radical scavenging effectiveness was purchased from Sigma-Aldrich (Slovenia).

Preparation of olive leaf extract

To obtain the olive leaf extract, olive leaves were extracted conventionally with mixing in solvent (water). Approximately 300 g of freshly grinded olive leaves and 3000 mL of water were added to a 4 L flask. The suspension was heated to a temperature of 80℃ while being constantly stirred. When the desired temperature was reached (after approximately 1 h) a timer was turned on. After 2 h of extraction, the suspension was cooled and filtered. The solvent was evaporated until dryness using a vacuum evaporator (Bűchi Labortechnik AG, Switzerland). The obtained extract was stored at −20℃ in darkness until further use.

Preparation of polymer solutions

Solutions of CMC, NaAlg and CaCl₂ were mixed in volume ratio of 12:4:1 (CMC:NaAlg:CaCl₂). Such a prepared solution was stirred for 15 minutes using a propeller stirrer with three blades (RE 10, IDL GmbH&CO KG, Nidderau, Germany). The solution was then combined with PEO solution (5% w/v) in the ratio of 50:50 v/v. During constant stirring, the 5.21 g (173.66 mg/mL) of olive leaf extract was progressively added to the 30 mL of solution, allowing smooth dissolving. The solution was then stirred for 24 h at room temperature, protected from light and evaporation.

Polymer blend characterization

Prior to the electrospinning process, the physical properties of the prepared blends, which significantly affect the nanofiber mat forming, were investigated. The conductivity measurements were made using a conductometer Seven Multi from Mettler Toledo International, Inc., Greifensee, Switzerland. Viscosity experiments were performed with a rotational viscometer from Fungilab (model Smart series, Barcelona, Spain). Surface tension was determined using a pedant drop method from DataPhysics Instruments GmbH (model OCA 35, Filderstadt, Germany). All measurements were performed at a room temperature and were repeated at least three times.

Formation of a 3D electrospun mat

The nanofibers from the polymer solution were spun onto a PP mat using a pilot-scale Nanospider NS 500 (ELMARCO; Czech Republic) apparatus. The scheme is presented in Figure 1.

Figure 1.

Scheme of Nanospider NS 500 (ELMARCO; Czech Republic) apparatus working principle. PP: polypropylene.

The electrospinning set consists of two electrodes. The rotating cylinder electrode, as a polymer solution feeding unit, is placed on the bottom and connected to a high-voltage distributor (0–75 kV). This electrode is partly immersed into the polymer solution placed in the tray, with the rotation speed of 3.8 rpm. The second electrode, called the collecting electrode, is placed on the top of the apparatus. The formed nanofibers are collected onto the PP mat placed before the collecting electrode. The distance from the feeding unit and the collector electrode to the PP mat was fixed at 16 cm. The stable polymer jets were maintained at a constant level at a voltage of 60 kV. The nanofibers were formed at room temperature (20 ± 2℃) and 30 ± 2% relative humidity. A total of 30 mL of polymer solution was used to cover the 49.7 cm × 56 cm PP mat within 30–40 minutes.

Scanning electron microscopy

The morphology of the electrospun nanofibers was investigated using a scanning electron microscope. The samples were fixed onto the sample holder using double-sided adhesive carbon tape. The SEM images were obtained by using a FE-SEM-ZEISS Gemini Supra 35 VP (Carl Zeiss AG, Oberkochen, Germany) scanning system with the acceleration voltage of 1 kV. In the basis of SEM images, the average electrospun fibers diameter was determined on 50 measurements per sample using ImageJ, an image processing program.

High-performance liquid chromatography

The content of HTS and OLE in olive leaf extract was analyzed by HPLC. The Agilent 1100 system (Agilent Technologies, Santa Clara, CA, USA) consisted of a binary pump, column heater, autosampler and variable wavelength detector (VWD). The chromatographic separation was performed on an Agilent Eclipse XDB-C18 (Agilent Technologies, Santa Clara, CA, USA) column (150 mm × 4.6 mm) with 5 µm particle size. The mobile phase consisted of two solvents, that is, methanol (eluent A) and 2% acetic acid in Milli-Q water (eluent B). The solvent gradient was as follows: 0 min 95%B, 10 min 35%B, 25 min 60%B, 30 min 75%B, 36 min 80%B, 37 min 5%B. The solvent flow rate was 1 mL/min, with column temperature set to 25℃. Detection of HTS and OLE was performed at 280 nm. Quantification was done by using calibration curves obtained with standards. For each sample, analysis was performed three times and results were expressed with their mean ± standard deviation.

Determination of free radical scavenging activity by DPPH•

Radical scavenging activities of samples were determined using the UV-VIS spectrophotometric DPPH• method.⁴⁰ A 6·10⁻⁵M solution of DPPH• in methanol was prepared prior to measurements. A total of 3 mL of this solution was added to 77 µL of extract solution (concentration = 1 mg/mL, dissolved in methanol) and incubated in a dark room for 15 min. After incubation the absorbance at 515 nm was measured using a Cary 50 UV-VIS spectrophotometer (Varian, USA). The reference solution was prepared similarly, but instead of sample solution, methanol was used, with absorbance being measured immediately at 515 nm. Antioxidant activity is expressed as percentage of inhibition towards the reference solution and is calculated using equation (1)

% inhibition = (\frac{A_{c}^{0} - A_{s}^{15}}{A_{c}^{0}}) \cdot 100 %

(1)

where $A_{c}^{0}$ represents the absorbance of the blank at 0 min and $A_{s}^{15}$ the absorbance of the sample after 15 min of incubation. Each sample was measured three times and results were expressed as mean values ± standard deviation.

The effective concentration of sample required to scavenge DPPH• radicals by 50% (IC₅₀ value) was obtained by linear regression analysis of the dose–response curve plotted as percentage inhibition versus concentration.

Determination of antibacterial activity

The antimicrobial activity of the polymer solutions and nanofibrous PP mats was determined using a modified version of the American Association of Textile Chemists and Colorists (AATCC-100-1999) test method. According to this, the antimicrobial activity of the sample is evaluated upon direct contact of microorganisms with the test substrate. According to the AATCC 100-1999 standard, the results are obtained quantitatively by comparing results from the test sample with the control sample.

The AATCC 100-1999 test was used to determine the antimicrobial activity of the test substrate. The samples of surface bound material were shaked in concentrated bacterial suspension (contact time – 1 h). In this way prepared suspensions were serially diluted before and after contact and cultivation. Viable organisms in the suspension were counted and the percent reduction was calculated based on initial counts or on retrievals from appropriate untreated controls. A more detailed description can be found elsewhere.⁴¹

A total of 0.5 mL of polymer solution and 1 g of electrospun nanofibers were used to determine the antimicrobial activity of four challenged microorganism species, that is, Staphylococcus aureus (G+) (DSM 799), Escherichia coli (G-) (DSM 1576), Enterococcus faecalis (G+) (ATCC 29212) and Pseudomonas aeruginosa (G-) (ATCC 27853). These microorganisms were chosen because they are the leading cause of healthcare-associated infections. S. aureus is the fourth-most-common hospital-acquired pathogen among older adults, following Escherichia coli, Pseudomonas aeruginosa and Enterococci, and it accounts for 9% of all nosocomial infections. In addition, S. aureus is the second-most-common cause of surgical site infection, accounting for 14% of the cases of this type of infection.⁴² Microorganisms routinely isolated from burn wounds include aerobic organisms like Staphylococcus aureus and E. coli.⁴³ Enterococci are common nosocomial pathogens that affect elderly patients with underlying disease and other immunocompromised patients. E. faecalis is consistently the second or third most common agent in wound infections and bacteraemia in hospitals.⁴⁴ Pseudomonas aeruginosa is an opportunistic pathogen that frequently causes hospital-acquired infections, particularly to burn patients, and patients with chronic debility. Pseudomonas flourishes on moist skin, such as improperly attended wounds.

Monitoring the olive leaf extract release using UV/VIS spectroscopy

The release study of olive leaf extract was undertaken on a tailor-made system, with the schematic presentation in Figure 2, which simulates the release of active compounds in an open wound system.

Figure 2.

Schematic overview of performed monitoring of olive leaf extracts release from the electrospun sample. PET: polyethylene terephthalate; PUR: polyurethane; PBS: phosphate.

The system consisted of a polyurethane (PUR) carrier with dimensions of 10 cm in length, 8 cm in width and 1 cm in thickness. Polyethylene terephthalate (PET) mesh was fixed on the top of this carrier using a silicon thread. The carrier was floating in the squared vessel containing 100 mL of phosphate (PBS) buffer (pH = 7.4). The vessel containing the floated sample was placed in the larger water-filled vessel. The temperature and stirring regulation apparatus was placed below the water-filled vessel, assuring an unchanged set-up of the temperature (37 ± 2℃) during the whole period of measuring. The two vessels contained a magnet allowing the constant stirring of both liquids.

The electrospun sample was placed on the PUR carrier with PET mesh. The vessel containing the sample was covered with parafilm and aluminum foil to minimize evaporation and oxidation. Using an electronic pipette, 1 mL of liquid sample was taken from the PBS-filled vessel in defined time intervals and put into a small plastic vessel and immediately replaced with 1 ml of fresh PBS buffer solution. The first sampling was immediately after sample–PBS contact, the second after 30 minutes and after that every 1 hour up to 24 hours. The total loaded amount of extract on the electrospun mat was determined by shaking the sample (1 cm × 1 cm; 1.47 mg) in PBS solution (7 mL) for 24 h. The amount of loaded and released extract components by the electrospun sample was determined spectrophotometrically. The UV/VIS measurements were performed using the Cary 60 UV-VIS Spectrophotometer (Agilent Technologies, Inc., Santa Clara, USA). The Xenon flash lamp and a double beam Czerny-Turner monochromator was used to measure absorbance at a wavelength of 230 nm at 1.5 nm fixed spectral bandwidth. The instrument has a submersible sensor enabling the measurement of UV/VIS spectra directly in liquid media with a wavelength accuracy of ±0.06 at 541.94 nm and a wavelength reproducibility of ±0.01 nm.

For fitting the results of the release performance of the as-prepared electrospun mats with incorporated olive leaf extract, several existing models were used. The zero-order, the first-order, the Higuchi and Korsmeyer–Peppas mathematical models for predictions of the release kinetics were used.^45–47

Zero-order model

To study the release kinetics of zero order, data obtained from in vitro release studies were plotted as the cumulative amount of extract components released versus time and can be represented by the following equation

Q_{t} = Q_{0} - K_{0} t

(2)

where Q_t is the amount of extract components dissolved in time t, Q₀ is the initial amount of extract components in the solution and K₀ is the zero-order release constant expressed in units of concentration/time.

First-order model

The data obtained were plotted as the log cumulative percentage of extract components remaining versus time for the first-order model, which would yield a straight line with a slope of –K/2.303

\log C = \log C_{0} - Kt / 2.303

(3)

where C₀ is the initial concentration of extract components, K is the first-order rate constant and t is the time.

Higuchi model

For the Higuchi model, data obtained were plotted as a cumulative percentage of extract component release versus square root of time

Q = {KHt}^{1 / 2}

(4)

where KH is the Higuchi dissolution constant.

Korsmeyer–Peppas model

The Korsmeyer–Peppas Model models the release of a molecule from a polymeric matrix, such as a hydrogel. To study the release kinetics by the Korsmeyer–Peppas model, data obtained from in vitro release studies were plotted as log cumulative percentage extract component release versus log time

Q / Q_{0} = Ktn

(5)

where Q/Q₀ is a fraction of the extract component release at time t, K is the constant and n is the diffusion constant that indicates the general operating release mechanism.

It is known that the Peppas model is widely used to confirm whether the release mechanism is Fickian diffusion or non-Fickian diffusion. The “n” (release exponent of the Korsmeyer–Peppas model) value could be used to characterize different release mechanisms. The interpretation of n values is as follows:

n < 0.5—quasi Fickian diffusion;

n = 0.5—diffusion mechanism;

0.5 < n < 1—anomalous (non-Fickian) diffusion—both diffusion and relaxation (erosion);

n = 1—case 2 transport (zero-order release);

n > 1—super case II transport (relaxation).

Results

Characterization of pure olive leaf extract

Several reports have been published on the active phenolic compounds in olive leaves^48–50 and their combined effect in terms of antioxidant and antimicrobial activities,^51,52 while the phenolics combined with polysaccharides have not been fully investigated. Therefore, prior to preparing the electrospun solutions for forming the nanofibrous mats, the pure extract was characterized to provide basic data to understand the role of olive leaf extract in the formed electrospun material.

Despite many known reports confirming the presence of phenolic components in olive leaves,²⁷ the HPLC analyses were performed on pure olive leaf extract used. Figure 3 presents the chromatogram and the amount of phenolic compounds of interest, that is, OLE and HTS, in extract.

Figure 3.

The chromatogram of pure olive leaf extract measured at 280 nm. HTS: hydroxytyrosol; OLE: oleuropein.

From Figure 3 it can be observed that the two main phenolic constituents, OLE and its hydrolytic derivative HTS, elute at the approximate retention times of 5.9 and 18.8 min, respectively, collectively contributing to 23.9% of mass of extract. Other peaks probably correspond to the other important phenolic compounds found in Olea europaea (e.g. tyrosol, verbascoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside, etc.)²⁷ that are visible at the same UV wavelength.

The radical scavenging DPPH• method was used to determine the antioxidative properties of the olive leaf extract. The extract concentration necessary for radical inhibition is shown in Figure 4.

Figure 4.

The percentage inhibition of 2,2-diphenyl-1-picrylhydrazyl radicals plotted against the concentration of olive leaf extract in water.

From Figure 4 it can be observed that the 50% inhibition of DPPH• radicals (IC₅₀) is obtained at an olive leaf extract concentration of approximately 0.96 mg/mL. The maximally obtainable radical inhibition (approximately 90%) is obtained at a concentration of >2 mg/mL. Further increase of the extract’s concentration does not increase its inhibitory effect.

The antimicrobial properties of pure olive leaf extract were determined using only two of the selected bacteria, that is, E. coli and P. aeruginosa, since several studies have been published reporting the minimal inhibitory concentration of olive leaf extract.⁵³ In order to use the same procedure as described in the AATCC 100-199 standard, the olive leaf extract was dissolved in Milli-Q water (173.66 mg/mL). The pure extract, in both cases, showed 100% reduction of the selected bacteria.

Characterization of polymer solutions

The nanofiber mats, as produced by the electrospinning process, result because of potential gradient between the electrode and collector . Since the biopolymers used were in the form of solution, the optimal properties of the blend are essential in order to obtain accurate control in producing nanofibers forming nonwoven webs. The solution properties that significantly affect electrospinning were determined and are listed in Table 1.

Table 1.

The properties of solution, as used for the nanofiber formation

Sample	Parameters influencing electrospinning			Parameters promoting wound healing
Sample	Conductivity (mS/cm)	Viscosity (mPa·s)	Surface tension (mN/m)	Phenolic content (mg/g blend)	IC₅₀ mg/mL	Antimicrobial activity %
Polymer solution without extract	4.4 ± 0.8	1499.0 ± 202.0	60.8 ± 0.7	0	No antioxidant activity	No reduction for all four tested bacteria
Polymer solution with extract	8.6 ± 0.7	2993.7 ± 389.5	48.6 ± 4.7	39.8 ± 0.0 _OLE 1.7 ± 0.0 _HTS	2.4 ± 0.4	100% reduction for all four tested bacteria

From the obtained results in Table 1, it can be seen that the addition of olive leaf extract markedly increased the spinning formulation conductivity as well as viscosity, while the surface tension, comparing with the spinning formulation without olive leaf extract, is significantly reduced.

In Figure 5, the obtained HPLC chromatograms of the polymer solution with and without extract are presented, while the quantitative amounts of phenolic components are listed in Table 1.

Figure 5.

The chromatograms of polymer solution without olive leaf extract (carboxymethyl cellulose (CMC)-NaAlg-CaCl₂/polyethylene oxide (PEO) 50:50) and polymer solution with olive leaf extract (CMC-NaAlg-CaCl₂-extract/PEO 50:50). HTS: hydroxytyrosol; OLE: oleuropein.

Figure 5 shows that the polymer solution (CMC-NaAlg-CaCl₂/PEO 50:50) has no visible peaks at the measured UV wavelength for the applied HPLC method, meaning that no other compounds interfere in the blend at the observed wavelength before the electrospinning process. The chromatogram of polymer solution with incorporated olive leaf extract (c = 173.66 mg/mL) therefore has the same characteristic peaks as the chromatogram of pure olive leaf extract (Figure 3).

The antioxidative and antimicrobial activities, as listed in Table 1, of both the polymer blends with and without extract were determined in order to predict the properties promoting wound healing of nanospun fibers. The antioxidative activity of the polymer solution without incorporated olive leaf extract equals zero regardless of measured concentration, meaning that the polymer solution does not exhibit any antioxidant activity, whereas with incorporated extract the effective concentration for inhibition of 50% of DPPH• radicals equaled 2.45 ± 0.36 mg/mL, which is reasonably higher than by the pure olive leaf extract. As expected, polymer solution without extract was not effective against the selected microorganisms, while a 100% reduction for all four tested bacteria was evidenced by the polymer solution with incorporated extract.

The morphology of electrospun mats

The electrospun mats were produced through a pilot electrospinning machine using fabrication parameters as follows: voltage 60 kV, distance 16 cm and fabrication duration 30–40 min. The nano-morphology of the electrospun polysaccharide fibers without and with incorporated olive leaf extract is shown in Figures 6(a) and (b), respectively.

Figure 6.

Electrospun mat (a) without olive leaf extract, (b) with olive leaf extract and (c) polypropylene support mat after the olive leaf extract release.

In the case of electrospun mats without extract, the fiber diameter was 157 ± 23 nm, while using the extract the diameter was 167 ± 42 nm. In addition, SEM figures were also obtained after the 24 h release of extract from the electrospun mats, as presented in Figure 6(c). According to the SEM micrographs, one can see that formation of a nanofiber layer on PP support material was obtained using polysaccharide solution without olive leaf extract present (see Figure 6(a)). In this case, the fiber morphology shows a bead-like structure on the overall length of the formed fibers, while with the addition of olive leaf extract formed fibers show more uniform morphology (see Figure 6(b)). The SEM analysis of the electrospun nanofiber layer on the PP support material after performing the 24 h release shows that the total degradation of polysaccharide nanofibers appeared. On supporting PP material only some residue (see Figure 6(c)) can be observed.

Phenolic content of electrospun mat

The contents of OLE and HTS in the electrospun mats with and without incorporated olive leaf extract were determined in order to define any loss of phenolic compounds during the electrospinning process. For that purpose, a solution of electrospun mat with the same concentration of extract as the polymer blend was prepared to enable analysis with HPLC. Representative chromatograms with given OLE and HTS concentrations are presented in Figure 7.

Figure 7.

The chromatograms of electrospun mats with and without incorporated olive leaf extract. HTS: hydroxytyrosol; OLE: oleuropein.

From Figure 7, it can be observed that the electrospinning process of the electrospun mat without incorporated extract does not result in any changes regarding formation of products that are visible at 280 nm, since no peaks can be noticed. On the other hand, the chromatogram of the electrospun mat with incorporated extract is different from that in Figure 5. The height of the characteristic peak at the retention time of 16 min increased, meaning that changes in composition of the extract occurred. Secondly, the amounts of HTS and OLE have changed (HTS concentration increased slightly, whereas OLE concentration decreased slightly). Nevertheless, the observed difference in HTS and OLE concentration is minimal, since their concentrations are equal to 1.85 ± 0.01 and 38.90 ± 0.04 mg/g mat, respectively, what are similar to concentrations in polymer solution before electrospinning (see Table 1).

Antioxidant activity of electrospun mats

The results of antioxidant activity are presented in Figure 8 and again the material without incorporated olive leaf extract shows no radical scavenging activity regardless of concentration, whereas the antioxidant activity of electrospun mats with incorporated olive leaf extract equals 81.04 ± 2.10%. This inhibition refers to olive leaf extract concentration of 14.89 mg/ml in electrospun mats, while the effective concentration of 50% of DPPH• radicals amounted to 2.43 ± 0.28 mg/mL.

Figure 8.

Inhibition of 2,2-diphenyl-1-picrylhydrazyl by electrospun mat without and with incorporated olive leaf extract.

This value is almost the same as the result obtained for the activity measured for the polymer blend with added extract before electrospinning. Both solutions were of the same concentration and, since no significant difference can be seen by inhibition of 50%, one can conclude that the electrospinning process did not worsen the antioxidative properties of olive leaf extract.

Antimicrobial efficiency of electrospun mats

The efficiency of natural extract of olive leaves, commonly used in botanic medicine to treat fever and overcome infections, incorporated into polysaccharide nanofibers, against pathogenic bacteria species was examined. Representative bacteria types were selected based on antimicrobial assessment of infected skin wounds. The rate of bacteria inhibition, expressed as reduction, is listed in Table 2.

Table 2.

The efficiency of olive leaf extract–polysaccharide electrospun mats on the reduction of pathogenic bacteria

Sample	Reduction (R) for pathogenic bacteria (%)
	Gram-positive bacteria		Gram-negative bacteria
	Staphylococcus aureus	Enterococcus faecalis	Escherichia coli	Pseudomonas aeruginosa
Electrospun mat without extract	No reduction	No reduction	No reduction	No reduction
Electrospun mat with extract	97	100	100	100

As well as by polymer solution without extract, also the electrospun mat sample without incorporated extract showed no antibacterial efficiency. The bacteria counts did not differ between electrospun mat and polymer solution when taking into account the olive leaf extract.

Release studies of phenolic compounds from electrospun mats

In addition to structural control of electrospun mats, loading of a natural additive for making the nanofibrous mat applicable in a biological environment was a significant parameter pointing the present study towards wound dressing applications. Since the growth rate of bacteria in a wound is highly dependent on the rate of the additive release from nanofiber electrospun mats, the release studies were carried out for a period of 24 h. The samples were floated on the PBS solution having a pH of 7.4 and kept at a constant temperature of 37 ± 2℃, simulating the infected wound environment. Namely, it is known⁵⁴ that the mean value of skin pH is 7.54 ± 0.09, while the pH of a chronic wound site can vary from 7.5 to 8.9,⁵⁵ whereas the presence of bacteria may cause a decrease in pH value. Bacteria can survive at a range of pH levels, while keeping their internal pH fairly constant. Therefore, it is possible that several strains of bacteria grow best in an environment of higher (or more alkaline) pH levels.⁵⁶ The skin temperature can also vary depending on the body location and the external conditions, averaging 2–4℃ less than the core temperature.⁵⁷

Prior to monitoring the 24 h release, all components were analyzed with UV/VIS spectroscopy in order to evaluate their absorption maxima in the wavelength region of 200–400 nm. The collected UV/VIS spectra are presented in Figure 9.

Figure 9.

Absorption measurements of individual components ((a) carboxymethyl cellulose (CMC); (b) NaAlg and (c) polyethylene oxide (PEO)) and (d) electrospun mats (with and without extract) in the region from 200 to 400 nm .

UV/VIS spectra from individual components present in the electrospun matrix (CMC, NaAlg and PEO), as well as electrospun mats, without olive leaf extract, exhibit no typical absorption maxima in this wavelength region. However, in absorption spectra of electrospun mats with olive leaf extract three characteristic absorption peaks are present at 320, 280 and 230 nm. Absorption maxima at 320 nm is characteristic for caffeic acid,⁵⁸ as well as probably for other compounds present in the extract, while the absorption maxima at 230 and 280 nm are characteristic for OLE and HTS. Since the absorption maxima at 230 nm is on the UV/VIS spectra detection limit, where also some spectra noise is observed, the absorption maxima at 280 nm was chosen to be used for monitoring the release kinetics. The absorption maxima at 280 nm are demonstrated to be purely olive leaf extract and the degradation of CMC, NaAlg and PEO, as matrix components, were not detected during the release study.

The amount of released olive leaf extract from the electrospun mat was determined by UV spectroscopy using a pre-determined calibration curve. In Figure 10 the release profile of extract from electrospun mats is presented, as a function of 24 h monitoring.

Figure 10.

The release profile of olive leaf extract from electrospun mats monitored at 280 nm.

The results evidence that 75% of herbal extract is released within the first hour, while the remaining 15% is released during the prolonged time of 24 hours.

In order to understand the kinetics and mechanism of extract release, the results of the in vitro release study are presented in Figure 11.

Figure 11.

The release kinetics model of olive leaf extract from electrospun mats (a) the zero-order model; (b) the first-order model; (c) the Higuchi model; and d) the Korsmeyer–Peppas model.

The obtained results were fitted using various models, such as the zero-order model (cumulative percentage release versus time) (see Figure 11(a)), the first-order model (log of percentage of extract compounds remaining versus time) (see Figure 11(b)), the Higuchi model (cumulative percentage release versus square root of time) (see Figure 11(c)) and the Korsmeyer–Peppas plot (log of cumulative percentage release versus log of time) (see Figure 11(d)). Moreover, R² (coefficient of correlation) values were calculated for the linear curves obtained by regression analysis of the above plots and are also shown in Figure 11. The zero-order model was used since it is appropriate for understanding the slow release, while the first-order and Higuchi models were used for water-soluble components incorporated in porous matrices and the Korsmeyer–Peppas model for interpreting the diffusional release mechanisms from the polymeric system.

The regression coefficient (R²) of the zero-order, first-order and Higuchi model curves varies from 0.41 to 0.72. Due to the deviation of regression coefficients, these curves preclude the possibility of the mentioned model kinetics. The highest value of regression coefficient suggested that olive leaf extract release is followed by the Korsmeyer–Peppas model release kinetics. The value of the regression coefficient is 0.96, while the n value for the Korsmeyer–Peppas model is 1.657, indicating a super case II transport release. For systems exhibiting super case II transport, the dominant mechanism for drug transport is polymer relaxation. For our materials, the super case II transport mechanism was obtained due to the relaxation of the polymer chain and dissolution of the polymeric matrix.

Discussion

Effect of polymer solution on the 3D formation of electrospun mats

Electrospun nanofibrous mats with their porous nature could represent excellent functional wound dressing materials due to desired properties associated in a single layer and obtained by a one-step procedure.⁵⁹ However, the polymer solution properties with regard to the process parameters are the two more important parameters affecting the spinning ability and fiber morphology, which should be taken into account.

Regarding the formation of electrospun mats with incorporated olive leaf extract, it was found that when comparing with and without olive leaf extract-loaded polymer solution properties, one can see that the addition of olive leaf extract caused an increase in polymer solution viscosity and conductivity. The increase in solution viscosity can be attributed to the van der Waals interactions (orientational interaction and H-bonding) between CMC, NaAlg and olive leaf extract. The conductivity increase can be explained by dissociation of carboxylic groups present in the molecular structure of olive leaf extract (OLE compound). The latter affected the electrospinning process in such a way that the intense stretching of the polymer solution jets, from the surface of the spinning electrode towards the collecting electrode, that is, PP support materials, occurred. Higher level of charges carried by the polymer solution contributed to intense stretching of the polymer solution jets, resulting in more uniform and bead-free fibers (see Figure 6(b)). Equally, the reduction of surface tension with the addition of olive leaf extract in the spinning formulation resulted in more uniform fibers, since the surface tension reduced the surface area per unit mass of a liquid, while the electric charges on the electrospinning jet increased the surface area through elongation. Therefore, the addition of olive leaf extract resulted in more fine and uniform fibers, which posess, due to their small diameter, high surface area, which would effect the dissolution rate of olive leaf extract.

Effect of phenolic compounds on antioxidant and antimicrobial properties

As anticipated, the antioxidant activity was observed only by samples with incorporated extract; a little lower activity resulted by electrospun mats compared to polymeric solution. The antioxidant activity could be explained by the extract composition. As shown in Figures 5 and 7, the HTS and the OLE are the two components mostly present in the olive leaf extract and are well-known as antioxidants.^60,61 Some research studies report that the quality and the quantity of phenolic compounds that could consequently manifest in different properties depend mainly on the geographic region of the olive tree plantation,^62,63 seasonal variations and environmental factors,⁶⁴ and its cultivation and harvesting techniques.^65,66. The results of the present study indicate the high antimicrobial activity of olive leaf extract incorporated in electrospun mats against both the Gram-positive and Gram-negative bacteria (see Table 2). A slightly lower activity (i.e. 97%) was obtained against S. aureus, a microorganism that has a capacity to produce enterotoxins and is known for its unique resistance (among Gram-positive bacteria) to natural phenolic compounds.

A study by Capasso and colleagues⁶⁷ proved that HTS exhibits antimicrobial properties and is non-toxic for human cells. OLE has been shown to possess an inhibitory effect against several microorganisms.⁶¹ However, the obstruction mechanism is still not well-defined, while some indices (also for HTS) pointed towards an o-diphenolic system.⁶⁸ Delaquis and co-workers⁶⁹ demonstrated that extract, as provided from nature, might even show better microorganism constraint compared to one particular active component. This fact might be explained due to synergistic effects between different active components present in the extract.

The safety issue is also important factor, especially when taking into account the use for human beings. Olive products are considered to be safe since we used them daily, mainly as food. The polyphenols have been reported to be non-toxic⁶⁷ and highly antioxidative;⁶⁰ however, a few studies indicated the pro-oxidant activity of natural polyphenols.⁷⁰ According to the presented results, it is interesting to predict that olive leaf extract could be a promising antioxidant/antimicrobial agent for treating infected wounds; however, some further studies in vivo are still needed to prove these expectations.

Effect of active compound release on wound healing

Different kinetic equations were used to monitor the active component release from the electrospun mat. In Figure 11, the regression coefficient values (R²) are presented for all four models used. The highest coefficient value, that is, R²= 0.96 and the slope with n = 1.657 was obtained by the Korsmeyer–Peppas model. The calculated “n” factor indicates super case II transport, where the process of relaxation dominates. This is also confirmed by the SEM images of electrospun extract loaded mats after the release (see Figure 6(c)), where nanofibers are not noticeable on the support PP mat any more. The results in Figure 10 show that 50% of active component release occurred immediately after the dry electrospun extract loaded mat came into contact with aqueous solution, while within the following 60 min the additional 25% of active component release was evident. After that, the continuous release of the remaining active components in the next 24 h is evident. It is known that the release of active components from the substrate is conditioned by several physical processes. Taking into account the obtained release results (see Figures 10 and 11), one could predict that the erosion followed by the diffusion would be one of more reasonable scenarios in the case when the mat contacts the wound. The fluid from the wound would penetrate into the mat. Consequently, the mat would relax and erosion followed by finally dissolution of the mat would occur. Swelling and dissolution is another set-up that might also occur simultaneously. Bearing this in mind, one could conclude that the rate of active component release depends on how quick the medium (i.e. PBS and wound fluids in the real environment) is able to penetrate into the material matrix. In addition, one could observe that erosion is the foremost factor in most of the release mechanisms, as also confirmed by the Korsmeyer–Peppas model results (see Figure 11(d)). The results evidenced that the active substances could be gradually delivered to the wound within the period of 24 h. The developed electrospun mats with included olive leaf extract is a naturally derived polymeric material that is biodegradable. This is of certain benefit once the active components are delivered to the wound, promoting the desired effects. Based on the above, the proposed electrospun mats could be possibly applied as dressing for treating infected wounds by preventing bacteria growth and thereby reducing patients’ complications that might lead to prolonged healing and, consequently, to increased treatment costs.

Conclusion

In the present study, the challenge to design a material, acting as a vehicle to deliver natural agents to promote antimicrobial and antioxidative properties and thus promoting wound healing, was introduced. The olive leaf, treated as waste, was used to prepare the natural extract, demonstrating the potential economic and social importance and, above all, the benefit to be derived from utilization of its by-products. The electrospinning technique was employed to fabricate the nanofibers from natural polymers with incorporated natural extract, thus making the designed matrix favored over a synthetic one due to its biocompatibility and biodegradability, as well for opening new possibilities for producing a new generation of dressing materials.

It was demonstrated that incorporation of olive leaf extract effects simultaneously on increase of polymer solution’s conductivity and decrease its surface tension, resulting in bead-free nanofiber formation. The presence of phenolic components in electrospun mats, mainly detected as OLE and HTS, was responsible for a noticeable antioxidant effect, which is desired as resistance towards oxidative damages having an impact on human health. The electrospun mat with incorporated natural agent successfully inhibited the growth of bacteria that are mostly responsible for infections by skin wounds. The nanofibers with the olive leaf extract showed a sustained release profile, since 50% of the active compounds were released immediately, while the remaining amount were released within 24 h. Applying various kinetic models, the release data were best fitted to the Korsmeyer–Peppas model, indicating erosion as a dominant release mechanism.

Overall, this study demonstrates for the first time the ability to electrospin carboxylmethylcellulose-alginate-olive leaf extract complex, used as effective, sustained and human-safe delivery of therapeutic agents to infected skin wounds. However, still some research regarding time-controlled release, aging studies and, above all, clinical trials to examine the safety and efficiency are needed and are planned to be carried out in the future.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research work was produced within the framework of the operation entitled “Centre of Open innovation and ResEarch UM”. The operation is co-funded by the European Regional Development Fund and conducted within the framework of the Operational Programme for Strengthening Regional Development Potentials for the period 2007–2013, development priority 1: “Competitiveness of companies and research excellence”, priority axis 1.1: “Encouraging competitive potential of enterprises and research excellence”. The study was co-produced within the project No. L2-5492, supported by the Slovenian Research Agency.

References

Kruse

. Evaluation and treatment of diabetic foot ulcers. Clin Diabetes 2006; 24: 91–93.

Posnett

Gottrup

Lundgren

. The resource impact of wounds on health-care providers in Europe. J Wound Care 2009; 18: 154–161.

Gottrup

Holstein

Jørgensen

. A new concept of a multidisciplinary wound healing center and a national expert function of wound healing. Arch Surg 2001; 136: 765–772.

AoA – American Administration of Ageing. A profile of older Americans, http://www.aoa.acl.gov/Aging_Statistics/Profile/index.aspx (2013, accessed 4 May 2015).

Wild

Roglic

Green

. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004; 27: 1047–1053.

Brown

Neville

Rhee

. The impact of obesity on the outcomes of 1,153 critically injured blunt trauma patients. J Trauma 2005; 59: 1048–1051.

Watanakunakorn

Glotzbecker

. In vitro activity of sisomicin and netilmicin alone and in combination with nafcillin, oxacillin and methicillin against enterococci. Chemotherapy 1978; 24: 236–239.

Lee

Huang

. Zinc sulphadiazines: novel topical antimicrobial agents for burns. J Pharm Pharmacol 1995; 47: 503–509.

Hart

Silcock

Gunnigle

. The role of oxidised regenerated cellulose/collagen in wound repair: Effects in vitro on fibroblast biology and in vivo in a model of compromised healing. Int J Biochem Cell Biol 2002; 34: 1557–1570.

10.

Gilbert

Moore

. Cationic antiseptics: Diversity of action under a common epithet. J Appl Microbiol 2005; 99: 703–715.

11.

Esteban-Cubillo

Pecharromán

Aguilar

. Antibacterial activity of copper monodispersed nanoparticles into sepiolite. J Mater Sci 2006; 41: 5208–5212.

12.

Zhang

. Preparation and antibacterial activity of electrospun chitosan/poly(ethylene oxide) membranes containing silver nanoparticles. Coll Polym Sci 2009; 287: 1425–1434.

13.

Fras Zemljič

Peršin

Stenius

. Improvement of chitosan adsorption onto cellulosic fabrics by plasma treatment. Biomacromolecules 2009; 10: 1181–1187.

14.

Madhumathi

Sudheesh Kumar

Abhilash

. Development of novel chitin/nanosilver composite scaffolds for wound dressing applications. J Mater Sci Mater Med 2010; 21: 807–813.

15.

Cady

Behnke

Strickland

. Copper-based nanostructured coatings on natural cellulose: nanocomposites exhibiting rapid and efficient inhibition of a multi-drug resistant wound pathogen, A. baumannii, and mammalian cell biocompatibility in vitro. Adv Funct Mater 2011; 21: 2506–2514.

16.

Kontogiannopoulos

Assimopoulou

Tsivintzelis

. Electrospun fiber mats containing shikonin and derivatives with potential biomedical applications. Int J Pharm 2011; 409: 216–228.

17.

Mikhaylova

Hradsky

Kreutz

. Between promiscuity and specificity: novel roles of EF-hand calcium sensors in neuronal Ca2+ signalling. J Neurochem 2011; 118: 695–713.

18.

Breitwieser

Spirk

Fasl

. Design of simultaneous antimicrobial and anticoagulant surfaces based on nanoparticles and polysaccharides. J Mater Chem B 2013; 1: 2022–2030.

19.

Fraser

Cuttle

Kempf

. Cytotoxicity of topical antimicrobial agents used in burn wounds in Australasia. ANZ J Surg 2004; 74: 139–142.

20.

Melchior

Bovine mastitis and biofilms. In: Percival

Cochrane

Costerton

(eds). Biofilms and veterinary medicine. Springer Series on Biofilms 6, Berlin, Heidelberg: Springer, 2011, pp. 205–221.

21.

Maver

Stana Kleinschek

. A review of herbal medicines in wound healing. Int J Dermatol 2015; 54: 740–751.

22.

Chen SR, Chen XP, Lu JJ, et al. Potent natural products and herbal medicines for treating liver fibrosis. Chin Med 2015; 10: 7.

23.

Wang

Que

Wang

. Chinese herbal medicines for treating skin and soft-tissue infections. Cochrane Db Syst Rev 2014; 7: CD010619–CD010619.

24.

Erbay

Icier

. The importance and potential uses of olive leaves. Food Rev Int 2010; 26: 319–334.

25.

Han

Talorete

Yamada

. Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotech 2009; 59: 45–53.

26.

Tuck

Hayball

. Major phenolic compounds in olive oil: metabolism and health effects. J Nutr Biochem 2002; 13: 636–644.

27.

Pereira

Ferreira

Marcelino

. Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobrancosa) leaves. Molecules 2007; 12: 1153–1162.

28.

Richards KL. The most powerful natural antioxidant discovered to date - hydroxytyrosol, www.ProHealth.com, http://www.prohealth.com/library/showarticle.cfm?libid=17054 (31 October 2014, accessed 25 April 2015).

29.

Xie

Huang

Zhang

. Reduced pressure extraction of oleuropein from olive leaves (Olea europaea L.) with ultrasound assistance. Food Bioprod Process 2013; 93: 29–38.

30.

Maver

Hribernik

Mohan

. Functional wound dressing materials with highly tunable drug release properties. RSC Advances 2015; 5: 77873–77884.

31.

Boateng

Matthews

Stevens

HNE

. Wound healing dressings and drug delivery systems: a review. J Pharm Sci 2008; 97: 2892–2923.

32.

Zhang

Ouyang

Lim

. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. J Biomed Mater Res B Appl Biomater 2005; 72: 156–165.

33.

Maver

Kurečič

Smrke

. Electrospun nanofibrous CMC/PEO as a part of an effective pain-relieving wound dressing. J Sol-Gel Sci Techn 2015; 10: 1–12.

34.

Lowe

Bills

Verma

. Electrospun nitric oxide releasing bandage with enhanced wound healing. Acta Biomater 2015; 13: 121–130.

35.

Liao

Unnithan

Joshi

. Electrospun bioactive poly (ɛ-caprolactone)–cellulose acetate–dextran antibacterial composite mats for wound dressing applications. Colloid Surface A 2015; 469: 194–201.

36.

Unnithan

Sasikala

ARK

Murugesan

. Electrospun polyurethane-dextran nanofiber mats loaded with Estradiol for post-menopausal wound dressing. Int J Biol Macromol 2015; 77: 1–8.

37.

Doshi

Reneker

. Electrospinning process and application of electrospun fibers. J Electrostat 1995; 35: 151–160.

38.

Dabney SE. The use of electrospinning technology to produce wound dressings. Thesis, University of Akron, 2002.

39.

Martindale

. Scar no more. Sci Am 2000; 283: 34–36.

40.

Majhenič

Škerget

Knez

. Antioxidant and antimicrobial activity of guarana seed extracts. Food Chem 2007; 104: 1258–1268.

41.

Persin

Maver

Pivec

. Novel cellulose based materials for safe and efficient wound treatment. Carbohydr Polym 2014; 100: 55–64.

42.

Emori

Banerjee

Culver

. Nosocomial infections in elderly patients in the United States, 1986–1990. Am J Med 1991; 91: 289–293.

43.

Revathi

Puri

Jain

. Bacteriology of burns. Burns 1998; 24: 347–349.

44.

Teixeira LM, Carvalho MGS, Facklam RR, et al. (eds) Manual of clinical microbiology. 11th ed. Washington, DC: ASM Press, 2007, pp.403–421.

45.

Grassi

. Mathematical modelling and controlled drug delivery: matrix systems. Curr Drug Deliv 2005; 2: 97–116.

46.

Korsmeyer

Gurny

Doelker

. Mechanisms of solute release from porous hydrophilic polymers. Int J Pharm 1983; 15: 25–35.

47.

Singhvi

Mahaveer

. Review: in-vitro drug release characterization models. Int J Pharma Stud Res 2011; 2: 77–84.

48.

Benavente-García

Castillo

Lorente

. Antioxidant activity of phenolics extracted from Olea europaea L. leaves. Food Chem 2000; 68: 457–462.

49.

Khayyal

Ghazaly

Abdallah

. Blood pressure lowering effect of an olive leaf extract (Olea europaea) in L-NAME induced hypertension in rats. Arzneimittelforschung 2002; 52: 797–802.

50.

Lee

. Assessment of phenolics-enriched extract and fractions of olive leaves and their antioxidant activities. Bioresour Technol 2009; 100: 6107–6113.

51.

Somova

Shode

Ramnanan

. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. J Ethnopharmacol 2003; 84: 299–305.

52.

Sudjana

D'Orazio

Ryan

. Antimicrobial activity of commercial Olea europaea (olive) leaf extract. Int J Antimicrob Agents 2009; 33: 461–463.

53.

Markin

Duek

Berdicevsky

. In vitro antimicrobial activity of olive leaves. Mycoses 2003; 46: 132–136.

54.

Harrison

Walker

. Micro-electrode measurement of skin pH in humans during ischaemia, hypoxia and local hypothermia. J Physiol 1979; 291: 339–350.

55.

Schneider

Korber

Grabbe

. Influence of pH on wound-healing: a new perspective for wound-therapy? Arch Dermatol Res 2007; 298: 413–420.

56.

. The relationship of pH of the granulation tissue and the take of the skin graft. Plast Reconstr Surg 1957; 19: 213–217.

57.

Lenhardt

Sessler

. Estimation of mean body temperature from mean skin and core temperature. Anesthesiology 2006; 105: 1117–1121.

58.

Lin

Yan

. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex. Microb Cell Fact 2012; 11: 42–64.

59.

Zahedi

Rezaeian

Ranaei Siadat

. A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages. Polymer Adv Tech 2010; 21: 77–95.

60.

Torres de Pinedo

Peñalver

Morales

. Synthesis and evaluation of new phenolic-based antioxidants: structure–activity relationship. Food Chem 2007; 103: 55–61.

61.

Visioli

Bellosta

Galli

. Oleuropein, the bitter principle of olives, enhances nitric oxide production by mouse macrophages. Life Sci 1998; 62: 541–546.

62.

Cerretani

Bendini

Rotondi

. Analytical comparison of monovarietal virgin olive oils obtained by both a continuous industrial plant and a low-scale mill. Eur J Lipid Sci Tech 2005; 107: 93–100.

63.

Gomez-Alonso

Salvador

Fregapane

. Phenolic compounds profile of cornicabra virgin olive oil. J Agric Food Chem 2002; 50: 6812–6817.

64.

Morelló

Motilva

Ramo

. Effect of freeze injuries in olive fruit on virgin olive oil composition. Food Chem 2003; 81: 547–553.

65.

Tovar

Motilva

Romero

. Changes in the phenolic composition of virgin olive oil from young trees (Olea europaea L. cv. Arbequina) grown under linear irrigation strategies. J Agric Food Chem 2001; 49: 5502–5508.

66.

Tovar

Romero

M-P

Girona

. L-Phenylalanine ammonia-lyase activity and concentration of phenolics in developing olive (Olea europaea L cv Arbequina) fruit grown under different irrigation regimes. J Sci Food Agric 2002; 82: 892–898.

67.

Capasso

Evidente

Schivo

. Antibacterial polyphenols from olive oil mill waste waters. J Appl Bacteriol 1995; 79: 393–398.

68.

Tranter

Tassou

Nychas

. The effect of the olive phenolic compound, oleuropein, on growth and enterotoxin B production by Staphylococcus aureus. J Appl Bacteriol 1993; 74: 253–259.

69.

Delaquis

Stanich

Girard

. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int J Food Microbiol 2002; 74: 101–109.

70.

Aeschbach

Löliger

Scott

. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem Toxicol 1994; 32: 31–36.