Human Cell Cytotoxicity, Anti-Microbial Effect and Chromatographic Analyses of Leaf and Fruit Extracts of Olea europaea (Olive) Collected from Different Distances to Yatagan Thermal Power Plant in Mugla, Türkiye

Human Cell Cytotoxicity, Anti-Microbial Effect and Chromatographic Analyses of Leaf and Fruit Extracts of Olea europaea (Olive) Collected from Different Distances to Yatagan Thermal Power Plant in Mugla, Türkiye | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human Cell Cytotoxicity, Anti-Microbial Effect and Chromatographic Analyses of Leaf and Fruit Extracts of Olea europaea (Olive) Collected from Different Distances to Yatagan Thermal Power Plant in Mugla, Türkiye Esra Gürbüz, Aytül Sandallı, Funda Bilgili Tetikoğlu, Enes Şeker, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5381441/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Thermal power plants (TPP) are important for meeting today's increasing energy needs. However, TPPs pose risks to the ecosystem and human health. Olea europea (olive) grows widely in the Aegean region and is commonly used in the human diet. The TPP in Yatağan is close to the agricultural and residential areas. However, the effect of closeness to TPP on olives and the cytotoxic effect of olives close to TPPs on human cells is unknown. This study showed 1) phenolic compounds, flavonoids, and fundamental olive contents changed in the fruit and leaf extracts collected far-medium-close to TPP, 2) extracts obtained from olives close to the TPP had cytotoxic effects on healthy human cells, and 3) the highest antimicrobial activity of extracts was found against Staphylococcus aureus regardless of distance to the TPP. The findings suggest that TPPs may affect the nutritional value of olives, and the viability of human cells. Olea europea olive cytotoxicity phenolic compounds thermal power plant anti-microbial activity HPLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Introduction There has been an increasing interest in medicinal plants due to their content for health promotion such as flavonoids (Tajner-Czopek et al. 2020 ). The fruits of Olea europea have been commonly used especially in the Mediterranean diet and olive oil is an indispensable product for gastronomy. Olive tree leaves are also widely used in traditional treatments in European and Mediterranean countries such as Greece, Spain, Italy, France, Turkey, Israel, Morocco, and Tunisia. They have been used as extracts, herbal teas, and powders in the human diet and contain many potential bioactive compounds that may have antioxidant, antihypertensive, antiatherogenic, anti-inflammatory, hypoglycemic, and hypocholesterolemic properties (El and Karakaya 2009). O. europea has been reported to be highly resistant to environmental stressors, such as drought (Marchioni et al. 2024 ; Nteve et al. 2024 ), water (Sánchez-Piñero et al. 2024 ), and salt stress (Regni et al. 2019 ). The salinity of the soil and irrigation water, as well as the lack of water and nutrient resources, have all impacted the recent expansion of olive farming beyond the Mediterranean region to several continents (Oteros et al. 2013 ). Climate change is one of the most important drawbacks globally. This may increase temperatures and accordingly water restraint. Mediterranean and Aegean regions are at risk of drought (Toreti et al. 2024 ). Thermal power stations can also contribute to increased temperature due to coal combustion (Faizan et al. 2021 ). Olives naturally resist drought and can thrive in environments with few resources, therefore they rank among the most productive crops. Nevertheless, the challenges posed by climate constraints necessitate investigating the high variability of species to one another in the direction of abiotic stress tolerance and identifying the genetic factors that govern how plants react to stress. Closeness to thermal power stations can be another environmental stress for olive plants, but there is no clear understanding of the possible effects on olive fruits and leaves. Besides, the effects of the olives around thermal power plants on human cells have not been explored before. The importance of olives in the human diet questions the potential harmful effects of thermal olives on human health. In this study, we investigated the quantitative and qualitative content of olive fruits and leaves grown in places close to the Yatağan thermal power plant compared to those far away from the station. We also treated four types of normal human cells (derived from the breast, retina, vein, and bronchus) with the extracts of olive fruits and leaves to conclude the cytotoxicity of the cells. 2. Materials and Methods 2.1. Sample collection Olive fruit and leaves (O. Europaea) were collected in October from Yatagan Center, Deştin Village, and Şahinler Village in Yatagan district of Mugla province ( Fig. 1 A ) at different distances from the thermal power station ( Fig. 1 B-C-D ) . The specific locations in the collected regions were recorded on Google Maps, and then their distances to the thermal power station were calculated by direct lines. Figure 1 E shows the representative morphological profiles of fruits and leaves from the plants located at different distances from the thermal power station. It is observed that the leaves and fruits of the samples closest to the thermal power station are quite small ( Fig. 1 E, left) . The largest fruit size belongs to the samples that are the farthest from the power station ( Fig. 1 E, right) . The abbreviations for the locations used throughout the study are as follows: D; Destin (the furthest location), M (the middle location), -T (thermal location) and F (fruit), L (leaves). For instance; TL (thermal-leaves), MF (middle-fruit) 2.2. Extraction protocol Olive fruits and leaves were dried at 25 o C for 20 days without direct sunlight. The leaves were passed ground into a fine powder with a grinder. The grinding process was repeated every 5 minutes to prevent friction heating of the sample. 40 grams of each sample was added to 150 mL of methanol into the Erlen mayer and covered with aluminum foil. Samples were incubated in a shaker at room temperature for 6 hours. The extracts were filtered into the balloons using Whatmann papers. Then the same amount of solvent was added to the remaining part for another 6 hours at the same condition. The filtrated extracts from samples were collected into the balloons, and the solvent was evaporated with a Rotary Evaporator. The obtained dense-liquid extracts were placed in Eppendorf tubes and stored in the refrigerator (Supplementary Fig. 1) . These liquid extract samples were dried using a lyophilizer, and lyophilized extracts were stored at + 4°C. The main stocks of the extracts were prepared with DMSO (mg/mL), aliquoted as 60–80 µl, and kept at – 20°C until use. 2.3. Cell culture and extract treatments The healthy cell lines used in this study were 1) MCF10A (ATCC, CRL-10317™) human mammary gland epithelial cells 2) ARPE-19 (ATCC, CRL-2302™) human eye retinal pigment epithelial cells, 3) HUVEC (ATCC, CRL-1730™) primary human umbilical vein endothelial cells, and 4) BEAS-2B (ATCC, CRL-3588™) epithelial cells isolated from normal human bronchial epithelium. Except for BEAS-2B cells cultured in DMEM, other cells were cultured in RPMI media including 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C with 5% CO 2 . After they reached at full confluency, cells were split into 96 well plates. Confluent cells in the wells were treated by the extracts with final concentrations of 500 µg/mL, 100 µg/mL, 25 µg/mL, 6.25 µg/mL, 1.56 µg/mL, 0.39 µg/mL, 0.097 µg/mL, 0.024 µg/mL, for 24 h or 48 h. Control cells were untreated. 2.4. MTT assay The principle of the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test is based on the metabolizing of MTT dye into a formazane crystal which indicates mitochondrial activity in the living cells. The MTT dye is yellow, and purple is formed after the tetrazolium ring was enzymatically broken in live cells. Tetrazolium crystals are then dissolved with DMSO followed by the measurement of color intensities as absorbance at 570 nm using a spectrophotometer. Live cells have more absorbance due to the purple color (Demir et al. 2021 ; Kumar, Nagarajan, and Uchil 2018 ). For the MTT experiment, the culture media was removed from the wells and 190 µl media and 10 µl 0.25 mg/mL MTT dye was added to each well, and incubated at 37 o C for 2 hours. After incubation, media including MTT was removed, and 200 µl of DMSO was added to each well to visualize color formation. Plates treated with DMSO were dark-incubated in a shaker at 120xg for 1 hour to dissolve the crystals. Absorbances of each well in the plates were read at 570 nm by a spectrophotometer. The absorbance of untreated cells was considered 100% viable, and the viability of treated cells was calculated relative to the absorbances of untreated control. 2.5. HPLC analyses Chemicals HPLC grade acetonitrile (ACN), acetic acid (AA), and standard phenolic compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA) but quercetin from Fluka Chemie GmbH (Switzerland). Methanol for analysis (Supelco®) was supplied by Merck (Darmstadt, Germany). Extraction of phenolic compounds from samples Dried extracts were dissolved in the methanol and diluted with 50% water for suitable concentrations for HPLC-DAD analysis (DL: 10, DF: 50, ML: 20, MF: 100, TL: 10, and TF: 57 mg/mL). HPLC-DAD and HPLC-MS conditions for separation of phenolic compounds The chromatographic analyses were performed using a Dionex (Thermo Scientific, Germering, Germany) Ultimate 3000 high-performance liquid chromatography (HPLC) system equipped with an Ultimate 3000 diode array detector (DAD). A Thermo acclaim C30 column (150mm. 3mm id. 3µm pd) was used with a Macherey Nagel (3mm id) guard column. Gradient elution was used with mobile phases; A: 2% acetic acid in water and B: 70%acetonitrile-30% water. The flow rate was 0.37 mL/min, and the injection volume was 10µL. Column temperature was 25°C. Following 25 phenolic standards were used to calibrate and validate HPLC-DAD analysis method: Gallic acid, protocatechuic acid, p-hydroxy benzoic acid (p-OH benzoic acid), chlorogenic acid, vanillic acid, caffeic acid, syringic acid, vanillin, epicatechin, p-coumaric acid, ferulic acid, rutin, luteolin-7-glycoside, naringin, hesperidin, apigenin-7-glycoside, rosmarinic acid, fisetin, eriodictyol, luteolin, quercetin, naringenin, hesperetin, apigenin, and kaempferol. They were diluted from their stock solution into nine different concentrations at 0.625; 1.25; 5.0; 10.0; and 20.0 µg.L − 1 in a 1:1 methanol-water solution for the external calibration. Repeatability of the retention time (RT) and peak areas were measured as coefficient of variation (CV) which was under 0.61 for retention times and under 3.60 for areas of the peaks. The limit of detection (LOD) and quantification (LOQ) values of all standards were under 0.18 and 0.52 µg.mL − 1 (Supp. Table S1). Chromatograms were processed at 254, 280, 315, and 370 nm with DAD which operated at 200–400 nm. Dried extracts were dissolved in 1:1 methanol-water solution for suitable concentrations for HPLC-DAD analysis (DL: 10, DF: 50, ML: 20, MF: 100, TL: 10, and TF: 57 mg.mL − 1 ). Extracts were centrifuged at 10,000 rpm for 15 min before HPLC-DAD analysis. The identification of the peaks was carried out by comparing the RT and UV spectra with those of standard phenolic compounds. Some peaks had the same or similar UV spectra as some standards but with different RTs. They were defined as derivatives of standards with similar UV spectra and quantified as the equivalent of those standards. The peaks with different spectra from all standard compounds were characterized using the results of the reports studied to identify the compound of this plant species. For instance, the spectrum of oleuropein aglycone, nüzhenide 11-methyl oleoside, and oleocanthal was reported in olive extracts (Cecchi et al. 2023 ; Spagnuolo et al. 2023 ). Peaks with spectra similar to those of these three compounds were characterized as their derivatives and measured as the equivalent of protocatechuic acid using peak areas at 254 nm. Calibration and validation parameters of HPLC-DAD method were presented in Supplementary Table 1 . 2.6. Antimicrobial and antifungal activity Collected and extracted six olive fruit and leaf samples (DL, DF, ML, MF, TL, TF) were assayed for their antimicrobial and antifungal activities. Common six pathogenic bacteria and yeast-like fungi were used for these tests. Pathogenic bacteria selected as gram-negative bacteria are Escherichia coli (ATCC 25922), Yersinia pseudotuberculosis (ATCC 911), and Pseudomonas aeruginosa (ATCC 17853). Other pathogenic bacteria selected as gram-positive bacteria are Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), and Bacillus subtilis (ATCC 10876). Also, another pathogenic microorganism selected as yeast-like fungi is Candida albicans (ATCC 10231). Minimum inhibitory concentration (MIC) values (µg/mL) of extracts were determined by microtiter broth dilution method using rapid INT (iodonitrotetrazolium chloride) colorimetric assay based on the Clinical and Laboratory Standards Institute (CLSI) guidelines (Kuete et al. 2012 ). First, the maximum soluble concentrations of the 6 extracts were determined by dissolving them in DMSO. Stock concentrations 50 mg/mL for DL, 50 mg/mL for TL, 55 mg/mL for ML, 50 mg/mL for MF, 40 mg/mL for TF and 60 mg/mL for DF. Stock concentrations were diluted 2-fold with Mueller–Hinton broth (MHB) and added to the 96 well plate as 100µl/well. Then, bacteria were added as 5 × 10 − 5 CFU (colony-forming units)/mL for each well. Microplates were incubated at 37°C for 24 h. After that, 40µl of 0.2 mg/mL INT was added to each well and incubated at 37°C for 30 mins. A representative microplate design is given in Supplementary Fig. 2 . The results were evaluated by whether the indicator resulted in a pink color. The pink color indicates bacterial growth. The colorless first dose gives the MIC value. To determine the MIC values, experiments were performed as three independent replicates and at least 2 replicates within the experiment. Ampicillin antibiotic (50 mg/mL) was used to inhibit bacteria, and kanamycin antibiotic (50 mg/mL) was used to inhibit yeast-like fungi as experimental positive control. An experiment was also designed to calculate MIC values ​​for antibiotics. An experiment was designed to determine the MIC value of DMSO on microorganisms as a negative control (Supplementary Fig. 3) . The MIC values of all extracts were determined according to the non-toxic DMSO dose. Toxic doses of DMSO in dissolved extracts were neglected in experiments. 2.7. Statistical Analyses Cell viabilities (%) were compared within and between the cells using the UNIANOVA test of the SPSS program (Version 13.0). p values less than 0.05 were considered significant. 3. Results 3.1. Chromatographic analyses of olive samples The leaves and fruits were collected from three different regions close to the Thermal power station, and their metabolites were extracted using methanol as mentioned in the method section. The chemical composition of the extracts was then determined by HPLC-DAD analysis. Fruit extracts mostly contained the major olive compounds such as oleuropein aglycone, nüzhenide 11-methyl oleoside, and oleocanthal (Figure 2A, Table 2) . The highest variety amount of compounds was found in DF extract, while their amounts were lowest in TF. Flavonoids were found to be the highest in the leaf extracts (Figure 2B, Table 3) . Apigenin-7-glucoside was the highest flavonoid in the leaf extracts while luteolin-7-glucoside was the highest flavonoid in the fruit extracts. Leaf and fruit extracts obtained from the farthest location – Destin Village had the highest amount of these bioactive compounds. In contrast, extracts obtained from the closest location to the thermal station had the lowest amount. Oleuropein aglycones are the main compound in the fruit extracts (Figures 5-7, Table 2) . Nüzhenide 11-methyl oleoside derivatives were found in high amount in the fruit extracts as well. Oleuropein aglycone D6 was found the most abundant compound followed by Nüzhenide 11-methyl oleoside D5 and D10 in the TF extract ( Figure 3 and Table 2) . MF extract had Oleuropein aglycone D7 at highest amount which was followed by Nüzhenide 11-methyl oleoside D4 ( Figure 4 and Table 2) . Oleuropein aglycone D7 was found to be highest in both DF and DL extracts and was also found to be highest in D extracts compared to other extracts. Oleuropein aglycone D8 was found in the highest amount in leaf extracts, followed by apigenin-7-glucoside and luteolin-7-glucoside (Figures 6-8, Table 3). Protocatechuic acid, gallic acid, oleuropein aglycone D5, and apigenin were detected in the fruits of olive trees around the thermal power station, TF, which were not present in the other two locations ( Table 2) . The most abundant compounds in the fruit extracts such as oleuropein aglycone D7 and Nüzhenide 11-methyl oleoside D5 were the highest in DF which is farthest from the thermal power station (Figure 9, Table 2). The most abundant compounds in leaf extracts were oleuropein aglycone D7, apigenin-7-glycoside and luteolin-7-glycoside, which were found at the highest levels in DL among the extracts. (Figure 10, Table 3). In addition, secondary metabolites such as ferulic acid, luteloin, luteloin glycoside, and apigenin glycoside were detected in the leaves of trees close to the thermal power station (Table 4) . Besides, chlorogenic acid and caffeic acid, which were not found in samples from other regions, were found in the leaf samples collected from Destin village, which is the farthest from the thermal power station. Oleocanthal phenolic compound was found in the fruit samples. Oleochantal is a monophenolic secoiridoid, a group of antioxidants in some plant-based foods. Since this compound could not be detected in the fruits of olives grown in two regions near the thermal power plant, it can be thought that the proximity of the olive plant to the thermal power plant has a negative effect in terms of nutritional value and quality. Since olive fruit is used directly as food, these negative effects may also have adverse effects on the human body. 3.2. Cytotoxic profiles of human cells after the treatments with olive extracts Olive fruit and leaf extracts collected from different locations were applied to healthy human cell lines at a range of doses (0 µg/mL, 0.032 µg/mL, 0.16 µg/mL, 0.8 µg/mL, 4 µg/mL, 20 µg/mL, 100 µg/mL, and 500 µg/mL) for 24 or 48 hours, and the cytotoxic effects of these samples were analyzed. The human cells used for this were 1) ARPE-19 (human retinal epithelial cells) (Figure 11) , 2) MCF10A (human mammary epithelial cells) (Figure 12) , 3) BEAS-2B (human bronchial epithelial cells) (Figure 13) and 4) HUVEC (human umbilical cord endothelial cells) (Figure 14) . In ARPE-19 (Figure 11) and MCF10A (Figure 12) cells, olive leaf extracts obtained from the closest location to the thermal power station showed more cytotoxic effects at high doses for 48h than fruit extracts at the conditions. The cytotoxic effect of the high dose treatment for 48h was also determined to be more than 24h incubation. The highest cytotoxicity (around 80% cell death) was shown in BEAS-2B cells after 100 µg/mL leaf extract from the thermal location for 48h compared to other locations (Figure 13) . There was a similar decrease in the cell viability of HUVEC cells after the treatment with 500 µg/mL leaf extracts of collected forms from both in the center and near the thermal station compared to the further Destin village ( Figure 14) . The p values analyzed by the UNIANOVA test of SPPS software for all cells and extracts are summarized in Table 5. In general, the cytotoxicity results suggest that 1) the leaf extract is more cytotoxic in ARPE-19 and MCF10A cells, and 2) the death rate is similar in all locations after the highest dose (500 µg/mL) of leaf extracts while 100 µg/mL of thermal leaf samples resulted in cytotoxicity of the cells compared to other locations. Unlike ARPE-19 and MCF10A2, fruit extracts showed more cytotoxic properties in BEAS-2B and HUVEC cells. Table 6 summarises the comparison of cell viability between the cells after treatments with 100µg/mL or 500µg/mL leaf or fruit extracts for 48h collected from the closest location to the thermal station. The cytotoxicity of BEAS-2B cells appears to be more sensitive to the leaf extracts at 100µg/mL compared to other cells. All cells responded to the highest concentrations (500µg/mL) of leaf extracts similarly, because no significant difference between the cells was found. However, after the treatment with the highest concentration (for 48h) of fruit extracts, there are statistically significant differences in the viability of the cells. 3.3. Anti-microbial effect of olive extracts We then assessed the antimicrobial and antifungal activities of extracts. For this, three gram-negative bacteria ( Escherichia coli, Yersinia pseudotuberculosis , and Pseudomonas aeruginosa ), three gram-positive bacteria ( Staphylococcus aureus , Enterococcus faecalis, and Bacillus subtilis ) and one of yeast-like fungi ( Candida albicans ) were studied. The effects of extracts on microorganisms and MIC values are shown in Table 7 . Some extracts did not show antimicrobial activity against the microorganisms used in the experiment (Supplementary Figure 1) . Antimicrobial activities of all olive fruits and leaf extracts against B. subtilis, E. faecalis, P. aeruginosa, Y. pseudotuberculosis, E. coli, and C. albicans were not found, and MIC values could not be determined. On the other hand, antimicrobial activities of olive fruits and leaves extract, except MF, against S. aureus were found . MIC values on the S. aureus were determined as 13.73 mg/ml for ML, 12.5 mg/ml for TL, 20 mg/ml for TF, 12.5 mg/ml for DL, and 30 mg/ml for DF extract. 4. Discussion This study aimed to analyze the effect of thermal power plants on Olea europea fruit and leaf extracts and to reveal the potential result of these extracts on the cell viability of normal human cells and antimicrobial activity. To the best of our knowledge, this study is the first to investigate the outcome of proximity to thermal power stations on the contents of the fruits and leaves of the olive plant, as well as the effects of extracts obtained from these samples on healthy human cells. One of these potentially bioactive compounds in the leaves is the secoiridoide oleuropein, which can account for 6–9% of the dry matter. Other bioactive components in olive leaves include related secoiridoides, flavonoids, and triterpenes (El and Karakaya 2009). There are five groups of phenolic compounds principally present in olive leaves: oleuropeosides (oleuropein and verbascoside); flavones (luteolin-7-glucoside, apigenin-7-glucoside, diosmetin-7-glucoside, luteolin, and diosmetin); flavonols (rutin); flavan-3-ols (catechin), and substituted phenols (tyrosol, hydroxytyrosol, vanillin, vanillic acid, and caffeic acid). The most abundant compound in olive leaves is oleuropein followed by hydroxytyrosol, the flavone-7-glucosides of luteolin and apigenin, and verbascoside (El and Karakaya 2009). The present study showed that fruit extracts contained mostly the major olive compounds such as oleuropein aglycone, nüzhenide 11-methyl oleoside, and oleocanthal. Nüzhenide 11-methyl oleoside is one of the secoiridoides commonly found in olive (Silva et al. 2010 ). Leaf and fruit extracts obtained from the farthest location – Destin Village– had the highest amount of these bioactive compounds. In contrast, extracts obtained from the closest location to the thermal station had the lowest amount. For instance, apigenin is one of the most common flavonoids in fruit extracts from thermal stations. Flavonoids can play different roles in plant metabolism, such as protecting them from sun-derived radiation, defending them against pathogens and herbivory, regulating plant metabolism, and “serving as visual attractors for pollinators” (Salehi et al. 2019 ). Apigenin has a function in the cell cycle arrest during different phases of proliferation, such as G1/S or G2/M by promoting several cyclin-dependent kinases and other genes (Iizumi et al. 2013 ; Maggioni et al. 2013 ; Takagaki et al. 2005 ). This may result in cell death suggesting that it is therefore considered to have an anti-cancer activity within the cells. Oleocanthal is one of the olive-specific constituents with strong anti-inflammatory activities (Pang and Chin 2018), and this was found only in the fruit extracts collected from the farthest location (Destin village). Thermal stations may prevent the production of oleocanthal and therefore can lower the nutritional yield of olive fruits. On the other hand, leaf extracts collected from around the thermal station have ferulic acid in content. Ferulic acid is a biologically active compound that plays roles in oxidative stress, inflammation, vascular endothelial injury, fibrosis, cell death, and even platelet aggregation (Li et al. 2021 ). Ferrulic acid was found in the leaf extracts of olives collected from around the thermal power station only. Benavente-Garcia et al. showed that ferulic acid was not a main content of leaves, however such apigenin-7-glycoside, vanillic acid, and caffeic acid were abundant in the leaves of O. europea (Benavente-García et al. 2000 ). Caffeic and chlorogenic acids were only found in the leaf extracts collected from Destin village, while oleochanthal was the component only found in fruit extracts from Destin village. Xie et al. found that one of the common contents of leaves and fruit is apigenin-7-β-D-glucose (Xie et al. 2015). In our study, fruits from O. europea located near to thermal power station consisted of apigenin itself while leaves consisted of apigenine glycoside. The results of this study suggest that the closeness to the thermal power station affected the quantitative and qualitative features of leaves and fruits. The fact that the olive is a long-lived plant indicates that it is resistant to negative environmental conditions, especially drought. Although olive trees are considered drought-resistant species, water stress; may be associated with a wide range of adverse effects and physiological processes, such as nutrient uptake, carbon assimilation, reduced flower and fruit formation, canopy size, and flower loss (Nteve et al. 2024 ). Physiological studies conducted with olive plants in the literature are related to drought (Marchioni et al. 2024 ; Nteve et al. 2024 ), water (Sánchez-Piñero et al. 2024 ), and salt stress (Regni et al. 2019 ). Besides, high concentrations of lead, copper, and zinc were found in the olive samples collected from industrial areas, and areas close to roads with heavy traffic (Şahan and Başoğlu 2009). It has been reported that the amount of copper and zinc in some garden soils with olive groves in Akhisar district of Manisa is significantly high, however, the amounts of cadmium, lead, and arsenic are below the pollution limits (Zincircioğlu 2015 ). Unal et al. found that the concentrations of chromium, lead, zinc, and copper in the leaves of olive trees close to a factory in Izmir Kemalpaşa industrial zone (Turkiye) were higher than in the leaves of olive trees farther from the region (Ünal et al. 2011 ). The in vitro antimicrobial activity of olive leaves against bacteria and fungi has been demonstrated before (Juven and Henis 1970 ; Malhadas et al. 2017 ; Markín, Duek, and Berdícevsky 2003). This may be an adjunct treatment in the cases of long-term use of antibiotics. In addition, olive fruit's antimicrobial and antioxidant activities and their phenolic compounds on pathogenic microorganisms have been extensively studied and effective results have been obtained (Borjan et al. 2020; Juven and Henis 1970 ; Šimat et al. 2022 ). In a study, phenolic compounds of olive leaves collected from Muğla province in Türkiye were extracted and their antioxidant and antimicrobial activities were determined. It was shown that olive leaves in this region have antimicrobial activities against S. aureus, L. monocytogenes, Salmonella , and E. coli microorganisms. In addition, the highest antimicrobial effect of Muğla olive leaves was determined against S. aureus bacteria (Baysal et al. 2021 ). In our study, the antimicrobial effect of olive leaves and fruits collected from the thermal power plant region of Muğla was investigated, and according to the literature, the antimicrobial activity decreased significantly. In this study, we determined the highest antimicrobial activity of olive fruits and leaves against S. aureus bacteria. The antimicrobial activity of olive leaves was higher than that of olive fruits. The antimicrobial effect could not be determined against the other 6 microorganisms. Leaves and fruits of O. europea are reported to have anti-cancer effects on different cancer cell lines, and leaves also have an anti-hypertension and anti-microbial potential (Alesci et al. 2022 ). Ozturk et al. found that olive leaf extracts induced cytotoxicity in colon cancer cells, while these did not significantly affect normal human fibroblast cells (Öztürk, Çalık, and Ulusoy 2022). There are also many studies showed the cytotoxicity on breast cancer cells (Han et al. 2009 ; Junkins, Rodgers, and Phelan 2023 ), liver cancer cells (using commercial olive plants) (Bektay et al. 2021 ), but not in normal cells such as human gingival and neutrophil cells (Han et al. 2009 ), normal liver cells (Bektay et al. 2021 ) and human mesenchymal stem cells (Işik et al. 2012 ). Işık et al. collected samples from three different suburbs of Balikesir City; Ayvalık, Domat, and Uslu (Türkiye) which thermal power plants are not around these. Han et al. declared that they collected samples in Tunisia without any specific location (Han et al. 2009 ). Many of these studies did not mention the location of the sample collection. However, these all suggest that olive plants are not harmful to normal human cells. Our study aimed to investigate the wellness of normal human cells only after extract treatments, and showed that extracts of leaf samples from the thermal area induced higher cytotoxicity on healthy cells, especially BEAS-2B bronchial cells compared to those collected from other locations. The findings of this study conclude that thermal power plants have negative effects on both olives and olive-treated human cells. A detailed investigation is needed for significant changes in genes or proteins of both O. europea and human cells to reveal the molecular effects of thermal power plants. Thus, all these findings will give important clues for the regions where thermal power plants should be built and/or will question how thermal power stations can be developed to minimize the potential effects on the ecosystem and living things. Declarations Acknowledgments This study was supported by a 2209-A grant fromTUBITAK (The Scientific and Technological Research Council of Türkiye) (Project ID: 1919B012306896) and by the Scientific Research Project Coordination Unit of Karadeniz Technical University (Project numbers: FLÖ-2024-11149, FLÖ-2024-11176 and FHD-2024-16036). The authors thank 1) Prof Nurettin YAYLI and Mrs. Gözde BOZDAL (Karadeniz Technical University, Faculty of Pharmacy) for assisting with extractions from plants, 2) Mrs. Zeliha BAYRAM (from Istanbul Metropolitan Municipality), a topographical engineer, for assisting the use of Google maps for finding locations, 3) Prof Zülal ATLI ŞEKEROĞLU (Ordu University, Department of Molecular Biology and Genetics), Assoc Prof Hatice SEVİM NALKIRAN (Recep Tayyip Erdoğan University, Department of Medical Biology) and Assit Prof Cihan INAN (Karadeniz Technical University, Department of Molecular Biology and Genetics) for providing a batch of BEAS-2B cells, ARPE-19 cells and MCF10A cells, respectively, and 6) Prof Kadriye INAN BEKTAS (Karadeniz Technical University, Department of Molecular Biology and Genetics) for assisting anti-microbial experiments, and 7) Mr. Ali GÜRBÜZ for collecting fruit and leaves. Conflict of interest The authors declare that no conflict of interest exists. Author contributions EG conceptualized the study, collected and extracted samples, and also performed cell culture and MTT experiments, EG was granted by TUBITAK(Project ID: 1919B012306896), AS performed the HPLC-DAD experiment, FBT planned andperformed anti-microbial experiments, ES and NNK performed cell culture applications and MTT tests, ST supported cell culture and anti-microbial experiments, HM managed anti-microbial experiments and was granted by the project (Karadeniz Technical University, project ID: FHD-2024-16036), EAT conceptualized, performed and analyzed the HPLC-DAD experiment SCU conceptualized and managed the study, and was granted by (Karadeniz Technical University, Project numbers: FLÖ-2024-11149 and FLÖ-2024-11176). References Alesci, Alessio, Anthea Miller, Roberta Tardugno, and Simona Pergolizzi. 2022. ‘Chemical Analysis, Biological and Therapeutic Activities of Olea Europaea L. Extracts’. Natural Product Research 36(11): 2932–45. Baysal, Gülay et al. 2021. ‘Determination of Theoretical Calculations by DFT Method and Investigation of Antioxidant, Antimicrobial Properties of Olive Leaf Extracts from Different Regions’. Journal of food science and technology 58(5): 1909–17. Bektay, Muhammed Yunus, Eray Metin Güler, Mustafa Gökçe, and Mustafa Volkan Kiziltaş. 2021. ‘Investigation of the Genotoxic, Cytotoxic, Apoptotic, and Oxidant Effects of Olive Leaf Extracts on Liver Cancer Cell Lines’. Turkish Journal of Pharmaceutical Sciences 18(6): 781–89. Benavente-García, O. et al. 2000. ‘Antioxidant Activity of Phenolics Extracted from Olea Europaea L. Leaves’. Food Chemistry 68(4): 457–62. Borjan, Dragana, Maja Leitgeb, Željko Knez, and Maša Knez Hrnčič. 2020. ‘Microbiological and Antioxidant Activity of Phenolic Compounds in Olive Leaf Extract’. Molecules (Basel, Switzerland) 25(24). Cecchi, Lorenzo et al. 2023. ‘Virgin Olive Oil By-Product Valorization: An Insight into the Phenolic Composition of Olive Seed Extracts from Three Cultivars as Sources of Bioactive Molecules’. Molecules 2023, Vol. 28, Page 2776 28(6): 2776. Demir, Elif Ayazoglu, A. Colak, S. Celik Uzuner, and O. Bekircan. 2021. ‘Cytotoxic Effect of a 3-(4-Chlorophenyl)-5-(4-Methoxybenzyl)-4H-1,2,4-Triazole Derivative Compound in Human Melanoma Cells’. International Journal of Biology and Chemistry 14(1): 139–48. El, Sedef N., and Sibel Karakaya. 2009. ‘Olive Tree (Olea Europaea) Leaves: Potential Beneficial Effects on Human Health’. Nutrition Reviews 67(11): 632–38. Faizan, Yusuf, Sandesh Mishra, Amritansu Khali, and Rishi Diwan. 2021. ‘Hazard Identification and Risk Assessment of 2 × 300 MW Thermal Power Plant with Their Control Measures to Optimize the Risk’. Journal of Failure Analysis and Prevention 21(1): 179–92. Han, Junkyu, Terence P.N. Talorete, Parida Yamada, and Hiroko Isoda. 2009. ‘Anti-Proliferative and Apoptotic Effects of Oleuropein and Hydroxytyrosol on Human Breast Cancer MCF-7 Cells’. Cytotechnology 59(1): 45–53. Iizumi, Yosuke et al. 2013. ‘The Flavonoid Apigenin Downregulates CDK1 by Directly Targeting Ribosomal Protein S9’. PLOS ONE 8(8): e73219. Işik, Sevim, Aysel Karagöz, Şeyda Karaman, and Cevdet Nergiz. 2012. ‘Proliferative and Apoptotic Effects of Olive Extracts on Cell Lines and Healthy Human Cells’. Food Chemistry 134(1): 29–36. Junkins, Katherine, Margaret Rodgers, and Shelley A. Phelan. 2023. ‘Oleuropein Induces Cytotoxicity and Peroxiredoxin Over-Expression in MCF-7 Human Breast Cancer Cells’. Anticancer Research 43(10): 4333–39. Juven, B., and Y. Henis. 1970. ‘Studies on the Antimicrobial Activity of Olive Phenolic Compounds’. The Journal of Applied Bacteriology 33(4): 721–32. Kuete, Victor et al. 2012. ‘Antibacterial Activities of the Extracts, Fractions and Compounds from Dioscorea Bulbifera’. BMC complementary and alternative medicine 12. Kumar, Priti, Arvindhan Nagarajan, and Pradeep D. Uchil. 2018. ‘Analysis of Cell Viability by the MTT Assay’. Cold Spring Harbor Protocols 2018(6): pdb.prot095505. Li, Dan et al. 2021. ‘Ferulic Acid: A Review of Its Pharmacology, Pharmacokinetics and Derivatives’. Life Sciences 284: 119921. Maggioni, Daniele et al. 2013. ‘Apigenin Impairs Oral Squamous Cell Carcinoma Growth in Vitro Inducing Cell Cycle Arrest and Apoptosis’. International Journal of Oncology 43(5): 1675–82. Malhadas, Cynthia et al. 2017. ‘Antimicrobial Activity of Endophytic Fungi from Olive Tree Leaves’. World journal of microbiology & biotechnology 33(3). Marchioni, Ilaria et al. 2024. ‘Comparative Effects of Drought Stress on Three Olive Cultivars Focusing on Older Leaves’. Scientia Horticulturae 332: 113234. Markín, D., L. Duek, and Israela Berdícevsky. 2003. ‘In Vitro Antimicrobial Activity of Olive Leaves’. Mycoses 46(3–4): 132–36. Nteve, Georgia Maria et al. 2024. ‘Adaptation Mechanisms of Olive Tree under Drought Stress: The Potential of Modern Omics Approaches’. Agriculture (Switzerland) 14(4): 1–18. Oteros, J. et al. 2013. ‘Modelling Olive Phenological Response to Weather and Topography’. Agriculture, Ecosystems & Environment 179: 62–68. Öztürk, Emre, Fatma Çalık, and Derya Ulusoy. 2022. ‘Investigation of Cytotoxic and Genotoxic Effects of Olive Leaf Extract on Colon Cancer Cells and Normal Cell Lines’. Eurasian Journal of Molecular and Biochemical Sciences 1(2): 26–31. Pang, Kok Lun, and Kok Yong Chin. 2018. ‘The Biological Activities of Oleocanthal from a Molecular Perspective’. Nutrients 2018, Vol. 10, Page 570 10(5): 570. Regni, Luca et al. 2019. ‘Behavior of Four Olive Cultivars during Salt Stress’. Frontiers in Plant Science 10: 436704. Şahan, Yasemin;, and Fikri Başoğlu. 2009. ‘Heavy Metal Pollution in Olives Grown in Bursa, Turkey’. Asian Journal of Chemistry 21(4): 3023–29. Salehi, Bahare et al. 2019. ‘The Therapeutic Potential of Apigenin’. International Journal of Molecular Sciences 20(6): 1305. Sánchez-Piñero, Marta et al. 2024. ‘Assessment of Water Stress Impact on Olive Trees Using an Accurate Determination of the Endocarp Development’. Irrigation Science 42(3): 461–76. Silva, Sandra et al. 2010. ‘Secoiridoids in Olive Seed: Characterization of Nüzhenide and 11-Methyl Oleosides by Liquid Chromatography with Diode Array and Mass Spectrometry’. Grasas y Aceites 61(2): 157–64. Šimat, Vida et al. 2022. ‘Antioxidant and Antimicrobial Activity of Hydroethanolic Leaf Extracts from Six Mediterranean Olive Cultivars’. Antioxidants (Basel, Switzerland) 11(9). Spagnuolo, Carmela et al. 2023. ‘Phenolic Extract from Extra Virgin Olive Oil Induces Different Anti-Proliferative Pathways in Human Bladder Cancer Cell Lines’. Nutrients 15(1). Tajner-Czopek, Agnieszka et al. 2020. ‘Study of Antioxidant Activity of Some Medicinal Plants Having High Content of Caffeic Acid Derivatives’. Antioxidants 2020, Vol. 9, Page 412 9(5): 412. Takagaki, Nobumasa et al. 2005. ‘Apigenin Induces Cell Cycle Arrest and P21/WAF1 Expression in a P53-Independent Pathway.’ International journal of oncology 26(1): 185–89. Toreti, A. et al. 2024. Drought in Western Mediterranean - Publications Office of the EU . Ünal, Dilek, Şenol Sert, Nuray Olcay Işık, and Ünal Kaya. 2011. ‘İzmir-Kemalpaşa Sanayi Bölgesinde Ağır Metal Kirliliğinin Biyoindikatör Olarak Zeytin (Olea Europaea) Bitkisi Kullanılarak Belirlenmesi’. Zeytin Bilimi 2: 59–64. Xie, Pu jun, Li xin Huang, Cai hong Zhang, and Yao lei Zhang. 2015. ‘Phenolic Compositions, and Antioxidant Performance of Olive Leaf and Fruit (Olea Europaea L.) Extracts and Their Structure-Activity Relationships’. Journal of Functional Foods 16: 460–71. Zincircioğlu, Nurdan. 2015. ‘Manisa-Akhisar’da Bulunan Bazı Zeytin Bahçelerinde Cu, Zn, Cd, Pb ve as Içeriklerinin Belirlenmesi.’ Zeytin Bilimi 5: 21–26. Table Table 1 is not available with this version Supplementary Figures and Tables The supplementary figures and tables are not available with this version. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5381441","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373421704,"identity":"1aa22220-bbcf-43fa-8646-f2506359e19e","order_by":0,"name":"Esra Gürbüz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Gürbüz","suffix":""},{"id":373421705,"identity":"6171d9fe-03d2-4f4e-9d30-32f2b8624d9e","order_by":1,"name":"Aytül 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(C), the distances from the station (red star) by calculation of direct lines (D), and representative morphological profiles of olive fruit and leaf from different locations, after drying (E).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/42514959ad11e79c64616518.png"},{"id":68293252,"identity":"c347737c-cd15-4ef3-848f-74c745ae12eb","added_by":"auto","created_at":"2024-11-05 17:52:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical composition of (A) the fruit and (B) leaf extracts of the olive samples as mg/100g extract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/d5f3b483f679a235bdba8d71.png"},{"id":68293826,"identity":"dabbc2dc-0f96-4371-8ea3-02e7cee4dcab","added_by":"auto","created_at":"2024-11-05 18:00:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC-DAD chromatogram of TF extract at 254 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/c9084c006d212a9ed55550cd.png"},{"id":68294210,"identity":"f3e5c6d9-f3d6-4181-947c-ff20d6d788d3","added_by":"auto","created_at":"2024-11-05 18:08:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC-DAD chromatogram of MF extract at 280 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/824b3f359b367e123f750865.png"},{"id":68292863,"identity":"2c1b15f3-4a12-47c5-a2d6-9aee0773dc84","added_by":"auto","created_at":"2024-11-05 17:44:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":73606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC-DAD chromatogram of DF extract at 280 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/a60c4e9d1909e20227b6faf8.png"},{"id":68292865,"identity":"bb39a5f3-5950-4f41-9a63-04bfa885a2fb","added_by":"auto","created_at":"2024-11-05 17:44:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC-DAD chromatogram of TL extract at 280 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/28b3f4c2821f7e2c30ab516d.png"},{"id":68294211,"identity":"8284cf03-93ef-45a8-8856-8e0294af1819","added_by":"auto","created_at":"2024-11-05 18:08:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":76580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC-DAD chromatogram of ML extract at 280 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/f88a833a88af7cf688f17595.png"},{"id":68293258,"identity":"de53edd6-bb06-4a8e-bda5-3b981d96804c","added_by":"auto","created_at":"2024-11-05 17:52:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":68698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC-DAD chromatogram of DL extract at 280 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/df4f3d193bd588d42f116b02.png"},{"id":68293821,"identity":"642168ad-5c3f-4037-b995-20d4188ec097","added_by":"auto","created_at":"2024-11-05 18:00:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":55625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanging the chemical composition of fruit extracts based on their region.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/8089f1e41dbadb13ab7f984a.png"},{"id":68294212,"identity":"326f6a49-c199-4c86-bb55-e0f01a48d1cc","added_by":"auto","created_at":"2024-11-05 18:08:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":58825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanging the chemical composition of fruit extracts based on their region.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/a8415f8def1fa6116f8ae730.png"},{"id":68292867,"identity":"bd9e39ad-63ee-4748-b7c9-390e81c3e4be","added_by":"auto","created_at":"2024-11-05 17:44:37","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":131888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cell viability in ARPE-19 human retinal epithelial cells after the treatments with fruit (left panel) and leaf (right panel) extracts at different concentrations for 24h and 48h which were collected from Destin Village (the farthest location), Central (the middle location) and Thermal station.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/d8b9f50cc3db1542a61929be.png"},{"id":68293825,"identity":"8193566b-a42c-4719-8f0d-f504ba3b96e8","added_by":"auto","created_at":"2024-11-05 18:00:37","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":130749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cell viability in MCF10A human breast epithelial cells after the treatments with fruit (left panel) and leaf (right panel) extracts at different concentrations for 24h and 48h which were collected from Destin Village (the farthest location), Central (the middle location) and Thermal station.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/5ceaecca54578c9dbb383051.png"},{"id":68293824,"identity":"040fffe8-e791-4962-92c3-86574a47fd2b","added_by":"auto","created_at":"2024-11-05 18:00:37","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":116527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cell viability in BEAS-2B human bronchial epithelial cells after the treatments with fruit (left panel) and leaf (right panel) extracts at different concentrations for 24h and 48h which were collected from Destin Village (the farthest location), Central (the middle location) and Thermal station.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/67da4a45a147e5e9ef15e1cf.png"},{"id":68292874,"identity":"9b03d243-29ed-4f6c-a0d4-c6acc1a971e9","added_by":"auto","created_at":"2024-11-05 17:44:37","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":118854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe cell viability in HUVEC human umbilical vein endothelial cells after the treatments with fruit (left panel) and leaf (right panel) extracts at different concentrations for 24h and 48h which were collected from Destin Village (the farthest location), Central (the middle location) and Thermal station.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/cb4ec3ced7185d355124feb1.png"},{"id":68294546,"identity":"5598ade6-283f-4c15-bf52-f9983a186fb2","added_by":"auto","created_at":"2024-11-05 18:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2938555,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5381441/v1/8ec95a01-9299-464a-bde1-041168fe21ed.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHuman Cell Cytotoxicity, Anti-Microbial Effect and Chromatographic Analyses of Leaf and Fruit Extracts of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOlea europaea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (Olive) Collected from Different Distances to Yatagan Thermal Power Plant in Mugla, Türkiye\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThere has been an increasing interest in medicinal plants due to their content for health promotion such as flavonoids (Tajner-Czopek et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The fruits of \u003cem\u003eOlea europea\u003c/em\u003e have been commonly used especially in the Mediterranean diet and olive oil is an indispensable product for gastronomy. Olive tree leaves are also widely used in traditional treatments in European and Mediterranean countries such as Greece, Spain, Italy, France, Turkey, Israel, Morocco, and Tunisia. They have been used as extracts, herbal teas, and powders in the human diet and contain many potential bioactive compounds that may have antioxidant, antihypertensive, antiatherogenic, anti-inflammatory, hypoglycemic, and hypocholesterolemic properties (El and Karakaya 2009).\u003c/p\u003e \u003cp\u003e \u003cem\u003eO. europea\u003c/em\u003e has been reported to be highly resistant to environmental stressors, such as drought (Marchioni et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nteve et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), water (S\u0026aacute;nchez-Pi\u0026ntilde;ero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and salt stress (Regni et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The salinity of the soil and irrigation water, as well as the lack of water and nutrient resources, have all impacted the recent expansion of olive farming beyond the Mediterranean region to several continents (Oteros et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Climate change is one of the most important drawbacks globally. This may increase temperatures and accordingly water restraint. Mediterranean and Aegean regions are at risk of drought (Toreti et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thermal power stations can also contribute to increased temperature due to coal combustion (Faizan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOlives naturally resist drought and can thrive in environments with few resources, therefore they rank among the most productive crops. Nevertheless, the challenges posed by climate constraints necessitate investigating the high variability of species to one another in the direction of abiotic stress tolerance and identifying the genetic factors that govern how plants react to stress. Closeness to thermal power stations can be another environmental stress for olive plants, but there is no clear understanding of the possible effects on olive fruits and leaves. Besides, the effects of the olives around thermal power plants on human cells have not been explored before. The importance of olives in the human diet questions the potential harmful effects of thermal olives on human health. In this study, we investigated the quantitative and qualitative content of olive fruits and leaves grown in places close to the Yatağan thermal power plant compared to those far away from the station. We also treated four types of normal human cells (derived from the breast, retina, vein, and bronchus) with the extracts of olive fruits and leaves to conclude the cytotoxicity of the cells.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample collection\u003c/h2\u003e \u003cp\u003eOlive fruit and leaves \u003cem\u003e(O. Europaea)\u003c/em\u003e were collected in October from Yatagan Center, Deştin Village, and Şahinler Village in Yatagan district of Mugla province \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e at different distances from the thermal power station \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C-D\u003cb\u003e)\u003c/b\u003e. The specific locations in the collected regions were recorded on Google Maps, and then their distances to the thermal power station were calculated by direct lines. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE shows the representative morphological profiles of fruits and leaves from the plants located at different distances from the thermal power station. It is observed that the leaves and fruits of the samples closest to the thermal power station are quite small \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cb\u003eleft)\u003c/b\u003e. The largest fruit size belongs to the samples that are the farthest from the power station \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cb\u003eright)\u003c/b\u003e. The abbreviations for the locations used throughout the study are as follows: D; Destin (the furthest location), M (the middle location), -T (thermal location) and F (fruit), L (leaves). For instance; TL (thermal-leaves), MF (middle-fruit)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Extraction protocol\u003c/h2\u003e \u003cp\u003eOlive fruits and leaves were dried at 25\u003csup\u003eo\u003c/sup\u003eC for 20 days without direct sunlight. The leaves were passed ground into a fine powder with a grinder. The grinding process was repeated every 5 minutes to prevent friction heating of the sample. 40 grams of each sample was added to 150 mL of methanol into the Erlen mayer and covered with aluminum foil. Samples were incubated in a shaker at room temperature for 6 hours. The extracts were filtered into the balloons using Whatmann papers. Then the same amount of solvent was added to the remaining part for another 6 hours at the same condition. The filtrated extracts from samples were collected into the balloons, and the solvent was evaporated with a Rotary Evaporator. The obtained dense-liquid extracts were placed in Eppendorf tubes and stored in the refrigerator \u003cb\u003e(Supplementary Fig.\u0026nbsp;1)\u003c/b\u003e. These liquid extract samples were dried using a lyophilizer, and lyophilized extracts were stored at +\u0026thinsp;4\u0026deg;C. The main stocks of the extracts were prepared with DMSO (mg/mL), aliquoted as 60\u0026ndash;80 \u0026micro;l, and kept at \u0026ndash; 20\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell culture and extract treatments\u003c/h2\u003e \u003cp\u003eThe healthy cell lines used in this study were 1) MCF10A (ATCC, CRL-10317\u0026trade;) human mammary gland epithelial cells 2) ARPE-19 (ATCC, CRL-2302\u0026trade;) human eye retinal pigment epithelial cells, 3) HUVEC (ATCC, CRL-1730\u0026trade;) primary human umbilical vein endothelial cells, and 4) BEAS-2B (ATCC, CRL-3588\u0026trade;) epithelial cells isolated from normal human bronchial epithelium. Except for BEAS-2B cells cultured in DMEM, other cells were cultured in RPMI media including 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After they reached at full confluency, cells were split into 96 well plates. Confluent cells in the wells were treated by the extracts with final concentrations of 500 \u0026micro;g/mL, 100 \u0026micro;g/mL, 25 \u0026micro;g/mL, 6.25 \u0026micro;g/mL, 1.56 \u0026micro;g/mL, 0.39 \u0026micro;g/mL, 0.097 \u0026micro;g/mL, 0.024 \u0026micro;g/mL, for 24 h or 48 h. Control cells were untreated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. MTT assay\u003c/h2\u003e \u003cp\u003eThe principle of the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test is based on the metabolizing of MTT dye into a formazane crystal which indicates mitochondrial activity in the living cells. The MTT dye is yellow, and purple is formed after the tetrazolium ring was enzymatically broken in live cells. Tetrazolium crystals are then dissolved with DMSO followed by the measurement of color intensities as absorbance at 570 nm using a spectrophotometer. Live cells have more absorbance due to the purple color (Demir et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar, Nagarajan, and Uchil \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For the MTT experiment, the culture media was removed from the wells and 190 \u0026micro;l media and 10 \u0026micro;l 0.25 mg/mL MTT dye was added to each well, and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 2 hours. After incubation, media including MTT was removed, and 200 \u0026micro;l of DMSO was added to each well to visualize color formation. Plates treated with DMSO were dark-incubated in a shaker at 120xg for 1 hour to dissolve the crystals. Absorbances of each well in the plates were read at 570 nm by a spectrophotometer. The absorbance of untreated cells was considered 100% viable, and the viability of treated cells was calculated relative to the absorbances of untreated control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. HPLC analyses\u003c/h2\u003e \u003cp\u003e \u003cb\u003eChemicals\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHPLC grade acetonitrile (ACN), acetic acid (AA), and standard phenolic compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA) but quercetin from Fluka Chemie GmbH (Switzerland). Methanol for analysis (Supelco\u0026reg;) was supplied by Merck (Darmstadt, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExtraction of phenolic compounds from samples\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDried extracts were dissolved in the methanol and diluted with 50% water for suitable concentrations for HPLC-DAD analysis (DL: 10, DF: 50, ML: 20, MF: 100, TL: 10, and TF: 57 mg/mL).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHPLC-DAD and HPLC-MS conditions for separation of phenolic compounds\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe chromatographic analyses were performed using a Dionex (Thermo Scientific, Germering, Germany) Ultimate 3000 high-performance liquid chromatography (HPLC) system equipped with an Ultimate 3000 diode array detector (DAD). A Thermo acclaim C30 column (150mm. 3mm id. 3\u0026micro;m pd) was used with a Macherey Nagel (3mm id) guard column. Gradient elution was used with mobile phases; A: 2% acetic acid in water and B: 70%acetonitrile-30% water. The flow rate was 0.37 mL/min, and the injection volume was 10\u0026micro;L. Column temperature was 25\u0026deg;C. Following 25 phenolic standards were used to calibrate and validate HPLC-DAD analysis method: Gallic acid, protocatechuic acid, p-hydroxy benzoic acid (p-OH benzoic acid), chlorogenic acid, vanillic acid, caffeic acid, syringic acid, vanillin, epicatechin, p-coumaric acid, ferulic acid, rutin, luteolin-7-glycoside, naringin, hesperidin, apigenin-7-glycoside, rosmarinic acid, fisetin, eriodictyol, luteolin, quercetin, naringenin, hesperetin, apigenin, and kaempferol. They were diluted from their stock solution into nine different concentrations at 0.625; 1.25; 5.0; 10.0; and 20.0 \u0026micro;g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in a 1:1 methanol-water solution for the external calibration. Repeatability of the retention time (RT) and peak areas were measured as coefficient of variation (CV) which was under 0.61 for retention times and under 3.60 for areas of the peaks. The limit of detection (LOD) and quantification (LOQ) values of all standards were under 0.18 and 0.52 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supp. Table S1). Chromatograms were processed at 254, 280, 315, and 370 nm with DAD which operated at 200\u0026ndash;400 nm. Dried extracts were dissolved in 1:1 methanol-water solution for suitable concentrations for HPLC-DAD analysis (DL: 10, DF: 50, ML: 20, MF: 100, TL: 10, and TF: 57 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Extracts were centrifuged at 10,000 rpm for 15 min before HPLC-DAD analysis. The identification of the peaks was carried out by comparing the RT and UV spectra with those of standard phenolic compounds. Some peaks had the same or similar UV spectra as some standards but with different RTs. They were defined as derivatives of standards with similar UV spectra and quantified as the equivalent of those standards. The peaks with different spectra from all standard compounds were characterized using the results of the reports studied to identify the compound of this plant species. For instance, the spectrum of oleuropein aglycone, n\u0026uuml;zhenide 11-methyl oleoside, and oleocanthal was reported in olive extracts (Cecchi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Spagnuolo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Peaks with spectra similar to those of these three compounds were characterized as their derivatives and measured as the equivalent of protocatechuic acid using peak areas at 254 nm. Calibration and validation parameters of HPLC-DAD method were presented in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Antimicrobial and antifungal activity\u003c/h2\u003e \u003cp\u003eCollected and extracted six olive fruit and leaf samples (DL, DF, ML, MF, TL, TF) were assayed for their antimicrobial and antifungal activities. Common six pathogenic bacteria and yeast-like fungi were used for these tests. Pathogenic bacteria selected as gram-negative bacteria are \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922), \u003cem\u003eYersinia pseudotuberculosis\u003c/em\u003e (ATCC 911), and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC 17853). Other pathogenic bacteria selected as gram-positive bacteria are \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 25923), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (ATCC 29212), and \u003cem\u003eBacillus subtilis\u003c/em\u003e (ATCC 10876). Also, another pathogenic microorganism selected as yeast-like fungi is \u003cem\u003eCandida albicans\u003c/em\u003e (ATCC 10231).\u003c/p\u003e \u003cp\u003eMinimum inhibitory concentration (MIC) values (\u0026micro;g/mL) of extracts were determined by microtiter broth dilution method using rapid INT (iodonitrotetrazolium chloride) colorimetric assay based on the Clinical and Laboratory Standards Institute (CLSI) guidelines (Kuete et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). First, the maximum soluble concentrations of the 6 extracts were determined by dissolving them in DMSO. Stock concentrations 50 mg/mL for DL, 50 mg/mL for TL, 55 mg/mL for ML, 50 mg/mL for MF, 40 mg/mL for TF and 60 mg/mL for DF. Stock concentrations were diluted 2-fold with Mueller\u0026ndash;Hinton broth (MHB) and added to the 96 well plate as 100\u0026micro;l/well. Then, bacteria were added as 5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e CFU (colony-forming units)/mL for each well. Microplates were incubated at 37\u0026deg;C for 24 h. After that, 40\u0026micro;l of 0.2 mg/mL INT was added to each well and incubated at 37\u0026deg;C for 30 mins. A representative microplate design is given in \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e. The results were evaluated by whether the indicator resulted in a pink color. The pink color indicates bacterial growth. The colorless first dose gives the MIC value. To determine the MIC values, experiments were performed as three independent replicates and at least 2 replicates within the experiment. Ampicillin antibiotic (50 mg/mL) was used to inhibit bacteria, and kanamycin antibiotic (50 mg/mL) was used to inhibit yeast-like fungi as experimental positive control. An experiment was also designed to calculate MIC values ​​for antibiotics. An experiment was designed to determine the MIC value of DMSO on microorganisms as a negative control \u003cb\u003e(Supplementary Fig.\u0026nbsp;3)\u003c/b\u003e. The MIC values of all extracts were determined according to the non-toxic DMSO dose. Toxic doses of DMSO in dissolved extracts were neglected in experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analyses\u003c/h2\u003e \u003cp\u003eCell viabilities (%) were compared within and between the cells using the UNIANOVA test of the SPSS program (Version 13.0). \u003cem\u003ep\u003c/em\u003e values less than 0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u0026nbsp;Chromatographic analyses of olive samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leaves and fruits were collected from three different regions close to the Thermal power station, and their metabolites were extracted using methanol as mentioned in the method section. The chemical composition of the extracts was then determined by HPLC-DAD analysis. Fruit extracts mostly contained the major olive compounds such as oleuropein aglycone, n\u0026uuml;zhenide 11-methyl oleoside, and oleocanthal \u003cstrong\u003e(Figure 2A, Table 2)\u003c/strong\u003e. The highest variety amount of compounds was found in DF extract, while their amounts were lowest in TF. Flavonoids were found to be the highest in the leaf extracts \u003cstrong\u003e(Figure 2B, Table 3)\u003c/strong\u003e. Apigenin-7-glucoside was the highest flavonoid in the leaf extracts while luteolin-7-glucoside was the highest flavonoid in the fruit extracts. Leaf and fruit extracts obtained from the farthest location \u0026ndash; Destin Village had the highest amount of these bioactive compounds. In contrast, extracts obtained from the closest location to the thermal station had the lowest amount.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOleuropein aglycones are the main compound in the fruit extracts \u003cstrong\u003e(Figures 5-7, Table 2)\u003c/strong\u003e.\u0026nbsp;N\u0026uuml;zhenide 11-methyl oleoside derivatives were found in high amount in the fruit extracts as well.\u0026nbsp;Oleuropein aglycone D6 was found the most abundant compound followed by\u0026nbsp;N\u0026uuml;zhenide 11-methyl oleoside D5 and D10\u0026nbsp;in the TF extract (\u003cstrong\u003eFigure 3 and Table 2)\u003c/strong\u003e. MF extract had Oleuropein aglycone D7 at highest amount which was followed by\u0026nbsp;N\u0026uuml;zhenide 11-methyl oleoside D4\u0026nbsp;(\u003cstrong\u003eFigure 4 and Table 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eOleuropein aglycone D7 was found to be highest in both DF and DL extracts and was also found to be highest in D extracts compared to other extracts. \u0026nbsp;Oleuropein aglycone D8 was found in the highest amount in leaf extracts, followed by apigenin-7-glucoside and luteolin-7-glucoside \u003cstrong\u003e(Figures 6-8, Table 3).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtocatechuic acid, gallic acid, oleuropein aglycone D5, and apigenin were detected in the fruits of olive trees around the thermal power station, TF, \u0026nbsp;which were not present in the other two locations (\u003cstrong\u003eTable 2)\u003c/strong\u003e. The most abundant compounds in the fruit extracts such as oleuropein aglycone D7 and N\u0026uuml;zhenide 11-methyl oleoside D5 were the highest in DF which is farthest from the thermal power station \u003cstrong\u003e(Figure 9, Table 2).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe most abundant compounds in leaf extracts were oleuropein aglycone D7, apigenin-7-glycoside and luteolin-7-glycoside, which were found at the highest levels in DL among the extracts. \u003cstrong\u003e(Figure 10, Table 3).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, secondary metabolites such as ferulic acid, luteloin, luteloin glycoside, and apigenin glycoside were detected in the leaves of trees close to the thermal power station \u003cstrong\u003e(Table 4)\u003c/strong\u003e. Besides, chlorogenic acid and caffeic acid, which were not found in samples from other regions, were found in the leaf samples collected from Destin village, which is the farthest from the thermal power station. \u0026nbsp;Oleocanthal phenolic compound was found in the fruit samples. Oleochantal is a monophenolic secoiridoid, a group of antioxidants in some plant-based foods. Since this compound could not be detected in the fruits of olives grown in two regions near the thermal power plant, it can be thought that the proximity of the olive plant to the thermal power plant has a negative effect in terms of nutritional value and quality. Since olive fruit is used directly as food, these negative effects may also have adverse effects on the human body.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.\u0026nbsp;Cytotoxic profiles of human cells after the treatments with olive extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOlive fruit and leaf extracts collected from different locations were applied to healthy human cell lines at a range of doses (0 \u0026micro;g/mL, 0.032 \u0026micro;g/mL, 0.16 \u0026micro;g/mL, 0.8 \u0026micro;g/mL, 4 \u0026micro;g/mL, 20 \u0026micro;g/mL, 100 \u0026micro;g/mL, and 500 \u0026micro;g/mL) for 24 or 48 hours, and the cytotoxic effects of these samples were analyzed. The human cells used for this were 1) ARPE-19 (human retinal epithelial cells) \u003cstrong\u003e(Figure 11)\u003c/strong\u003e, 2) MCF10A (human mammary epithelial cells) \u003cstrong\u003e(Figure 12)\u003c/strong\u003e, 3) BEAS-2B (human bronchial epithelial cells) \u003cstrong\u003e(Figure 13)\u003c/strong\u003e and 4) HUVEC (human umbilical cord endothelial cells) \u003cstrong\u003e(Figure 14)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn ARPE-19 \u003cstrong\u003e(Figure 11)\u003c/strong\u003e and MCF10A \u003cstrong\u003e(Figure 12)\u003c/strong\u003e cells, olive leaf extracts obtained from the closest location to the thermal power station showed more cytotoxic effects at high doses for 48h than fruit extracts at the conditions. The cytotoxic effect of the high dose treatment for 48h was also determined to be more than 24h incubation. The highest cytotoxicity (around 80% cell death) was shown in BEAS-2B cells after 100 \u0026micro;g/mL leaf extract from the thermal location for 48h compared to other locations \u003cstrong\u003e(Figure 13)\u003c/strong\u003e. There was a similar decrease in the cell viability of HUVEC cells after the treatment with 500 \u0026micro;g/mL leaf extracts of collected forms from both in the center and near the thermal station compared to the further Destin village \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFigure 14)\u003c/strong\u003e. The \u003cem\u003ep\u003c/em\u003e values analyzed by the UNIANOVA test of SPPS software for all cells and extracts are summarized in \u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003eIn general, the cytotoxicity results suggest that 1) the leaf extract is more cytotoxic in ARPE-19 and MCF10A cells, and 2) the death rate is similar in all locations after the highest dose (500 \u0026micro;g/mL) of leaf extracts while 100 \u0026micro;g/mL of thermal leaf samples resulted in cytotoxicity of the cells compared to other locations. Unlike ARPE-19 and MCF10A2, fruit extracts showed more cytotoxic properties in BEAS-2B and HUVEC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u003c/strong\u003e summarises the comparison of cell viability between the cells after treatments with 100\u0026micro;g/mL or 500\u0026micro;g/mL leaf or fruit extracts for 48h collected from the closest location to the thermal station. The cytotoxicity of BEAS-2B cells appears to be more sensitive to the leaf extracts at 100\u0026micro;g/mL compared to other cells. All cells responded to the highest concentrations (500\u0026micro;g/mL) of leaf extracts similarly, because no significant difference between the cells was found. However, after the treatment with the highest concentration (for 48h) of fruit extracts, there are statistically significant differences in the viability of the cells. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Anti-microbial effect of olive extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then assessed the antimicrobial\u0026nbsp;and antifungal activities of extracts. For this, three gram-negative bacteria (\u003cem\u003eEscherichia coli, Yersinia pseudotuberculosis\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e), three gram-positive bacteria (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis,\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e) and one of yeast-like fungi (\u003cem\u003eCandida albicans\u003c/em\u003e) were studied. \u0026nbsp;The effects of extracts on microorganisms and MIC values are shown in \u003cstrong\u003eTable 7\u003c/strong\u003e. Some extracts did not show antimicrobial activity against the microorganisms used in the experiment \u003cstrong\u003e(Supplementary Figure 1)\u003c/strong\u003e. Antimicrobial activities of all olive fruits and leaf extracts against \u003cem\u003eB. subtilis, E. faecalis, P. aeruginosa, Y. pseudotuberculosis, E. coli,\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e were not found, and MIC values could not be determined. On the other hand, antimicrobial activities of olive fruits and leaves extract, except MF, against\u0026nbsp;\u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003ewere found\u003cem\u003e.\u0026nbsp;\u003c/em\u003eMIC values on the\u0026nbsp;\u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003ewere determined as 13.73 mg/ml for ML, 12.5 mg/ml for TL, 20 mg/ml for TF, 12.5 mg/ml for DL, and 30 mg/ml for DF extract.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study aimed to analyze the effect of thermal power plants on \u003cem\u003eOlea europea\u003c/em\u003e fruit and leaf extracts and to reveal the potential result of these extracts on the cell viability of normal human cells and antimicrobial activity. To the best of our knowledge, this study is the first to investigate the outcome of proximity to thermal power stations on the contents of the fruits and leaves of the olive plant, as well as the effects of extracts obtained from these samples on healthy human cells.\u003c/p\u003e \u003cp\u003eOne of these potentially bioactive compounds in the leaves is the secoiridoide oleuropein, which can account for 6\u0026ndash;9% of the dry matter. Other bioactive components in olive leaves include related secoiridoides, flavonoids, and triterpenes (El and Karakaya 2009). There are five groups of phenolic compounds principally present in olive leaves: oleuropeosides (oleuropein and verbascoside); flavones (luteolin-7-glucoside, apigenin-7-glucoside, diosmetin-7-glucoside, luteolin, and diosmetin); flavonols (rutin); flavan-3-ols (catechin), and substituted phenols (tyrosol, hydroxytyrosol, vanillin, vanillic acid, and caffeic acid). The most abundant compound in olive leaves is oleuropein followed by hydroxytyrosol, the flavone-7-glucosides of luteolin and apigenin, and verbascoside (El and Karakaya 2009). The present study showed that fruit extracts contained mostly the major olive compounds such as oleuropein aglycone, n\u0026uuml;zhenide 11-methyl oleoside, and oleocanthal. N\u0026uuml;zhenide 11-methyl oleoside is one of the secoiridoides commonly found in olive (Silva et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Leaf and fruit extracts obtained from the farthest location \u0026ndash; Destin Village\u0026ndash; had the highest amount of these bioactive compounds. In contrast, extracts obtained from the closest location to the thermal station had the lowest amount. For instance, apigenin is one of the most common flavonoids in fruit extracts from thermal stations. Flavonoids can play different roles in plant metabolism, such as protecting them from sun-derived radiation, defending them against pathogens and herbivory, regulating plant metabolism, and \u0026ldquo;serving as visual attractors for pollinators\u0026rdquo; (Salehi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Apigenin has a function in the cell cycle arrest during different phases of proliferation, such as G1/S or G2/M by promoting several cyclin-dependent kinases and other genes (Iizumi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Maggioni et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Takagaki et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This may result in cell death suggesting that it is therefore considered to have an anti-cancer activity within the cells. Oleocanthal is one of the olive-specific constituents with strong anti-inflammatory activities (Pang and Chin 2018), and this was found only in the fruit extracts collected from the farthest location (Destin village). Thermal stations may prevent the production of oleocanthal and therefore can lower the nutritional yield of olive fruits. On the other hand, leaf extracts collected from around the thermal station have ferulic acid in content. Ferulic acid is a biologically active compound that plays roles in oxidative stress, inflammation, vascular endothelial injury, fibrosis, cell death, and even platelet aggregation (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ferrulic acid was found in the leaf extracts of olives collected from around the thermal power station only. Benavente-Garcia \u003cem\u003eet al.\u003c/em\u003e showed that ferulic acid was not a main content of leaves, however such apigenin-7-glycoside, vanillic acid, and caffeic acid were abundant in the leaves of \u003cem\u003eO. europea\u003c/em\u003e (Benavente-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Caffeic and chlorogenic acids were only found in the leaf extracts collected from Destin village, while oleochanthal was the component only found in fruit extracts from Destin village. Xie \u003cem\u003eet al.\u003c/em\u003e found that one of the common contents of leaves and fruit is apigenin-7-β-D-glucose (Xie et al. 2015). In our study, fruits from \u003cem\u003eO. europea\u003c/em\u003e located near to thermal power station consisted of apigenin itself while leaves consisted of apigenine glycoside. The results of this study suggest that the closeness to the thermal power station affected the quantitative and qualitative features of leaves and fruits.\u003c/p\u003e \u003cp\u003eThe fact that the olive is a long-lived plant indicates that it is resistant to negative environmental conditions, especially drought. Although olive trees are considered drought-resistant species, water stress; may be associated with a wide range of adverse effects and physiological processes, such as nutrient uptake, carbon assimilation, reduced flower and fruit formation, canopy size, and flower loss (Nteve et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Physiological studies conducted with olive plants in the literature are related to drought (Marchioni et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nteve et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), water (S\u0026aacute;nchez-Pi\u0026ntilde;ero et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and salt stress (Regni et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Besides, high concentrations of lead, copper, and zinc were found in the olive samples collected from industrial areas, and areas close to roads with heavy traffic (Şahan and Başoğlu 2009). It has been reported that the amount of copper and zinc in some garden soils with olive groves in Akhisar district of Manisa is significantly high, however, the amounts of cadmium, lead, and arsenic are below the pollution limits (Zincircioğlu \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Unal \u003cem\u003eet al.\u003c/em\u003e found that the concentrations of chromium, lead, zinc, and copper in the leaves of olive trees close to a factory in Izmir Kemalpaşa industrial zone (Turkiye) were higher than in the leaves of olive trees farther from the region (\u0026Uuml;nal et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e antimicrobial activity of olive leaves against bacteria and fungi has been demonstrated before (Juven and Henis \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Malhadas et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mark\u0026iacute;n, Duek, and Berd\u0026iacute;cevsky 2003). This may be an adjunct treatment in the cases of long-term use of antibiotics. In addition, olive fruit's antimicrobial and antioxidant activities and their phenolic compounds on pathogenic microorganisms have been extensively studied and effective results have been obtained (Borjan et al. 2020; Juven and Henis \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Šimat et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In a study, phenolic compounds of olive leaves collected from Muğla province in T\u0026uuml;rkiye were extracted and their antioxidant and antimicrobial activities were determined. It was shown that olive leaves in this region have antimicrobial activities against \u003cem\u003eS. aureus, L. monocytogenes, Salmonella\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e microorganisms. In addition, the highest antimicrobial effect of Muğla olive leaves was determined against \u003cem\u003eS. aureus\u003c/em\u003e bacteria (Baysal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In our study, the antimicrobial effect of olive leaves and fruits collected from the thermal power plant region of Muğla was investigated, and according to the literature, the antimicrobial activity decreased significantly. In this study, we determined the highest antimicrobial activity of olive fruits and leaves against \u003cem\u003eS. aureus\u003c/em\u003e bacteria. The antimicrobial activity of olive leaves was higher than that of olive fruits. The antimicrobial effect could not be determined against the other 6 microorganisms.\u003c/p\u003e \u003cp\u003eLeaves and fruits of \u003cem\u003eO. europea\u003c/em\u003e are reported to have anti-cancer effects on different cancer cell lines, and leaves also have an anti-hypertension and anti-microbial potential (Alesci et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ozturk \u003cem\u003eet al.\u003c/em\u003e found that olive leaf extracts induced cytotoxicity in colon cancer cells, while these did not significantly affect normal human fibroblast cells (\u0026Ouml;zt\u0026uuml;rk, \u0026Ccedil;alık, and Ulusoy 2022). There are also many studies showed the cytotoxicity on breast cancer cells (Han et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Junkins, Rodgers, and Phelan \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), liver cancer cells (using commercial olive plants) (Bektay et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but not in normal cells such as human gingival and neutrophil cells (Han et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), normal liver cells (Bektay et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and human mesenchymal stem cells (Işik et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Işık \u003cem\u003eet al.\u003c/em\u003e collected samples from three different suburbs of Balikesir City; Ayvalık, Domat, and Uslu (T\u0026uuml;rkiye) which thermal power plants are not around these. Han \u003cem\u003eet al.\u003c/em\u003e declared that they collected samples in Tunisia without any specific location (Han et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Many of these studies did not mention the location of the sample collection. However, these all suggest that olive plants are not harmful to normal human cells. Our study aimed to investigate the wellness of normal human cells only after extract treatments, and showed that extracts of leaf samples from the thermal area induced higher cytotoxicity on healthy cells, especially BEAS-2B bronchial cells compared to those collected from other locations.\u003c/p\u003e \u003cp\u003eThe findings of this study conclude that thermal power plants have negative effects on both olives and olive-treated human cells. A detailed investigation is needed for significant changes in genes or proteins of both \u003cem\u003eO. europea\u003c/em\u003e and human cells to reveal the molecular effects of thermal power plants. Thus, all these findings will give important clues for the regions where thermal power plants should be built and/or will question how thermal power stations can be developed to minimize the potential effects on the ecosystem and living things.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a 2209-A grant fromTUBITAK (The Scientific and Technological Research Council of Türkiye) (Project ID:\u0026nbsp;1919B012306896) and by the Scientific Research Project Coordination Unit of Karadeniz Technical University (Project numbers: FLÖ-2024-11149, FLÖ-2024-11176 and FHD-2024-16036). The authors thank 1) Prof Nurettin YAYLI and Mrs. Gözde BOZDAL (Karadeniz Technical University, Faculty of Pharmacy) for assisting with extractions from plants, 2) Mrs. Zeliha BAYRAM (from Istanbul Metropolitan Municipality), a topographical engineer, for assisting the use of Google maps for finding locations, 3) Prof Zülal ATLI ŞEKEROĞLU (Ordu University, Department of Molecular Biology and Genetics), Assoc Prof Hatice SEVİM NALKIRAN (Recep Tayyip Erdoğan University, Department of Medical Biology) and Assit Prof Cihan INAN (Karadeniz Technical University, Department of Molecular Biology and Genetics) for providing a batch of BEAS-2B cells, ARPE-19 cells and MCF10A cells, respectively, and 6) Prof Kadriye INAN BEKTAS (Karadeniz Technical University, Department of Molecular Biology and Genetics) for assisting anti-microbial experiments, and 7) Mr. Ali GÜRBÜZ for collecting fruit and leaves.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no conflict of interest exists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEG\u0026nbsp;\u003c/strong\u003econceptualized the study, collected and extracted samples, and also performed cell culture and MTT experiments,\u003cstrong\u003e\u0026nbsp;EG\u0026nbsp;\u003c/strong\u003ewas granted by TUBITAK(Project ID:\u0026nbsp;1919B012306896), \u003cstrong\u003eAS\u0026nbsp;\u003c/strong\u003eperformed the HPLC-DAD experiment, \u003cstrong\u003eFBT\u0026nbsp;\u003c/strong\u003eplanned andperformed anti-microbial experiments, \u003cstrong\u003eES\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;NNK\u0026nbsp;\u003c/strong\u003eperformed cell culture applications and MTT tests, \u0026nbsp;\u003cstrong\u003eST\u0026nbsp;\u003c/strong\u003esupported cell culture and anti-microbial experiments, \u003cstrong\u003eHM\u0026nbsp;\u003c/strong\u003emanaged anti-microbial experiments and was granted by the project (Karadeniz Technical University, project ID: FHD-2024-16036), \u003cstrong\u003eEAT\u0026nbsp;\u003c/strong\u003econceptualized, performed and analyzed the HPLC-DAD experiment\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSCU\u0026nbsp;\u003c/strong\u003econceptualized and managed the study, and was granted by (Karadeniz Technical University, Project numbers: FLÖ-2024-11149 and FLÖ-2024-11176).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlesci, Alessio, Anthea Miller, Roberta Tardugno, and Simona Pergolizzi. 2022. \u0026lsquo;Chemical Analysis, Biological and Therapeutic Activities of Olea Europaea L. 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Antioxidants 2020, Vol. 9, Page 412 9(5): 412.\u003c/li\u003e\n \u003cli\u003eTakagaki, Nobumasa et al. 2005. \u0026lsquo;Apigenin Induces Cell Cycle Arrest and P21/WAF1 Expression in a P53-Independent Pathway.\u0026rsquo; International journal of oncology 26(1): 185\u0026ndash;89.\u003c/li\u003e\n \u003cli\u003eToreti, A. et al. 2024. \u003cem\u003eDrought in Western Mediterranean - Publications Office of the EU\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003e\u0026Uuml;nal, Dilek, Şenol Sert, Nuray Olcay Işık, and \u0026Uuml;nal Kaya. 2011. \u0026lsquo;İzmir-Kemalpaşa Sanayi B\u0026ouml;lgesinde Ağır Metal Kirliliğinin Biyoindikat\u0026ouml;r Olarak Zeytin (Olea Europaea) Bitkisi Kullanılarak Belirlenmesi\u0026rsquo;. Zeytin Bilimi 2: 59\u0026ndash;64.\u003c/li\u003e\n \u003cli\u003eXie, Pu jun, Li xin Huang, Cai hong Zhang, and Yao lei Zhang. 2015. \u0026lsquo;Phenolic Compositions, and Antioxidant Performance of Olive Leaf and Fruit (Olea Europaea L.) Extracts and Their Structure-Activity Relationships\u0026rsquo;. Journal of Functional Foods 16: 460\u0026ndash;71.\u003c/li\u003e\n \u003cli\u003eZincircioğlu, Nurdan. 2015. \u0026lsquo;Manisa-Akhisar\u0026rsquo;da Bulunan Bazı Zeytin Bah\u0026ccedil;elerinde Cu, Zn, Cd, Pb ve as I\u0026ccedil;eriklerinin Belirlenmesi.\u0026rsquo; Zeytin Bilimi 5: 21\u0026ndash;26.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is not available with this version\u003c/p\u003e"},{"header":"Supplementary Figures and Tables","content":"\u003cp\u003eThe supplementary figures and tables are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"d31cdb2c-bd9c-4758-94ac-5454b02714b1","identifier":"10.13039/501100004410","name":"Türkiye Bilimsel ve Teknolojik Araştirma Kurumu","awardNumber":"2209-A","order_by":0},{"identity":"f84009cd-082c-457c-b406-095fab7da4f4","identifier":"10.13039/501100004045","name":"Karadeniz Teknik Üniversitesi","awardNumber":"FLÖ-2024-11149, FLÖ-2024-11176 and FHD-2024-16036","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Karadeniz Technical University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Olea europea, olive, cytotoxicity, phenolic compounds, thermal power plant, anti-microbial activity, HPLC","lastPublishedDoi":"10.21203/rs.3.rs-5381441/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5381441/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThermal power plants (TPP) are important for meeting today's increasing energy needs. However, TPPs pose risks to the ecosystem and human health. \u003cem\u003eOlea europea\u003c/em\u003e (olive) grows widely in the Aegean region and is commonly used in the human diet. The TPP in Yatağan is close to the agricultural and residential areas. However, the effect of closeness to TPP on olives and the cytotoxic effect of olives close to TPPs on human cells is unknown. This study showed 1) phenolic compounds, flavonoids, and fundamental olive contents changed in the fruit and leaf extracts collected far-medium-close to TPP, 2) extracts obtained from olives close to the TPP had cytotoxic effects on healthy human cells, and 3) the highest antimicrobial activity of extracts was found against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e regardless of distance to the TPP. The findings suggest that TPPs may affect the nutritional value of olives, and the viability of human cells.\u003c/p\u003e","manuscriptTitle":"Human Cell Cytotoxicity, Anti-Microbial Effect and Chromatographic Analyses of Leaf and Fruit Extracts of Olea europaea (Olive) Collected from Different Distances to Yatagan Thermal Power Plant in Mugla, Türkiye","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-05 17:44:32","doi":"10.21203/rs.3.rs-5381441/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52b1572b-d171-411a-8493-148d7c5abb28","owner":[],"postedDate":"November 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-05T17:44:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-05 17:44:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5381441","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5381441","identity":"rs-5381441","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}