Characterization of PLA nanofiber structures containing herbal extracts European Journal of Chemistry 13 (1) (2022) 99-108 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2022 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.13.1.99-108.2213 European Journal of Chemistry View Journal Online View Article Online Characterization of PLA nanofiber structures containing herbal extracts Nilsen Sunter Eroglu 1,* and Suat Canoglu 2 1 Department of Textile and Fashion Design, Faculty of Fine Arts, Halic University, Istanbul, 34421, Turkey 2 Department of Textile Engineering, Faculty of Technology, Marmara University, Istanbul, 34730, Turkey * Corresponding author at: Department of Textile and Fashion Design, Faculty of Fine Arts, Halic University, Istanbul, 34421, Turkey. e-mail: nilseneroglu@halic.edu.tr (N.S. Eroglu). 10.5155/eurjchem.13.1.99-108.2213 Received: 15 November 2021 Received in revised form: 14 January 2022 Accepted: 26 January 2022 Published online: 31 March 2022 Printed: 31 March 2022 The use of renewable, sustainable, and biocompatible products without chemical side effects is increasing day by day in antibacterial applications instead of materials that harm nature and humans. In biomedicine, antibacterial nanofiber composite surfaces with generally produced from materials with antibacterial properties such as chitosan, hyaluronic acid, collagen, and silver nanoparticles. In this study, olive leaf, terebinth, and fumitory plants and biocompatible, biodegradable, and environmentally friendly polylactic acid (PLA) polymer were used to obtain nanofiber structures with 100% plant extracts. Viscosity and conductivity of solutions prepared with optimum properties were analysed, the nanofiber material was produced in solution with electrospinning method, and the morphological evaluation and mechanical measurement of the nanofiber material were performed. Finally, bacterial exchange analyses were performed before and after incubation in the UV-VIS spectrophotometer. As a result of the study, the thinnest and the most uniform fiber materials were found in CFO (consist of PLA (C1) and fumitory (FO)) coded nanofiber material, the best strength values were found in COE (consist of PLA (C1) and olive leaf (OE)) coded nanofiber structure, and the highest bacterial exchange was observed in CFO coded nanofiber material. Based on these results, it has been suggested that the CFO coded nanofiber structure can be used in biomedicine. It has been observed that olive leaf, terebinth, and fumitory plant extracts, which can be grown easily in every region in Turkey, have a significant level of bacterial resistance. In conclusion, fumitory and terebinth plants can be used in antibacterial agent applications since they allow obtaining smooth and uniform nanofiber structures, and thanks to their high bacteria nullification properties. Fumitory Olive leaf Terebinth Nanofiber Polylactic acid Electrospinning Cite this: Eur. J. Chem. 2022, 13(1), 99-108 Journal website: www.eurjchem.com 1. Introduction In nature, animals, humans, and plants are in an ecological balance. Plants are the source of nutrients for all living things. According to archaeological findings, humans have been using plants for nutrition and health since ancient times. Plants can transform water, minerals, and some other substances (essential oils, alkaloids, tannins, etc.) they take from the soil in their metabolism and turn them into other compounds (carbohydrates, water, proteins, fats, vitamins, and minerals) that people can digest. This strengthens the defence mechanism of the human body, supports the function of organs, and facilitates the healing of scar tissues. The importance and industrial use of medicinal and aromatic plants have increased with the development of modern medical science. In Turkey, 347 different plant species grow in nature and are traded, and nearly 30% of them are exported [1]. Plants have some beneficial properties such as antibacterial, anti-inflammatory, antiviral, antitumor, antioxidant, and anticancer activity. It is thought that the use of various therapeutic plants will increase in several fields in medical science. Nanofiber production through electrospinning has attracted attention for its applications in many fields such as tissue engineering, drug release, filtration, food industry, cosmetics, agriculture, and bio- detection. Although synthetic-based polymer materials are generally used in these applications, approaches of using biocompatible, biodegradable, and toxic-free natural agents with few side effects are becoming more popular [2-5]. Polylactic acid (PLA) used in this study is the most popular polymer in the medical applications due to its biostructure and biocompatible and biodegradable properties [6]. It is a thermo- plastic polymer, structurally from the a-hydroxy acid family. PLA is obtained as a product of starch fermentation of plants such as corn, sugar cane, potatoes, and beets. In general, PLA has high mechanical strength and modulus, but low impact strength, low temperature of use, and relatively low machin- ability [7]. PLA has various molar masses, microstructures, and crystallinity and is used in the preparation of drug carriers and temporary medical implants [6]. Otherwise, absorbable sutures are used in many biomedical applications, including stents and orthopaedic plates and screws, anti-adhesive films, drug tissue engineering scaffolds, and absorbable implantable devices and coatings [8]. PLA was chosen for this research because of its promising biological properties in the field of biomedicine. The first plant used in this study is the olive leaf. Mostly produce in the Mediterranean region, the olive (Olea europaea) has attracted more attention in recent years due to its positive effects on human health. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.13.1.99-108.2213 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.13.1.99-108.2213 mailto:nilseneroglu@halic.edu.tr http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.13.1.99-108.2213&domain=pdf&date_stamp=2022-03-31 100 Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 (a) (b) (c) Figure 1. Olive leaf, terebinth, and fumitory samples. In the Mediterranean diet, the nutritional model of olive oil is known to decreased rates of risks coronary heart disease risks [9]. The fact that olive, which is the most important element of the Mediterranean diet, has phenolic compounds are highly bioavailable [10], has spread its use as an active agent. Many researchers [11-17] have observed the ability of oleuropein, which is abundant in the olive leaf, to delay and inhibit the growth rate of microorganisms. Researchers report that apart from oleuropein, hydroxytyrosol, 4-hydroxybenzoic acid, vanillic acid, and p-coumaric acid components also have antibacterial effects. The antibacterial effect of olive leaf has been proven by many studies. Electrospun structures formed by olive leaf extract showed strong resistance against E. coli, P. aeruginosa and B. subtilis bacteria [18]. It is possible to increase the efficiency of E. coli, S. aureus and C. albicans bacteria by changing the extract ratio of olive leaf [12]. Similarly, in another study, antibacterial capacity was observed in the form of B. cereus ~ C. albicans > E. coli > S. aureus > C. neoformans ~ K. pneumoniae ~ P. aeruginosa > B. subtilis with the change of extract concentration. It has been stated that this difference in the effect of oleurepein on bacteria is due to the difference in the cell structure of each bacterium [14]. Different parts of terebinth, which is the second type of plant used in this study, are used as an aphrodisiac, anti- inflammatory, antiseptic, and antihypertensive, and in the treatment of dental and gastrointestinal diseases, wounds and burns, rheumatism, liver, urinary tract, and respiratory tract diseases [19-20]. Terebinth is the most common plant species of the family because it is resistant to cold and drought, and it turns into pistachio with grafting [21]. Many researchers have observed that terebinth fruit has antioxidant, antimicrobial, antiviral, anticholinesterase, anti-inflammatory, antinocicep- tive, antidiabetic, antitumor, antihyperlipidemic, antiathe- rosclerotic, and hepatoprotective effects [19,22-23]. It has been observed that the nanofiber structures formed by the polyvinyl alcohol (PVA) and polycaprolactone (PCL) polymers of the Trenbinth plant show resistance against bacteria in the order of C. albicans > E. coli > P. aeruginosa > B. subtilis [18]. The third and last plant species used in this examination, fumitory (Fumaria officinalis), is in the Fumariaceae sub-family of the Papaveraceae family. Fumaria officinalis contains some chemical constituents like alkaloids, carbohydrates, phenolic compounds, flavonoids, glycosides, terpenoids, phytosterols, proteins, amino acids, saponins, fixed oils, steroids, tannins. The plant is contained alkaloids isoquinoline-type [24]. It has therapeutic properties against stomach and digestive tract disorders thanks to this alkaloid content [25]. Bactericidal activities against the Gram-positive organisms of Fumaria officinalis have been known [24]. In the studies of Dülger and Gönuz, it was determined that the extract of fumitory was active against E. coli, S. aureus, K. pneumoniae, P. aeruginosa, P. vulgaris, B. cereus, M. luteus, M. smegmatis, C. albicans, K. fragilis, and R. rubra bacteria [26]. In the literature, there are several researches on obtaining nanofiber materials by mixing different herbal extracts with polymer materials and using them in the medical field. For example, olive leaves have a high potential for tissue scaffolding in biomedical applications thanks to its high antioxidant effect [12] Electrospun mats with olive leaf therapeutic agents to infected skin wounds [27], coaxial nanofiber design by using olive leaf extract as a bioactive agent [28]. It is known that fumitory and trenbinth plants form electrospun structure with PVA and PCL and show bacterial resistance [18]. However, there is still a need for some research on multifunctional membranes that enable antipathogen activities, drug delivery systems with time-controlled release, and sterilized food packaging materials and the materials to be used for these researches. Based on this thought, within the scope of the case, olive leaves and lesser-known fumitory and terebinth plants, which can be grown easily in Turkey due to the climate, have been used. The most significant issue that emerged in these studies is the macromolecule compatibility between the herbal extract and the polymer material and the formation of a smooth surface. The rate of plant material used in this experimental study was determined as 15%. The cause for this is the ability to observe the bacterial exchange effect by using the highest possible plant ratio. Herbal extracts were obtained with the Soxhlet extraction method and mixed with PLA until they became a homogeneous solution. Then, the nanofiber material was produced by using the electrospinning method in the prepared solutions, and characterization, mechanical, and bacterial change tests were performed on these structures. By analysing these measurements, the optimum level of herbal extract/polymer substance was determined. The nanofiber material suitable for use in the biomedical field has been proposed after examining the values obtained. 2. Experimental 2.1. Materials PLA polymer was used by making it homogeneous. PLA ((C3H4O2)n) in 2003D coded bead structure was obtained from NatureWorks LLC (Minnetonka, USA), MN. In addition, dimethyl formamide (Merck), ethanol (Merck), methanol (Merck), chloroform (Merck), acetone (Merck) and distilled water are used to dissolve polymer and herbal extracts used. Herbal extraction was prepared with olive leaf plant (Figure 1a) in leaf form, terebinth plant (Figure 1b) in seed form, and fumitory plant (Figure 1c) in leaf and stem form. All herbs used were packaged within the last 3 months (2020) and purchased from a local brand, Aktar Diyarı (Izmir, Turkey). 2.2. Preparation of herbal extracts Plant parts were washed two times with distilled water to avoid dust and similar residues and dried in an oven at 70 °C for 10 minutes. Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 101 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 Table 1. Preparation of herbal extraction. Herbal extract Concentration of extracted part (%wt) Solvent type (v/v) Temperature of mixing (°C) Stir of mixing (rpm) Time of mixing (h) Reference Olive leaf 25 Methanol/Water (4:1) Room 300 24 [30] Fumaria officinalis 10 Methanol Room 300 4 [31] Pistacia terebinthus 30 Ethanol Room 300 6 [21] Table 2. Experimental design for electrospinning process *. Polymer structure Herbal extract Feeding rate (mL/h) Applied voltage (kV) Distance between syringe needle and collector (cm) Temperature of process (°C) Time of production (h) C1 - 1.6 28 20 22 3 CFO Fumaria officinalis (FO) 1.6 29 20 26 3 CPT Pistacia terebinthus (PT) 1.6 29 20 24 3 COE Olive leaf (OE) 1.6 32 20 20 3 * PLA Polymer Code: C. Soxhlet extraction could extract more herbal sample mass than the latest alternatives and subject to no matrix effects. For this reason, it was preferred in this study. Extraction was repeated for each plant species with the Soxhlet extraction method, which is a method of extracting essential oil from plants [29]. To prevent the loss of solvent by evaporation, a condenser was used. The herbal extracts were filtered using 2 pieces of filter paper and the solvent was removed using an evaporator at 38 °C at 120 rpm rotation. Herbal extraction preparation parameters are given in Table 1. 2.3. Preparation of solutions for electrospinning In the study, firstly, the polymer solution was ensured that 8% (percentage wt.) PLA was dissolved at room temperature in three hours at a ratio of dimethylformamide:chloroform (1:9, v:v) at 600 rpm until it became homogeneous. Homogeneous mixing and concentration of the herbal extract/polymer material are two essential factors to reveal the activity of the antibacterial components in the herbal extract. As a result of the tests conducted, the herbal substance ratio was determined as 15%, since the concentration of the herbal extract in the polymer had more efficient results in terms of determining the fiber morphology and chemical properties. The prepared mixture was stirred in a magnetic stirrer for 45 minutes to ensure homogeneity. 2.4. Viscosity and conductivity measurement parameters of polymer/herbal extract solutions 2.4.1. The viscosity measurement of polymer/herbal extract solutions The viscosities of polymer solutions were measured using a viscometer device (Brookfield DV-E Viscometer, USA). Viscosity measurement was made using S21 spindle type at 100 rpm. The viscosity values of all prepared solutions according to the shear rate are expressed in cP (centipoise). Two replicate tests were performed for each measurement. 2.4.2. The conductivity of polymer/herbal extract solutions The conductivity values of polymer solutions were measured with a portable electrical conductivity meter (WTW Cond 3110, Germany). The conductivity measurement probe was immersed in the prepared solution and measured in mS/cm. Viscosity and conductivity measurements were made under laboratory conditions (23±2 °C and 45±10 % RH). 2.5. Production of PLA based electrospun fibers In the study, after the solution was prepared and the characterization measurements were made, 8 mL of solution was taken with a 10 mL syringe. Then, nanofiber structure production was achieved by providing the values specified in Table 2 at a rotation speed of 100 rpm on oil paper with a syringe placed in the Inovenso NE 300 Nano Spinner brand electrospinning device. 2.6. Morphology and structure characterization of herbal extracts-based nanofiber structures The morphologies of the herbal-based nanofibers produced in the study were investigated with JEOL JSM-IT100 Scanning Electron Microscopes (SEM). The morphologies of herbal-based nanofiber structures were analysed using SEM device at 1000- and 20000-times magnification. Image J software was used to measure the fiber diameters of the images with 15 repetitions, and SPSS 24 statistical program was used to obtain the diameter distribution charts. Nicolet is10, Thermo Scientific brand device was used in the Fourier Transformed Infrared Spectrum (FTIR) examination of the obtained nanofiber structures. For both measurements, samples of 10×10 mm were taken. 2.7. Mechanical tests of herbal extract-based nanofiber structures Thickness, tensile strength, and elongation at break measurements were made to measure the effect of vegetable matter on strength in herbal-based nanofiber structures produced in the study. Thickness measurements were made with Mitutoyo Digital Thickness Comparator (Mitutoyo, Kawasaki, Japan) device in “mm” from 10 different points in vertical and horizontal directions. These values were used to measure strength measurements in megapascals (MPa). Tensile strength and elongation at break values of samples taken from 50×10 mm (length×width) dimensions were measured in Instron 4411 Universal tester (Instron, Norwood, MA, USA). 2.8. UV-VIS bacteria change measurements In the bacterial change measurement, dilutions were made in the broth (mL) which decreased by two times [32] (Table 3). A standard final concentration of the dilution was prepared, and the herbal extracts were added in equal proportions (1 mL) to each tube containing various dilutions [32]. Bacterial growths were observed by absorbance measurements before and after the incubation process. In the study, the first operation was bacterial cultivation. For this step, the liquid medium was prepared by using 250 mL of distilled water and 2 g (Nutrient broth). 50 mL of medium was taken from the bottle, some bacteria were scraped from the petri dish with a sterile disposable stick, and the bacteria were cultivated into falcon tubes and it is incubated. For reference measurement, 0.1 g Nutrient Broth medium was prepared with 12.5 mL distilled water, solutions containing reference mixture in the st cuvette, P. aeruginosa (PA) in the 2nd cuvette, B. subtilis (BS) in the 3rd cuvette, S. aureus (SA) in the 4th cuvette, E. coli (EC) bacteria in the 5th cuvette were placed in 102 Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 Table 3. Bacteria measurement values. Ingredients Medium (1st cuvette) P. aeruginosa (2nd cuvette) B. subtilis (3rd cuvette) S. aureus (4th cuvette) E. coli (5th cuvette) 1 mL medium + bacteria 0 0.090 0.066 0.267 0.231 1 mL medium + bacteria + pure water 0 0.756 0.125 0.442 0.460 Table 4. Mixing ratio of tubes. Tube Polymer structure liquid (mL) Isotonic NaCl (mL) Bacteria (mL) 1 1 4.0000 5.0000 2 1 6.5000 2.5000 3 1 7.7500 1.2500 4 1 8.3750 0.6250 5 1 8.6875 0.3125 6 1 8.8432 0.1568 7 1 8.9220 0.0780 8 1 8.9610 0.0390 Table 5. The viscosity and conductivity of C group solutions. Polymer samples Viscosity (cP) Torque rating (%) Conductivity (µS/cm) Temperature (°C) C1 155 31.1 0.2 22 CFO 81.5 16.3 35.9 26 CPT 71.5 14.3 1.6 24 COE 170 34.0 18 20 a UV-VIS spectrophotometer and measurements were perfor- med at 600 nm. Tubes without herbal extract but containing microorganisms and medium (1st cuvette) were used as a positive control. The presence of bacteria was confirmed by comparing the values seen in other cuvettes (Table 3). For the preparation process in the study, the first 4.5 g isotonic NaCl to be used in the bacteria mixture, and 500 mL distilled water mixture was prepared. In order to obtain the polymer structure liquid, autoclaved isotonic NaCl solution 100 times the polymer structure weight was transferred to the Falcon tube and the prepared polymer liquid solution was incubated at a shaking incubator. Then, the water of the polymer structure, isotonic NaCl, and bacteria were added to 8 Mc Farland tubes for each bacterium, and the mixture was prepared at the rates indicated in the table. In this process, the aim was to mix the antibacterial effect of the nanofiber structures into the solution, and dilution was applied for each tube, and bacteria were added at different concentrations (Table 4). In the investigation, bacteria measurements of the prepared mixtures were performed. UV-VIS spectrophoto- meter measurements were performed, and it was observed how the antibacterial factors in the nanofiber structures affected the bacteria with the change of time. 3. Results and discussion 3.1. Results of viscosity and conductivity measurement parameters of solutions The resistance of a solution to flow under surface tension determines its viscosity. Viscosity is the most important parameter that determines the flow rate of the solution. Viscosity is related to the degree of entanglement of polymer molecule chains in solution. Beaded fibers are more likely to be obtained rather than straight fibers at low viscosity, where there is usually a polymer chain. In general, since the polymer chain is more difficult to synthesize with each other [33], less chain entanglement occurs, and jet stability is lost. As a result, the fibers are collected into the collector as droplets, and the droplets first turn into spindle-like structures and then into beaded nanofibers [12]. Therefore, the factors affecting the viscosity of the solution also affect the electrospinning process and the resulting fibers [34]. As a result of the study, the viscosity values of the nanofiber structure groups are listed as COE > C1 < CFO > CPT (Table 5). When the results were examined, it was seen that the COE group fibers formed more regular and uncomplicated structures. The low conductivity value leads to the formation of beaded nanofiber structures with larger sizes [35]. Correspondingly, more regular and finer nanofibers are formed as the conduc- tivity of polymer solution increases [36]. It is known that this situation results from the increase in the attraction of the polymer jet in the electric field with the increase in charge density [37]. In the conductivity tests, it was observed that the highest values belonged to the CFO (255.6 nm) group nanofiber structures. The fact that CFO group nanofiber structures have the most uniform, smooth, and beadless structures and have lower fiber diameters from the four nanofiber structure groups supports this idea (C1 (359.73 nm), COE (441.87 nm), and CPT (513.87 nm). 3.2. Determination of morphology and structure characterization of herbal extract-based nanofiber structures 3.2.1. Surface characterization of herbal extract-based nanofiber structures In the study, the fiber diameter fineness (nm) of group C nanofiber structures is listed as CPT > COE > C1 > CFO (Figures 2 and 3). The smooth and uniform structure, which has the thinnest fibers among the C group nanofiber structures, was observed in the CFO nanofiber structure. It was observed that fumitory plant and PLA polymer provided good morphological compatibility and fine, smooth, uniform nanofiber formation was obtained. It is known that the fumitory plant, which has limited literature, is used in many treatment methods with its high alkaloid content (60-70%) [38]. It is thought that the nanofiber structure containing fumitory will also have good properties in terms of fineness and antibacterial properties. Since there are two different components in plant-based nanofiber structures, nanofiber surfaces were formed in more branched structures compared to the pure polymer C1 sample without plant content. This situation is clearly observed in the SEM images. 3.2.2. FTIR characterization of herbal extract-based nanofiber structures FTIR analysis was used in order to define the internal bonds of the molecular structures of the obtained nanofibers. Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 103 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 Figure 2. SEM images (1000 and 20,000×) and distribution of diameter (20,000×) of C1 and CFO nanofiber structures. Figure 3. SEM images (1000 and 20,000×) and distribution of diameter (20,000×) of COE and CPT nanofiber structures. The method is based on the absorption of infrared rays falling on the bonds with vibration and rotational motion in correlation. In the infrared spectra obtained, the presence and effectiveness of herbal extracts and polymer material were analyzed. In all FTIR spectra of the surfaces obtained as a result of the study, the wavelength is in the range of 450-4000 cm-1 for all C group samples. In the analyses, the effects of characteristic bands in herbal extracts on the obtained nanofiber structures were investigated. Therefore, comparative analyses were carried out with the pure polymer (C1) that did not contain the extract. According to this, =C-H bonds are seen in C1 polymer at absorbance values of 754 and 867 cm-1, C–O bond related to −CH(C H3) –OH at 1084 and 1181 cm-1, asymmetrical C–H bond at 1359 cm-1, symmetrical –CH3 bond at 1451 cm-1, carbonyl C=O bond at 1750 cm-1, and vibration of C-H bonds at 2995 and 3005 cm-1 [39]. The cause for the 1022 cm-1 peak of the FO extract in the FTIR spectrum with the fumitory plant content is due to the presence of =C-H bonds [40]. The fact that the wavelength of the CFO polymer is different at 1005 cm-1 compared to the C1 polymer is interpreted as the effect of the fumitory extract. In the FTIR spectrum of the terebinth plant, a broad absorption peak was observed at approximately 3327 cm-1, corresponding to the –OH stretch. Polyphenolic structures have an absorption peak with C–H symmetric stretching vibrations around 2974 cm-1. C=O stretching vibrations are observed at 1655 cm-1. The peaks around 1380 cm-1 are formed by the contribution of aromatic C=C bonds. Out-of-plane C–H bending vibrations of the aromatic ring were observed in the range of 623-879 cm-1 [41]. The peak at a wavelength of 1045 cm-1 in terebinth extract indicates the stretching of C-N bonds [42]. This peak affected on group C nanofibers. The presence of PT extract is seen at 1023 cm-1 in CPT nanofiber structures. Olive leaf contains main ingredients such as oleuropein and hydroxytyrosol. The primary components of the compound can be determined from the structures of the phenol O-H, carboxylic acid C=O, and alkene C=C stretch nodes observed at 3307 cm-1. In addition, the 1651 cm-1 peak corresponds to the presence of amide I [43]. It indicates the CH stretches of the characteristic bands at 2835-2946 cm-1 peaks, the C-O stretching of phenols in the 1111-1411 cm-1 absorption region, and the presence of aromatic rings of olive leaf polyphenols at 1449 cm-1 [39]. Conclusively, the peak observed at 1017 cm-1 is considered to be indicative of the CN stretching vibration of the amine group [43]. The subsistence of olive leaf extract is seen at 1015 cm-1 in the COE nanofiber structure. 104 Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 Figure 4. Tensile strength in the machine direction of C groups nanofiber structures. Figure 5. Tensile Strength in the material width direction of C groups nanofiber structures. Figure 6. Elongation in the machine direction of C group nanofiber structures (%). 3.3. Mechanical tests result of herbal extracts-based nanofiber structures 3.3.1. Tensile Strength Results of herbal extracts-based nanofiber structures The strength of a material increases as the length of its polymer chain and the number of cross-links between its polymer chain increase [37]. At this stage of the study, it was aimed to measure and evaluate the effects of plant materials on strength in nanofiber constructions. In this sense, vertical (machine direction) and horizontal (material width) breaking strength values were restrained and analyzed. The order of machine direction and material width strength averages of C group nanofiber structures was COE > C1> CFO > CPT (Figures 4 and 5). When the sequence was examined, it was observed that the CFO and CPT nanofibers decreased the breaking strength compared to the C1 nanofiber. PLA, the main material of group C nanofibers, is a polymer with high biodegradability, biocompatibility, thermoplastic process- ability, and mechanical properties [44]. When the chemical crosslink structure is defined in PLA, the material first hardens compared to pure PLA, but then it becomes more brittle over time and there is a sudden decrease in strength. After PLA hardens, it becomes brittle with increasing bond density [45]. It is thought that this structural change of PLA may have reduced the nanofiber strength by making the nanofiber formation brittle, depending on the addition of herbal extracts in CFO and CPT nanofibers and the binding status of the extract. In the COE nanofiber structure, an increase in strength is observed with the hardening of the PLA polymer. 3.3.2. Elongation at break tests of herbal extract-based nanofiber structures Elongation at break tests was performed in two different positions, similar to the tensile strength tests, by considering the machine direction (vertical) and the material width direction (horizontal) (Figures 6 and 7). 1.37 0.77 0.68 1.64 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 C1 CFO CPT COE Te ns ile s tre ng th (M pa ) Polymer 2.12 1.00 0.84 2.99 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 C1 CFO CPT COE Te ns ile s tre ng th (M pa ) Polymer 71.21 49.04 62.37 89.32 0 10 20 30 40 50 60 70 80 90 100 C1 CFO CPT COE El on ga tio n (% ) Polymer Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 105 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 Table 6. UV-VIS bacterial exchange measurements of P. aeruginosa and B. subtilis bacteria for COE nanofiber structures. First measurement Second measurement PA codes Absorbance BS codes Absorbance PA codes Absorbance BS codes Absorbance OE-PA-1 0.043 OE-BS-1 0.038 OE-PA-1 0.042 OE-BS-1 0.041 OE-PA-2 0.024 OE-BS-2 0.019 OE-PA-2 0.027 OE-BS-2 0.016 OE-PA-3 0.008 OE-BS-3 0.009 OE-PA-3 0.012 OE-BS-3 0.033 OE-PA-4 0.003 OE-BS-4 0.003 OE-PA-4 0.005 OE-BS-4 0.006 OE-PA-5 0.002 OE-BS-5 0.001 OE-PA-5 0.000 OE-BS-5 0.000 OE-PA-6 0.000 OE-BS-6 0.001 OE-PA-6 0.000 OE-BS-6 0.000 OE-PA-7 0.001 OE-BS-7 0.000 OE-PA-7 0.000 OE-BS-7 0.001 OE-PA-8 0.001 OE-BS-8 0.000 OE-PA-8 0.000 OE-BS-8 0.000 Figure 7. Elongation in the material width direction of C group nanofiber structures (%). Figure 8. Nanofiber structures thicknesses of group C. In the measurements performed, it was observed that there was no direct relationship between the herbal extract addition and the flexibility of the nanofiber structures. 3.3.3. Thickness of herbal extract-based nanofiber structures The thicknesses of the obtained nanofibers in the horizontal and vertical directions were measured in mm. In the fiber fineness distributions of the SEM measurements made within the scope of the study, it was observed that the CFO nanofiber structure consisted of thinner fibers than other nanofiber structures. This situation is associated with the combination of fiber groups consisting of fine fibers and forming a surface in a thinner layer. In the results examined, it was not possible to make a clear inference about the effect of herbal extracts on the nanofiber structure thickness. While the addition of herbal extract in the CFO nanofiber structure increased the structure thickness, it decreased the structure thickness in CPT and CFO structures. This is related to the morphological compatibility between the polymer and the herbal extract and does not form a link between the plant ingredients (Figure 8). 3.4. UV-VIS bacteria change results of herbal extract-based nanofiber structures In the study, UV-VIS spectrophotometer measurements of the mixtures of COE, CPT, and CFO nanofiber samples were performed before and after the incubation process. Shaking in the incubator was performed for 1 hour at 37 °C. It was recog- nized how much bacteria were affected over time. Eight replicates and two parallel absorbance measurements were made for each bacterium. The absorbance values were measured at 600 nm before and after incubation with P. aeruginosa, B. subtilis, S. aureus, and E. coli bacteria used in the study and the values given in Tables 6-11 were obtained. The first measurement indicated in the tables refers to the pre- incubation, the second measurement refers to the post- incubation. As seen in Table 4, a decrease was observed in the number of bacteria prepared at disparate concentrations. In the first and second measurements before and after incubation, absorbance measurements deteriorated as the bacterial concentration decreased. When the bacterial optical density (OD) values obtained from the UV-VIS spectrophotometer of COE, CPT, and CFO nanofibers are examined, it is seen that they are listed as CFO > CPT > COE. 46.37 38.40 42.47 70.80 0 10 20 30 40 50 60 70 80 C1 CSH CPT COE El on ga tio n (% ) Polymer 0.09 0.05 0.12 0.11 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 C1 CFO CPT COE Th ic kn es s (m m ) Polymer 106 Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 Table 7. UV-VIS bacterial exchange measurements of S. aureus and E. coli bacteria for COE nanofiber structures. First measurement Second measurement SA codes Absorbance EC codes Absorbance SA codes Absorbance EC codes Absorbance OE-SA-1 0.168 OE-EC-1 0.121 OE-SA-1 0.163 OE-EC-1 0.144 OE-SA-2 0.078 OE-EC-2 0.070 OE-SA-2 0.074 OE-EC-2 0.071 OE-SA-3 0.049 OE-EC-3 0.035 OE-SA-3 0.032 OE-EC-3 0.033 OE-SA-4 0.011 OE-EC-4 0.021 OE-SA-4 0.009 OE-EC-4 0.015 OE-SA-5 0.008 OE-EC-5 0.007 OE-SA-5 0.010 OE-EC-5 0.006 OE-SA-6 0.009 OE-EC-6 0.016 OE-SA-6 0.004 OE-EC-6 0.004 OE-SA-7 0.001 OE-EC-7 0.005 OE-SA-7 0.010 OE-EC-7 0.003 OE-SA-8 0.000 OE-EC-8 0.003 OE-SA-8 0.000 OE-EC-8 0.000 Table 8. UV-VIS bacterial exchange measurements of P. aeruginosa and B. subtilis bacteria for CPT nanofiber structures. First measurement Second measurement PA codes Absorbance BS codes Absorbance PA codes Absorbance BS codes Absorbance PT-PA-1 0.101 PT-BS-1 0.024 PT-PA-1 0.112 PT-BS-1 0.031 PT-PA-2 0.058 PT-BS-2 0.015 PT-PA-2 0.059 PT-BS-2 0.041 PT-PA-3 0.027 PT-BS-3 0.006 PT-PA-3 0.047 PT-BS-3 0.015 PT-PA-4 0.015 PT-BS-4 0.000 PT-PA-4 0.009 PT-BS-4 0.007 PT-PA-5 0.008 PT-BS-5 0.002 PT-PA-5 0.007 PT-BS-5 0.001 PT-PA-6 0.005 PT-BS-6 0.001 PT-PA-6 0.003 PT-BS-6 0.001 PT-PA-7 0.001 PT-BS-7 0.000 PT-PA-7 0.000 PT-BS-7 0.001 PT-PA-8 0.001 PT-BS-8 0.000 PT-PA-8 0.000 PT-BS-8 0.003 Table 9. UV-VIS bacterial exchange measurements of S. aureus and E. coli bacteria for CPT nanofiber structures. First measurement Second measurement SA codes Absorbance EC codes Absorbance SA codes Absorbance EC codes Absorbance PT-SA-1 0.030 PT-EC-1 0.133 PT-SA-1 0.045 PT-EC-1 0.132 PT-SA-2 0.015 PT-EC-2 0.080 PT-SA-2 0.042 PT-EC-2 0.093 PT-SA-3 0.012 PT-EC-3 0.045 PT-SA-3 0.036 PT-EC-3 0.055 PT-SA-4 0.013 PT-EC-4 0.025 PT-SA-4 0.015 PT-EC-4 0.027 PT-SA-5 0.017 PT-EC-5 0.015 PT-SA-5 0.011 PT-EC-5 0.018 PT-SA-6 0.011 PT-EC-6 0.037 PT-SA-6 0.005 PT-EC-6 0.015 PT-SA-7 0.001 PT-EC-7 0.001 PT-SA-7 0.002 PT-EC-7 0.006 PT-SA-8 0.000 PT-EC-8 0.002 PT-SA-8 0.004 PT-EC-8 0.007 Table 10. UV-VIS bacterial exchange measurements of P. aeruginosa and B. subtilis bacteria for CFO nanofiber structures. First measurement Second measurement PA codes Absorbance BS codes Absorbance PA codes Absorbance BS codes Absorbance FO-PA-1 0.229 FO-BS-1 0.078 FO-PA-1 0.250 FO-BS-1 0.078 FO-PA-2 0.144 FO-BS-2 0.055 FO-PA-2 0.154 FO-BS-2 0.055 FO-PA-3 0.170 FO-BS-3 0.038 FO-PA-3 0.168 FO-BS-3 0.038 FO-PA-4 0.075 FO-BS-4 0.021 FO-PA-4 0.081 FO-BS-4 0.021 FO-PA-5 0.056 FO-BS-5 0.016 FO-PA-5 0.051 FO-BS-5 0.016 FO-PA-6 0.042 FO-BS-6 0.013 FO-PA-6 0.038 FO-BS-6 0.013 FO-PA-7 0.011 FO-BS-7 0.005 FO-PA-7 0.011 FO-BS-7 0.005 FO-PA-8 0.007 FO-BS-8 0.001 FO-PA-8 0.007 FO-BS-8 0.001 Table 11. UV-VIS bacterial exchange measurements of S. aureus and E. coli bacteria for CFO nanofiber structures. First measurement Second measurement SA codes Absorbance EC codes Absorbance SA codes Absorbance EC codes Absorbance FO-SA-1 0.216 FO-EC-1 0.232 FO-SA-1 0.216 FO-EC-1 0.234 FO-SA-2 0.138 FO-EC-2 0.144 FO-SA-2 0.180 FO-EC-2 0.148 FO-SA-3 0.075 FO-EC-3 0.110 FO-SA-3 0.105 FO-EC-3 0.102 FO-SA-4 0.014 FO-EC-4 0.077 FO-SA-4 0.074 FO-EC-4 0.067 FO-SA-5 0.000 FO-EC-5 0.030 FO-SA-5 0.078 FO-EC-5 0.051 FO-SA-6 0.006 FO-EC-6 0.020 FO-SA-6 0.064 FO-EC-6 0.036 FO-SA-7 0.015 FO-EC-7 0.015 FO-SA-7 0.009 FO-EC-7 0.019 FO-SA-8 0.008 FO-EC-8 0.006 FO-SA-8 0.008 FO-EC-8 0.008 This means that the antibacterial inhibitory effect of the CFO nanofiber was higher, and it nullified the number of bacteria at a higher rate. Absorbance measurements for COE and CFO nanofibers were reset after eight replicate assess- ments. Even though the absorbance value for the CPT nanofiber decreased, it was not reset. Hereby, the lowest absorbance value without any visible turbidity, which prevents the growth of bacteria, was taken as the final value. 4. Conclusion We tried to demonstrate nanofiber structures were obtained by incorporating olive leaf, fumitory, and terebinth herbal extract into PLA electrospun fibers by using the electrospinning method. The herbal extracts were included in the polymer solutions at a 15% rate and the conductivity and strength characterization measurements of the solutions were performed. Characterization measurement results determined the structural properties of nanofiber formations. The presence of herbal extracts in the nanofibers was confirmed by the appearance of the peaks in the FTIR analysis and the morpho- logical appearances in the SEM images. In this study, it was observed that the thinnest fiber structures were CFO, the best strength values were COE, and the nanofiber structure with the highest bacterial exchange was CFO. In the bacterial exchange measurements, it was seen that the ability of the fumitory extract to nullify the number of bacteria was stronger than the extracts of olive leaf and terebinth. It has been concluded that the use of fumitory and terebinth plants, which are relatively less known than olive leaves and can easily grow in Turkey, as a source of strong environmentally friendly antibacterial substance has a great potential for future research. Further studies regarding the role of fumitory, and terebinth extracts in Eroglu and Canoglu / European Journal of Chemistry 13 (1) (2022) 99-108 107 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.99-108.2213 nanofiber structures would be worthwhile. In this study, it was used that instead of chemical and synthetic antibacterial agents, which are known to be harmful to nature and human health, herbal extracts, and the PLA, which is mostly used in biomedical applications. Herbal extracts have the potential to be a resource for future biomedical research because they are environ- mentally friendly, biocompatible, sustainable, and economical. In addition, the production of herbal extracts by creating different ratios/ variations with different natural or chemical nanofiber structures in future research may allow for various purposes such as tissue engineering, wound dressing, drug release mechanisms in the medical field. Acknowledgements The authors would also like to thank for supporting by the Scientific Research Project Unite (BAP) (Project number: FEN-C-DRP-090518-0246), Marmara University, Istanbul, Turkey. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Nilsen Sunter Eroglu, Suat Canoglu; Methodology: Nilsen Sunter Eroglu, Suat Canoglu; Software: Nilsen Sunter Eroglu; Validation: Nilsen Sunter Eroglu, Suat Canoglu; Formal Analysis: Nilsen Sunter Eroglu; Investigation: Nilsen Sunter Eroglu; Resources: Nilsen Sunter Eroglu; Data Curation: Nilsen Sunter Eroglu, Suat Canoglu; Writing - Original Draft: Nilsen Sunter Eroglu, Suat Canoglu; Writing - Review and Editing: Nilsen Sunter Eroglu; Visualization: Nilsen Sunter Eroglu. ORCID and Email Nilsen Sunter Eroglu nilseneroglu@halic.edu.tr https://orcid.org/0000-0002-8403-7809 Suat Canoglu scanoglu@marmara.edu.tr https://orcid.org/0000-0002-1604-9875 References [1]. Göktaş, B.; Gidik, B. Bayburt ilinde doğadan toplanan tibbi ve aromatik bitkilerin tüketimi. Res. Stud. Anatolia J. 2019, 303–311. [2]. Yao, C.-H.; Yeh, J.-Y.; Chen, Y.-S.; Li, M.-H.; Huang, C.-H. 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This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Materials 2.2. Preparation of herbal extracts 2.3. Preparation of solutions for electrospinning 2.4. Viscosity and conductivity measurement parameters of polymer/herbal extract solutions 2.4.1. The viscosity measurement of polymer/herbal extract solutions 2.4.2. The conductivity of polymer/herbal extract solutions 2.5. Production of PLA based electrospun fibers 2.6. Morphology and structure characterization of herbal extracts-based nanofiber structures 2.7. Mechanical tests of herbal extract-based nanofiber structures 2.8. UV-VIS bacteria change measurements 3. Results and discussion 3.1. Results of viscosity and conductivity measurement parameters of solutions 3.2. Determination of morphology and structure characterization of herbal extract-based nanofiber structures 3.2.1. Surface characterization of herbal extract-based nanofiber structures 3.2.2. FTIR characterization of herbal extract-based nanofiber structures 3.3. Mechanical tests result of herbal extracts-based nanofiber structures 3.3.1. Tensile Strength Results of herbal extracts-based nanofiber structures 3.3.2. Elongation at break tests of herbal extract-based nanofiber structures 3.3.3. Thickness of herbal extract-based nanofiber structures 3.4. UV-VIS bacteria change results of herbal extract-based nanofiber structures 4. Conclusion Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: