In vitro and in silico analyses of cytoprotective, antigenotoxic and antimutagenic potential of Phlomis pungens var. hirta extracts against oxidative damage

In vitro and in silico analyses of cytoprotective, antigenotoxic and antimutagenic potential of Phlomis pungens var. hirta extracts against oxidative damage | 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 In vitro and in silico analyses of cytoprotective, antigenotoxic and antimutagenic potential of Phlomis pungens var. hirta extracts against oxidative damage Franziska Johanna WILD KORKMAZ, Serap DOGAN, Begumhan YILMAZ KARDAS, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4780456/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2024 Read the published version in Genetic Resources and Crop Evolution → Version 1 posted 9 You are reading this latest preprint version Abstract Cellular integrity depends mainly on the stability of DNA, which can be disrupted by genetic mutations and aberrations. Here, we show that the cell-protective activity and DNA damage prevention ability of Phlomis pungens var. hirta have been investigated. We found cell protective activity, healthy cell proliferation promoter activity and DNA damage preventing capacity in the controlled in vitro assays. Additionally, P. pungens was shown to possess the ability to suppress the genotoxicity of H 2 O 2 as an antigenotoxic agent. Rutin hydrate and quercetin are the major flavonoids in P. pungens , which exhibit a wide range of biological activities, including antioxidant, neuro- and hepatoprotective and Aβ-oligomer reducing activities. P. pungens has shown impressive activity against Gram-positive and Gram-negative bacteria as well as strong scavenging activity against various radicals. Subsequently, in silico software tools PubChem, pkCSM and PASS Online were used for biological activity profiling and toxicity predictions of the major compounds in P. pungens . All evaluations of P. pungens could be suggested as a potential source for dietary supplements and therapeutic products. Phlomis pungens cytotoxicity genotoxicity 8-OhdG in silico Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Cellular integrity mainly depends on DNA stability that can be disrupted by genetic mutations and aberrations. Reactive oxygen species (ROS) induced oxidative stress scan threaten the DNA stability and this can lead vigorous results on biological pathways governed by DNA (Dumont & Monari 2015 ). ROS (superoxide anion, hydroxyl radical, hydrogen peroxide) can induce both cytotoxic and mutagenic damages (Benhusein et al. 2010 , Ray et al. 2012 ). Hydrogen peroxide (H 2 O 2 ) is known to act as a spreader by transporting itself to regions where the extremely toxic HO• radical can form in the presence of free iron(Lenzen et al. 2022 ). Therefore, H 2 O 2 and HO• are showing their toxic effects in concert and dependent on each other in oxidative stress (Lenzen et al. 2022 ). In fact, the main reason of the cell death is the iron-mediated oxidative DNA damage caused by the hydroxyl radical in eukaryotes/ prokaryotes(Perron et al. 2008 ).The reactive molecules can cause destruction of the bases and sugar-phosphate backbones of DNA, so base modifications (bulky like cyclo purine/etheno adducts and non-bulky like 8-oxoguanine/form amido pyrimidine), DNA crosslinks, a-basic sites, protein-DNA adducts and single-strand breaks can occur(Berquist &Wilson 2012 ). The oxidative DNA damages should be repaired before the replication or transcription processes in order to prevent mutagenesis. Otherwise, several pathologies like carcinogenesis and neurodegeneration can occur. In order to protect the DNA damage, cells have important defense and repair strategies against ROS (Berquist &Wilson 2012 ). Homologous recombination, single strand annealing and non-homologous/microhomology mediated end joining pathways are the repair mechanisms for double strand DNA breaks while mismatch repair, base and nucleotide excision repairs (BER/NER) are the repair mechanisms for single strand DNA breaks (Kaur &Mojumdar 2021 ). NER and BER mechanisms are also responsible for the protection of DNA from the detrimental effects of base modifications like 8-oxo-7,8-dihydroguanine caused by oxidative stress such as ROS, ultraviolet light or genotoxic agents. 8-oxo-7,8-dihydroguanine is an oxidized form of guanine and it can cause transversion mutation such as Guanine-Thymine or Gunanine-Adenine bonding(Ock et al. 2012 ). Therefore, the 8-hydroxydeoxyguanosine (8-OHdG), a nucleoside form of 8-oxo-7,8-dihydroguanine, is considered as a well-known biomarker of the oxidative damage (Emam et al. 2014 ).8-OHdG accumulation was detected in various carcinoma cells and mutated genes(Kanazawa et al. 2006 ). Extensive DNA damages can also lead to the production of acentric chromatid/chromosome fragments which in turn cause tiny extra-nuclear bodies called micronuclei (MN) and the identification of the MN is frequently used to determine genomic instability and genotoxic exposure levels in cells(Luzhna et al. 2013 , Xu et al. 2014 ).Moreover, somatic mutation recombination test (SMART) is able to identify point mutations, deletions, rarely seen mitotic recombinations (a consequence of DNA damage repair process resulting in genome instabilities because of the heterozygosity loss), chromosomal aberrations and gene conversions(Lafave &Sekelsky 2009 , Yilmaz Kardas et al. 2023 ). Thus, MN, 8-OHdG test and SMART are frequently used in literature for the detection of mutagens (Ceker et al. 2012a , Demir et al. 2013 , Dirican et al. 2012 , Haverić et al. 2018 , Marković et al. 2020 , Okada &Okada 2015 , Patenkovic et al. 2009 ). In addition to their usage for the nutrition, herbaceous plants have been consumed as medicine by people to take advantage of its useful features like protection against DNA damage and carcinogenesis (Lin et al. 2013 ). On the other hand, plants are known to have several phytochemicals or secondary metabolites identified as toxins which help plants to protect themselves against bacteria, fungi, insects or predators (Duan et al. 2019 ). Therefore, detailed toxicology analyzes should be done so that toxic constituents of the plant extracts are not ignored while investigating the beneficial effects. Phlomis pungens var. hirta (Lamiaceae) is a member of Phlomis L. genus whose members are known to have medicinal properties like ulcerogenic, anti-diabetic / microbial / inflammatory / nociceptive / mutagenic and immunosuppressive (Ulukanli &Akkaya 2011 ). Although P. pungens is known to be used by local people, there are limited number of studies in literature about it (Özcelik et al. 2010 , Özkan et al. 2009 , Ulukanli &Akkaya 2011 ). In one of those studies, researchers investigated the antibacterial activity of hexane, methanol and acetone extracts of P. pungens and found that hexane extract of the plant was antibacterial (Ulukanli &Akkaya 2011 ). In another studies, it was shown that petroleum ether and methanol extracts of P. pungens had antibacterial and antifungal effects (Özcelik et al. 2010 , Özkan et al. 2009 ). Although it has a therapeutic importance, the cytotoxicities and antigenotoxic/mutagenic properties of this medicinal plant have never been tested. The cytotoxicity and genotoxicity of P. pungens were aimed to be evaluated in this study by MTS assay, micronucleus assay, 8-OHdG ELISA test and SMART. Other biochemical characterization techniques like liquid chromatography (LC), antibacterial and antioxidant activity tests of P. pungens extracts were also revealed here. 2. MATERIALS AND METHODS 2.1. Materials Standards used for liquid chromatography and cell growth media were purchased from Merck-Sigma Aldrich. MTS assay reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-inner-salt) was obtained from the Promega Company. Bacterial growth medium was obtained from Biomark. Bacteria (kwik-stik) were obtained from Microbiologics of France. P. pungens (the aerial parts) were collected when they are in flowering period between Lice and Kulp, Diyarbakir (Turkey) districts at 1100 m (altitude of ca.) in June 2020. In addition, a voucher specimen coded as MA 2019is preserved in the Munzur University (Turkey). 2.2. Preparation of the plant extracts Plant extraction was performed using standard methods found in literature using water, methanol and ethanol as the solvents (Dirican et al. 2012 , Yilmaz Kardas et al. 2023 ). 2.3. Biochemical Characterization Techniques 2.3.1. LC analysis of the plant extracts LC system (1260 Series, Agilent, USA) with UV-Visible detector was used for the analysis of phenolic compounds according to previous studies (De Villiers et al. 2004 , Yilmaz Kardas et al. 2023 ). 2.3.2. Antioxidant activities of plant extracts The antioxidant activity of the plant extracts were determined by three methods. Firstly, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test was performed according to the literature(Re et al. 1999 , Yilmaz Kardas et al. 2023 ). The plant extract (0- 4000 mg/L) and ABTS + solution were combined (1.66% v/v) and the IC 50 values were calculated according to the absorbances obtained at 734 nm. Secondly, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) test was performed using standard methods (Alothman et al. 2009 , Aminjafari et al. 2016 , Yilmaz Kardas et al. 2023 , Zhang et al. 2013 ). The plant extracts (0- 4000 µg mL − 1 ) and the control group were then mixed with 10% DPPH reagent (v/v). The mixture was kept in dark for 1 hour and the absorbances were measured at 517 nm. IC 50 values were calculated, accordingly. Thirdly, ferric reducing/antioxidant power (FRAP) assay was performed according to the literature(Benzie &Strain 1996 ). The absorbances were taken at 593 nm after the interaction of the plant extracts with the oxidant solution because it is known that the Fe(III)-2,4,6-tripyridyl s-triazine complex was reduced to the Fe(II) form at pH:3.6. With the help of the calibration curve obtained by Trolox (hydrophilic vitamin E analogue) the “µg g − 1 Trolox” values were calculated (Melilli et al. 2020 ). 2.3.3. Antibacterial activities Standard disc diffusion method was used with Escherichia coli (ATCC-8739), Aeromonas sp. (ATCC-51107), Pseudomonas aeruginosa (ATCC-9027), Enterobacter aerogenes (ATCC-13048) and Staphylacoccus aureus (ATCC-6538)(Yilmaz Kardas et al. 2023 ). 6 mm diameter discs were soaked in 4 mg ml − 1 of plant extracts or gentamicin and they were placed onto the agar (Muller Hinton) inoculated with the bacteria (10 5 CFU mL − 1 ). After incubation at 37°C for 24h, area of the inhibitions were measured (Basile et al. 2006 ). 2.4. Cytotoxicity and Genotoxicity Tests 2.4.1. Lymphocyte isolation and cell culture conditions The lymphocyte cells are important model systems for in vitro cytotoxicity and genotoxicity tests (Amani et al. 2019 , Bailon-Moscoso et al. 2016 ). Therefore, the lymphocyte cells were used in this study and they were isolated from healthy human blood samples (obtained in heparinized tubes) by Ficoll-Paque technique (Jaatinen & Laine 2007 ). The cells were dissolved in a complete cell culture medium containing RPMI 1640 (500 mL), 2.5 mL of Penicillin-Streptomycin, fetal bovine serum (50 mL) and 1 g/L phytohemagglutinin. 37° Cis used for the cell growth. 2.4.2. MTS assay MTS test was performed to test cytotoxicity with healthy human lymphocytes. 24 h after the isolation step, the plant extracts prepared with cell culture media (0-100 mg/L final concentrations, no treatment for negative control) were added into the lymphocyte cells (2 × 10 6 cells mL − 1 ). Then, the treated cells were kept in the incubator further for 24 and 48 h. MTS was added into the cells (1:5) at the end of the periods. After 4 h, the absorbances were measured at 490 nm. 2.4.3. Micronucleus assay For the genotoxicity analysis the micronucleus assay was used with healthy human lymphocyte cells(Ari et al. 2015 , Celikler Kasimogullari et al. 2014 , Yilmaz Kardas et al. 2023 ). The plant extracts were added into the lymphocyte cell cultures at the 24th hour of the culture (50 mg/L final concentrations, no treatment for negative control, 6.5 µg mL − 1 H 2 O 2 for positive control). The micronuclei were identified from the microscopic images (Fenech et al. 2003 ). The NDI (nuclear division index)was calculted using the Eq. (1) and micronuclei ‰ observed among the two nuclei cells was calculated (Ari et al. 2015 ). \(\:\text{N}\text{D}\text{I}=\frac{(\text{O}\text{n}\text{e}\:\text{n}\text{u}\text{c}\text{l}\text{e}\text{u}\text{s}\:\text{c}\text{e}\text{l}\text{l}+(2\text{*}\text{T}\text{w}\text{o}\:\text{n}\text{u}\text{c}\text{l}\text{e}\text{i}\:\text{c}\text{e}\text{l}\text{l})+\left(3\text{*}\:\text{T}\text{h}\text{r}\text{e}\text{e}\:\text{n}\text{u}\text{c}\text{l}\text{e}\text{i}\:\text{c}\text{e}\text{l}\text{l}\right)\:+(4\text{*}\:\text{F}\text{o}\text{u}\text{r}\:\text{n}\text{u}\text{c}\text{l}\text{e}\text{i}\:\text{c}\text{e}\text{l}\text{l})}{200}\) Eq. (1) 2.4.4. 8-hydroxydeoxyguanosine assay (8-OHdG) Assay The oxidative DNA damage levels in the lymphocyte cells treated with 50 mg/Lplant extracts and 6.5 µg/mL H 2 O 2 for 24 h was analyzed with an oxidative DNA damage ELISA kit (Oxiselect TM, Cell Biolabs STA-320, USA). No treatment was applied for negative control. Genomic DNA was isolated from the cells using the DNA extraction kit (Monarch Genomic DNA Purification Kit, New England Biolabs) and the concentration of nucleic acids was determined by measuring the absorbance at 260 nm against a blank. The 8-OHdG assay was performed according to the supplier's instructions by coating the plate with 8-OHdG conjugate, blocking with the assay diluent, adding DNA samples and treating them with primary/secondary antibodies. At the end the substrate was added (100 µL) and after 2 minutes of incubations the absorbance was measured at 450 nm. 2.4.5. The somatic mutation recombination test (SMART ) SMART was performed using the standard protocols with flare-3, flr 3 / In (3LR) TM3, Bd s ) D. melanogaster strain and the multiple wing hairs strain with the genetic constitution y; mwh j ) (Graf et al. 1984 , Patenkovic et al. 2009 ). All flies were fed with standard growth medium and kept at 22 °C(Chung et al. 2009 , Yakovleva et al. 2016 ). flr3 female flies (10 adults) were crossed with the mwh males (10 adults). Then, the trans heterozygous larvae ( mwh+/+flr3 ) were collected after 72 ± 4 h of incubation and washed with distilled water. Plant extracts (50 mg/L), solvent controls (5%, v/v) and H 2 O 2 (6.5 mg/L) were added into separate growth media with the larvae. The adult wings were examined under microscope(Sarikaya et al. 2016 ). The wing spots were classified as small single spots ( flr3 or mwh phenotypes observed in ≤ 2 regions), large single spots ( flr3 or mwh phenotypes observed in > 2 regions) or twin spots (both of the phenotypes observed). The spot frequencies ( Fr. ) were determined as the spots (n) per number of wings (N) (Graf et al. 1984 , Patenkovic et al. 2009 , Sarikaya et al. 2016 ). The inhibition % observed in the cotreatment groups were calculated by Eq. (2) (Abraham 1994 , Mezzoug et al. 2006 ). \(\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\text{\%}=\frac{(\text{F}\text{r}.\:\text{o}\text{f}\text{H}2\text{O}2\:\text{a}\text{l}\text{o}\text{n}\text{e}-\:\text{F}\text{r}.\:\text{o}\text{f}\:\text{P}\text{l}\text{a}\text{n}\text{t}\:\text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t})}{\text{F}\text{r}.\:\text{o}\text{f}\:\text{H}2\text{O}2\:\text{a}\text{l}\text{o}\text{n}\text{e}}\text{x}100\) Eq. (2) 2.5. Statistics All of the experiments were performed in at least three biological repeats and standard errors (SE) were calculated according to them. Student’s (paired) t -test was applied using Excel software and p < 0.05 was determined as significant. 2.6. In silico analysis Prediction of Activity Spectra for Substance (PASS) ( https://www.way2drug.com/passonline/predict.php ) is a publicly available internet-based software tool that predicts the biological activity profiles of drug-like organic compounds, such as organic or phytochemicals. The results of the software tool are given as "Pa; probably active" and "Pi; probably inactive". If the Pa value is greater than 0.7, the compound is predicted to have a high probability of experimental, biological, and pharmacological activity. Ames toxicity, skin sensitisation, hepatotoxicity, etc. the pkCSM ( https://biosig.lab.uq.edu.au/pkcsm/ ) software tool was used to estimate toxicity-related values. The in silico analyses were done by adding SMILES formats from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ) to these software tools. 3. RESULTS AND DISCUSSION 3.1 LC Results Plant phenolics are the most abundant secondary metabolites in plants and have important roles in defense against pathogens, UV radiation, parasites and predators (Dai &Mumper 2010 ). It is also known that phenolic acids are the significant factors of odor and taste in plants and they are involved in the browning reactions, so it is important to identify those that occur in crops that have economic importance(Torres et al. 1987 ). The most significant property of the phenolic compounds is the protective role from the oxidative damage and related diseases like stroke, coronary heart disease and cancer. Thus, many analytical methods like LC–DAD,LC–MS and GC have been used for phenolic compound quantifications in plants(Matei et al. 2015 ). In this study, LC was performed to unveil the phenolic compounds of P. pungens According to the findings (Table 1 ), rutin hydrate (560.27 ± 6.18µg/g) and quercetin (110.22 ± 63.14 µg/g) were found in highest concentrations. Ferulic acid (7.03 ± 0.78 µg/g), vanillic acid (1.61 ± 0.36 µg/g) and p -coumaric acid (0.45 ± 0.05 µg/g) were also found in the plant. Although their concentrations differ, it can be clearly concluded that P. pungens is very rich in phenolic compounds (Table 1 ). In many studies, these compounds were analyzed and their protective properties were revealed. For example, rutin hydrate was classified as an antioxidant flavonoid and its extraordinary ability to prevent neurotoxicity by inhibiting the endoplasmic reticulum stress was proven (Mostafa et al. 2019 , Noon et al. 2020 , Ojha et al. 2016 , Sana et al. 2014 ). Rutin is a glycoside of quercetin. Because of its various protective biological properties, quercetin has been the focus of attention in many genotoxicity/carcinogenicity studies (Ozyurt et al. 2014 ). It was shown that quercetin has anti-inflammatory, antihypertensive, vasodilatory, antihypercholesterolemic, anti-atherosclerotic, antioxidant and anti-cancer properties (Lagunas-Rangel &Bermudez-Cruz 2020 ). Quercetin is known to protect DNA from oxidative damage and enhance DNA repair by modulating the DNA repair enzyme expressions (Min &Ebeler 2009 ). It is known that quercetin and rutin are able to interact with the hydrated electron at close to diffusion control rate as efficient scavengers (Cai et al. 1999 , Zhao et al. 2003 ). Thus, quercetin and rutin can protect DNA from hydrated electron attack in addition to their capacity to repair DNA damage caused by radiation (Zhao et al. 2003 ). Moreover, ferulic acid is a well-known antioxidant molecule and it is known to suppress homologous recombination during DNA repair by inhibiting RAD51 production in breast cancer cells (Choi &Park 2015 , Rahman et al. 2021 ). Oxidative damage protection capacities of vanillic acid and p -coumaric acid were also reported previously (Sevgi et al. 2015 , Shen et al. 2019 ). To conclude, it was determined by the LC results that P. pungens has an excellent phenolic content and the protective effect of this plant on living systems may be due to this rich content. Table 1 LC results of Phlomis pungens . Compounds Amounts (µg g − 1 ) Caffeic acid L.O.D.* Gallic acid L.O.D.* Vanillic acid 1.61 ± 0.36 Rutin hydrate 560.27 ± 6.18 p -coumaric acid 0.45 ± 0.05 Ferulic acid 7.03 ± 0.78 Quercetin 110.22 ± 3.14 * L.O.D. indicates the values below the limits of detection. 3.2 In silico Results Biological activity profiles and toxicity analysis of rutin hydrate and quercetin, which are the major phenolic ingredients detected in P. pungens , were determined in silico (Table 2 ). In particular, the results of the PASS software tool used to estimate the biological activity profile show that the two components have high effects in terms of antioxidant, free radical scavenger, lipid peroxidase inhibitor, hepatoprotectant, hemostatic, cardioprotectant, anticarcinogenic, apoptosis agonist, vasoprotector, antineoplastic, and chemopreventive (Filimonov et al. 2014 , DE et al. 2015 ). Table 2 Prediction of biological activity and toxicity based on major phenolic compounds in P. pungens via in silico analysis. Activity In silico analyses Rutin hydrate Quercetin Prediction of biological activity Antioxidant Pa: 0,923 Pi: 0,003 Pa: 0,872 Pi: 0,003 Free radical scavenger Pa: 0,988 Pi: 0,001 Pa: 0,811 Pi: 0,003 Lipid peroxidase inhibitor Pa: 0,987 Pi: 0,001 Pa: 0,788 Pi: 0,004 Hepatoprotectant Pa: 0,986 Pi: 0,001 Pa: 0,706 Pi: 0,007 Hemostatic Pa: 0,993 Pi: 0,001 Pa: 0,771 Pi: 0,003 Cardioprotectant Pa: 0,988 Pi: 0,001 Pa: 0,833 Pi: 0,003 Anticarcinogenic Pa: 0,984 Pi: 0,001 Pa: 0,757 Pi: 0,007 Apoptosis agonist Pa: 0,747 Pi: 0,011 Pa: 0,887 Pi: 0,005 Vasoprotector Pa: 0,980 Pi: 0,001 Pa: 0,824 Pi: 0,004 Antineoplastic Pa: 0,849 Pi: 0,007 Pa: 0,797 Pi: 0,012 Chemopreventive Pa: 0,968 Pi: 0,001 Pa: 0,717 Pi: 0,006 Prediction of toxicity Mutagenic (AMES toxicity) No No Max. tolerated dose (human) 0.451 (log mg/kg/day) 0.499 (log mg/kg/day) hERG I inhibitor No No hERG II inhibitor Yes No Oral Rat Acute Toxicity (LD50) 2.491 (mol/kg) 2.471 (mol/kg) Oral Rat Chronic Toxicity (LOAEL) 3.672 (log mg/kg_bw/day) 2.612 (log mg/kg_bw/day) Hepatotoxicity No No Skin Sensitisation No No T. Pyriformis toxicity 0.285 (log ug/L) 0.288 (log ug/L) Minnow toxicity 7.893 (log mM) 3.721 (log mM) Pa = Probably active (> 0,70), Pi = Probably inactive, ERG: human Ether-a-go-go-Related Gene; LD50: lethal dose of 50%; LOAEL: lowest observed adverse effect level 3.3 Antioxidant Activity Results Antioxidants constitute the molecules that are able to inhibit or quench the free radical reactions which lead to delay or prevention of cell damages. Every species has its own antioxidant mechanism and plants are known to be extremely rich in compounds with antioxidative activities (Dumanovic et al. 2020 ). The antioxidant capacities of water, methanol and ethanol extracts of P. pungens were investigated in this study and the data obtained as a result of the analyzes are given in Table 3 . According to the IC 50 values obtained in the results of both DPPH and ABTS tests, the methanol extract of the plant showed higher antioxidant capacities (DPPH: 0.18± 0.001 µg/µL, ABTS: 0.45± 0.004 µg/µL) than other extracts (Ethanol extract:0.28± 0.002 µg/µL for DPPH and 0.68± 0.009 µg/µL for ABTS; Water extract: 0.77± 0.009 µg/µL for DPPH and 1.03± 0.031 µg/µL for ABTS; Table 3 ) and these differences were statistically significant (p < 0.05). FRAP data also showed similar results (Table 3 ). Methanol extract of P. pungens showed the highest metal chelating capacity (92.58± 0.005 µg/g), while ethanol extract (79.74± 0.007 µg/g) and water extract (30.71± 0.007 µg/g) showed lower chelating capacities (p < 0.05). When the previous studies with different species belong to the Phlomis genus are examined, it can be seen that similar antioxidant activity results were obtained. For example, Firuzi et al. used DPPH and FRAP methods to determine the antioxidant and metal chelation capacity of methanol extracts of Phlomis elliptica , Phlomis olivieri , Phlomis persica and Phlomis bruguieri species and found that Phlomis olivieri had the highest radical scavenging activity (0.42 µg/µL), while Phlomis persica showed the lowest radical scavenging activity (1.19 µg/µL)(Firuzi et al. 2010 ). The researchers also determined that Phlomis elliptica had the highest metal chelating capacity (23.1 µM/g DW), while Phlomis bruguieri had the lowest (11.0 µM/g DW) according to the FRAP results (Firuzi et al. 2010 ). In another study, it was observed that ethanol extract of P. pungens L. started to scavenge DPPH radical at the lowest concentration studied (10 µg/L)(Isik et al. 2017 ). Taskin et al. also used DPPH, ABTS and FRAP tests to investigate the antioxidant activities of the methanol extract of P. pungens and IC 50 values were observed as 0.064 ± 0.005 mg/mL, 23.36 ± 0.005 mM trolox/mg and 8.60 ± 0.008 mM Fe 2+ /mg, respectively (Taşkın et al. 2018 ). Researchers also revealed the IC 50 values of P. pungens determined by DPPH and ABTS tests as 2.41mg/mL and 3.32mg/mL, respectively (Okur et al. 2021 ). These results showed that our plant P. pungens , showed better antioxidant activity than the common form of the plant and other Phlomis species. In addition, P. pungens has very high antioxidant capacity similar to the famous antioxidant plant species like sage ( Salvia sp.), lemon balm ( Melissa officinalis ), peppermint ( Menttha piperita ) and thyme ( Thymus serpyllum , Origanum vulgare ) reported before (Dogan et al. 2022 , Mekinic et al. 2014 ). Considering the fact that flavonoids act as antioxidants, free radical scavengers and radio protectors, it is not surprising that the antioxidant activity of P. pungens was very high because of the generous concentrations of quercetin (a well-known flavonoid) observed in this plant (Table 1 ). Quercetin is known to function as a free radical quencher to prevent lipid peroxidation because of its diffuse ability into the membranes and scavenging the oxyradicals in lipid bilayer or it can act as a metal ion chelator by orthodihydroxyphenolic structure so it scavenges alkoxyl/peroxyl radicals (Muthukumaran et al. 2008 ). Table 3 Antioxidant activities of P. pungens extracts (Mean ± SE) . ABTS (µg/µL) DPPH (µg/µL) FRAP (µg/g Trolox) Water Extract 1.03 ± 0.031 a,c 0.77 ± 0.009 a,c 30.71 ± 0.007 a,c Methanol Extract 0.45 ± 0.004 a,b,c 0.18 ± 0.001 a,b,c 92.58 ± 0.005 a,b,c Ethanol Extract 0.68 ± 0.009 a,b 0.28 ± 0.002 a,b 79.74 ± 0.007 a,b Gallic Acid (Positive Control) 0.001885 ± 0.000004 b,c 0.005 ± 0.000005 b,c - Trolox (Positive Control) - - 1.25 ± 0.007 b,c a statistically significant (p < 0.05) according to positive control (gallic acid for ABTS and DPPH tests, trolox for FRAP test), b statistically significant (p < 0.05) according to water extract, c statistically significant (p < 0.05) according to ethanol extract 3.4 Antibacterial Activity Results Drug resistance has been accelerated because of the extensive use of antibiotics and it is possible to discover new drugs with potential antibacterial activities using plants extracts which are known to have various secondary metabolites(Bhatia et al. 2021 ). In this study, the antibacterial activity of P. pungens was investigated (Fig. 1 ). The largest inhibition zones were observed against Aeromonas sp. (12.67 ± 0.32 mm) and E. aerogenes (12.26 ± 0.47 mm). The plant also caused inhibition zones against E. coli, P. aeruginosa and S. aureus as 10.97± 0.12, 11.14 ± 0.33 and 11.70± 0.38 mm, respectively (Fig. 1 ). There are similar results in previous studies. For example, Alpay et al. investigated the antibacterial activities of the ethanol extract of Phlomis russliana species against E. coli , P. aeruginosa and S. aureus and the inhibition zones were 10 mm, 11.2 mm and 12.4 mm, respectively (Alpay et al. 2019 ). The antibacterial activities of hexane, acetone and methanol extracts of P. pungens were also studied before and it was determined that acetone and methanol extracts of the plant did not show antibacterial activity, but hexane extract (1000 µg per disc) showed the highest antibacterial activity against meat-isolated S. aureus , S. aureus ATTC 6538 and S. epidermidis strains (diameter of inhibition: 10 mm) (Ulukanli &Akkaya 2011 ). Ozcelik et al. investigated the antimicrobial activity of petroleum ether and methanol extracts of P. pungens and found that the extracts had antibacterial and antifungal activities but they didn’t show any antiviral activity (Özcelik et al. 2010 ). In another study, methanol extract of P. pungens was shown to have an antibacterial effect against Pseudomonas aeruginosa and Bacillus subtilis (Özkan et al. 2009 ). The emergence and spread of multidrug-resistant bacterial strains has become a significant public health threat due to the availability of fewer or sometimes no effective antimicrobial agents for infection by pathogenic bacteria. Therefore, the discovery of new antimicrobial agents is of great importance. Many plants offer an effective alternative in the treatment of these problematic bacterial infections, as they can be natural antimicrobial compounds (Manandhar et al. 2019 ). One of these plants, P. pungens , has proven to be an important candidate for drug studies or biotechnological applications with its high antibacterial activity capacity. Figure 1 Antibacterial activity results of Phlomis pungens shown as inhibition zones (mm ± SE).Inhibition zones observed with gentamicin were 21.97 ± 0.28, 22.60 ± 0.22, 22.68 ± 0.09, 24.52 ± 0.15 and 24.71 ± 0.21 mm for Aeromonas sp., E. coli , E. aerogenes , P. aeruginosa and S. aureus , respectively. * is statistically significant according to gentamicin. Figure 2 MTS results of healthy human lymphocyte cells treated with (a) water, (b) methanol and (c) ethanol extracts of Phlomis pungens . * indicates significancy compared to negative control (p < 0.05). 3.5 MTS Assay Results Cytotoxicity studies are known to be a useful first step to determine the potential toxicity of a test substance like plant extract and minimal toxicity is needed for the development of a pharmaceutical or cosmetic product (McGaw et al. 2014 ).In order to test the cytotoxicity of P. pungens extracts, MTS assay was used in this study and the results are given in Fig. 2 . After 24 h of incubation, none of the plant extracts caused any cytotoxicity and there were statistically significant increases in ethanol and methanol groups (50 and 100 mg/L, p < 0.05, Fig. 2 ). When the cells were treated with the samples for 48 h, the absorbances started to decrease and these decreases were statistically significant for water extracts (50 and 100 mg L − 1 ), methanol extract (100 mg/ L) and ethanol extract (100 mg/ L) but the 50 mg/L doses of methanol and ethanol extracts didn’t cause any significant decrease (p > 0.05, Fig. 2 ). It is seen that the growth of the lymphocyte cells were supported in all of the extract groups (Fig. 2 ), but the increased incubation time for the samples treated with the higher dose of the extracts (100 mg/ L) negatively affected the viability of the cells which is an expected situation as primary cells have limited life spans (Sultan &Haagsman 2009 ). There isn’t any study in literature showing the cytotoxicity of P. pungens but there are some cytotoxicity studies performed with other Phlomis species. Mamadalieva et al . investigated the cytotoxic effect of P. bucharica on HeLa and HL-60 leukemia cell lines using the MTT method and it was observed that methanol, hexane, chloroform and water extracts of P. bucharica showed cytotoxic effects on cells due to increased concentrations of the extracts (Mamadalieva et al. 2015 ). It was also shown that methanol, hexane and water extracts of P. bucharica showed lower cytotoxic activity against HL-60 and HeLa cells than the chloroform extract (Mamadalieva et al. 2015 ). Sarkhail et al. investigated the cytotoxic effects of the methanol extracts of P. anisodontea , P. bruguieri , P. caucasica , P. olivieri , P. persica and P. kurdica against tumor and normal cell lines using the MTT test and all of the plants were shown to have cytotoxic activity at the concentrations above 309 .15 mg L − 1 (Sarkhail et al. 2017 ). Thoppil et al. also showed that P. platystedia was cytotoxic on human hepatocellular carcinoma cells after a 48-hour incubation period (Thoppil et al. 2013 ). Plant extract and phytochemical usage is very important in therapeutic treatments but medicinal plants in nature are known to synthesize toxic substances as a defense mechanism against insects, herbivores and infections which affects all of the organisms eating them. Therefore, evaluation of the cytotoxicity is necessary for safe use of the medicinal plants(Varalakshmi et al. 2011 ). In conclusion, P. pungens supported the healthy human cells in a dose dependent manner and can be considered as a potential candidate for therapeutic biomedical applications. 3.6 Micronucleus Assay Results In order to analyze chromosomal damage resulted from the exposure of mutagens, the MN assay is a common technique and it detects micronuclei structures which are the chromosomal fragments separated during mitosis (Cik & Jurzak 2007 ).At the same time, the MN assay performed with peripheral blood lymphocytes is considered as a standard method to monitor chromosome damage in human populations (Bonassi et al. 2001 ). In this study, MN test with the healthy human lymphocyte cells treated with P. pungens extracts was used and the results are given in Table 4 . According to the results, none of the extracts showed a significant change compared to the negative control (p > 0.05, Table 3 ). On the contrary, when MN ‰ of the extracts were compared with the cells treated with H 2 O 2 (6.5 µg/mL), all of the extracts showed significant decreases (p < 0.05). The water extract of P. pungens caused approximately 6 fold lower MN ‰ (5.18 ± 0.33) than the one that H 2 O 2 caused (p 0.05). In addition, methanol and ethanol extracts of the plant caused 2.15 and 2.58 fold lower MN ‰ than the one that H 2 O 2 caused (p 0.05, Table 3 ). These results showed that none of the extracts was genotoxic against healthy human cells and this effect can’t be attributed to the solvents used for the extractions. In addition, the nuclear division index (NDI) values were determined in this study (Table 3 ). NDI value is known to increase or decrease abnormally as it shows the cellular proliferative capability (Ipek et al. 2017 ). According to the results, there wasn’t any significant difference in NDI values between samples showing the fact that the micronucleus test in this study was conducted using the cells dividing in similar rhythm (Table 4 ). To our best knowledge, there isn’t any study in literature about the genotoxicity of P. pungens . However, there are some studies about other members of the Lamiaceae family. For example, Dirican et al. investigated the antigenotoxic effects of water, methanol and ethanol extracts of Thymbra spicata L. species belonging to Lamiaceae plant family on mercury-induced human lymphocyte cells by micronucleus method and they found that T. spicata extracts were able to protect the mercury-induced micronuclei formation (Dirican et al. 2012 ). In another study, the antigenotoxic activity of the essential oil isolated from Origanum vulgare L. (Lamiaceae) against the genotoxic effect of aflatoxin B1 on human lymphocyte cells was investigated by micronucleus test and the essential oils in 1 µL, 1.5 µL and 2.0 µL concentrations showed the highest antigenotoxic effects against aflatoxin B1 (Ceker et al. 2012b ). Table 4 Micronucleus assay results of P. pungens extracts. Treatments MN ‰ ± SE NDI ± SE Negative Control 11.44 ± 2.88 b 1.29 ± 0.04 H 2 O 2 (6.5 µg/mL) 30.12 ± 1.63 a 1.32 ± 0.01 Water Solvent Control (5% v/v) 4.87 ± 0.69 b 1.32 ± 0.005 Methanol Solvent Control ( 5% v/v) 15.48 ± 0.56 b 1.35 ± 0.003 Ethanol Solvent Control ( 5% v/v) 14.01 ± 0.71 b 1.35 ± 0.001 P. pungens Water Extract (50 mg/L) 5.18 ± 0.33 b 1.33 ± 0.0002 P. pungens Methanol Extract (50 mg/ L) 14.00 ± 2.32 b 1.41 ± 0.1 P. pungens Ethanol Extract (50 mg/ L) 11.66 ± 2.78 b 1.19 ± 0.01 a statistically significant (p < 0.05) according to negative control, b statistically significant (p < 0.05) according to H 2 O 2 (6.5 µg/ mL) 3.7 8-OHdG Assay Results ROS are known to have an ability to attack DNA and as a result of this attack, the guanine (G) base is modified into 8-OHdG which lead to mutations during DNA replication as polymerases can’t identify it as guanine (Emam et al. 2014 ). Thus, 8-OHdG was used as an indicator of oxidative DNA damage in this study and the results of 8-OHdG ELISA test are given in Fig. 3 . When the 8-OHdG amounts (ng/mL) of the samples treated with plant extracts were compared to the ones treated with an oxidative damage source, H 2 O 2 , there were dramatic decreases (Fig. 3 ). Although the water extract of P. pungens showed only 17% decrease, the methanol and ethanol extracts showed 41 and 50% decreases, respectively and all of those decreases were statistically significant (p 0.05) but it was lower than the one observed in the cells treated with H 2 O 2 (p < 0.05). On the other hand, the methanol and ethanol solvent controls (Fig. 3 b &Fig. 3 c) caused statistically significant increases in 8-OHdG level compared to negative control (p 0.05). These results showed that methanol and ethanol extracts of P. pungens were able to decrease the oxidative DNA damage (41 and 50%, respectively) and this effect can’t be attributed to the solvents used for the extractions which couldn’t decrease the 8-OHdG levels when applied alone. Although there isn’t any study in literature showing the preventive effect of P. pungens against 8-OHdG but various studies with different Phlomis species showed that this genus generally has antigenotoxic and antimutagenic properties. For example, Uysal et al. showed that methanol, ethyl acetate and water extracts of P. nissoli , P. pungens var. pungens and P. armeniaca had significant antigenotoxic effects according to the Ames test results(Uysal et al. 2016 ). Dellai et al. also demonstrated the antigenotoxic effects of ethyl acetate, chloroform and methanol extracts of P. crinita Cav. by Ames test (Dellai et al. 2009 ). In another study, researchers used Ames test to show that methanol and ethyl acetate extracts of P. mauritanica had the capacity to inhibit mutations (Limem et al. 2010 ). It is known that unrepaired DNA lesions can inhibit replication and transcription processes which potentially lead to mutations and/or cell death (Knezevic-Vukcevic et al. 2007 ). Therefore, the DNA damage prevention capacity of P. pungens shown in this study revealed significant potential for its use in the development of therapeutic products and applications. 3.8 SMART Results SMART is an assay used for the detection of mutagenic and recombinogenic activities in somatic cells induced by xenobiotics (Yilmaz Cetinkaya &Yurtsever 2021 ). It is based on the genetic damage induction in dividing wing disc cells of Drosophila melanogaster larvae which results in heterozygosity loss during development and this can be observed on the adult wings as mutant wing spots(Pitchakarn et al. 2021 ). SMART is considered as a very sensitive genotoxicity test because the imaginal disc cells are known to duplicate every 10 hour during larval development so the genotoxins have a bigger chance of interacting with the larval genome(Pitchakarn et al. 2021 ). Therefore, in this study SMART was used for the detection of in vivo antigenotoxic effects of P. pungens extracts and the results are given in Table 4 .As a genotoxic agent H 2 O 2 caused significant increases in small single, large single and twin spots (p 0.05) and there weren’t any large single or twin spots. Therefore, it can be concluded that the P. pungens extracts weren’t genotoxic. In order to evaluate the influence of the solvents used to prepare the extracts, solvent control groups were also investigated (Table 4 ). The results showed that, ethanol and methanol solvents caused higher frequencies of large single spots compared to negative control and the small single spot frequencies were similar to the ones observed with H 2 O 2 , while the water solvent control was non-genotoxic. Thus, the solvents are not responsible for the antigenotoxic effects of the P. pungens extracts. When the extracts were coadministered with H 2 O 2 , all of the extracts caused inhibitions in frequencies of different spot types (Table 4 ). In fact, ethanol and methanol extracts of P. pungens caused more than 74% of inhibitions. Therefore, P. pungens extracts can be considered as the antigenotoxic substances with an ability to suppress the genotoxic effects of H 2 O 2 . There isn’t any SMART data observed with P. pungens in literature. However, there are many studies showing the protective characteristics of the members of Lamiaceae family against the genotoxic agents shown by SMART (Guterres et al. 2022 , Magombe et al. 2022 , Yilmaz Kardas et al. 2023 ).To sum up, it can be said that mutations and mitotic recombination, which is known to be stimulated by H 2 O 2 induced DNA breaks, were inhibited by the P. pungens extracts used in this study(Qi et al. 2019 ). P. pungens is one of the medicinal plants that has been used by local people to treat diabetes, ulcer and infections. However, there are limited number of studies in literature about this plant and cyto/genotoxicity analyses have not been done before. Therefore, this study aimed to find out cell protective activity, DNA damage prevention capacity and antioxidant/antibacterial properties of the water, methanol and ethanol extracts of P. pungens. According to the LC results of this study, rutin hydrate (560.27 ± 6.18 µg g − 1 ) and quercetin (110.22 ± 63.14 µg g − 1 ) were found in highest concentrations. Antioxidant activity results showed that the methanol extract of the plant showed higher antioxidant capacities than other extracts (p < 0.05). The plant was found as antibacterial against all of the strains tested but the largest inhibition zones were observed against Aeromonas sp. (12.67 ± 0.32 mm) and Enterobacter aerogenes (12.26 ± 0.47 mm). Although the increased incubation time for the samples treated with the higher dose of the extracts negatively affected the viability of the cells, it was clearly seen that the growth of healthy human lymphocyte cells were supported by all of the extracts. Micronucleus assay showed that none of the extracts was genotoxic against healthy human cells. 8-OHdG ELISA test also showed that methanol and ethanol extracts of P. pungens were able to decrease the oxidative DNA damage (41 and 50%, respectively). According to the SMART results, P. pungens extracts were shown as the antigenotoxic substances with an ability to suppress the genotoxic effects of H 2 O 2 . As a result of the in vitro analyses, the biological activity profiles and toxicity of routine hydrate and quercetin, which discovered to be the main constituents of the plant, were investigated in silico analyses, and the obtained prediction results support our experimental findings. To conclude, this is the first study which demonstrated the in vitro cell protective activity and DNA damage prevention capacity of P. pungens which should be regarded as an important medicinal plant that can be used for nutritional supplements and therapeutic-products. Table 4 Results of the somatic mutation recombination test (SMART). Treatments Average Number of wings (N) Small single spots (S) Large single spots (L) Twin spots (T) Inhibition % (S,L,T, Total) n Fr. SE n Fr. SE n Fr. SE Negative Control 70 2 0.03 b 0 0 0 b 0 0 0 b 0 H 2 O 2 68 3.7 0.05 a 0.005 4.7 0.069 a 0.005 15.7 0.23 a 0.02 Water Solvent Control 70 2 0.03 b 0 1 0.014 b 0 0 0 b 0 Ethanol Solvent Control 69.3 2.7 0.04 0.005 4.7 0.067 a 0.005 7.3 0.11 ab 0.01 Methanol Solvent Control 64 3.7 0.06 a 0.005 9 0.141 b 0 7.3 0.11 ab 0.01 P. pungens Water 70 2 0.03 b 0 0 0 b 0 0 0 b 0 P. pungens Ethanol 75.3 2 0.03 b 0.0002 0 0 b 0 0 0 b 0 P. pungens Methanol 68 1.7 0.02 b 0.005 0 0 b 0 0 0 b 0 P. pungens Water + H 2 O 2 67.3 2 0.03 b 0.001 0 0 b 0 0 0 b 0 44.7, 100, 100, 91.6 P. pungens Ethanol + H 2 O 2 73.3 1 0.01 ab 0.0001 0.7 0.009 b 0.005 0 0 b 0 74.7, 86.7, 100, 93.5 P. pungens Methanol + H 2 O 2 72 1 0.01 b 0 0.7 0.009 b 0.005 0 0 b 0 74.2, 86.5, 100, 93.4 a statistically significant (p < 0.05), according to negative control, b statistically significant (p < 0.05), according to H 2 O 2 (6.5 mg/L), Fr. : Frequency, n: Average spot Declarations ACKNOWLEDGEMENTS This work is produced from a part of F. 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(2016) New Prospective Materials for Chemoprevention: Three Phlomis. British Journal of Pharmaceutical Research 10: 1–13. Varalakshmi K, Sangeetha C, Samee U, Irum G, Lakshmi H, Prachi S. (2011) In Vitro Safety Assessment of the Effect of Five Medicinal Plants on Human Peripheral Lymphocytes. Tropical Journal of Pharmaceutical Research 10. Xu B, Wang W, Guo H, Sun Z, Wei Z, Zhang X, Liu Z, Tischfield JA, Gong Y, Shao C. (2014) Oxidative stress preferentially induces a subtype of micronuclei and mediates the genomic instability caused by p53 dysfunction. Mutat Res 770: 1–8. Yakovleva EU, Naimark EB, Markov AV. (2016) Adaptation of Drosophila melanogaster to unfavorable growth medium affects lifespan and age-related fecundity. Biochemistry (Moscow) 81: 1445–1460. Yilmaz Cetinkaya A, Yurtsever S. (2021) Somatic mutations and recombination test in Drosophila melanogaster used for investigating the genotoxicity of some food additives. International Journal of Agriculture, Environment and Food Sciences: 65–73. Yilmaz Kardas B, Diken ME, Bayhan H, Acar M, Dogan S. (2023) Cytoprotective, antimutagenic/antirecombinogenic and antibacterial properties of Lallemantia iberica extracts. J Sci Food Agric 103: 1901–1911. Zhang W, Chen H, Wang Z, Lan G, Zhang L. (2013) Comparative studies on antioxidant activities of extracts and fractions from the leaves and stem of Epimedium koreanum Nakai. Journal of Food Science and Technology 50: 1122–1129. Zhao C, Shi Y, Wang W, Jia Z, Yao S, Fan B, Zheng R. (2003) Fast repair of deoxythymidine radical anions by two polyphenols: rutin and quercetin. Biochem Pharmacol 65: 1967–71. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2024 Read the published version in Genetic Resources and Crop Evolution → Version 1 posted Editorial decision: Revision requested 09 Oct, 2024 Reviews received at journal 15 Aug, 2024 Reviews received at journal 31 Jul, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviewers invited by journal 24 Jul, 2024 Editor assigned by journal 24 Jul, 2024 Submission checks completed at journal 24 Jul, 2024 First submitted to journal 22 Jul, 2024 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-4780456","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":340717904,"identity":"62f4a7fc-004b-4f5f-b020-e567baaa2871","order_by":0,"name":"Franziska Johanna WILD KORKMAZ","email":"","orcid":"","institution":"Balıkesir University","correspondingAuthor":false,"prefix":"","firstName":"Franziska","middleName":"Johanna WILD","lastName":"KORKMAZ","suffix":""},{"id":340717905,"identity":"a5ce0b2d-d867-4579-9810-7eda806a588b","order_by":1,"name":"Serap DOGAN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYFCCBBBhk8DAztgMZEiAeAbEaElLYGBG0ZJAUMthoBYghAL8WuTbkw+/+LnjfB5/M3OzwYc/FvkM7M3bJBh/3MOpxeDMszTL3jO3iyUOMzYnzmyTsGzgOVYmwZBQjFuLRI6ZAW/b7cQGoJbDvA0SBgxAEaAW3C6Tn5FjZvi37VzifJCWP3+AWuTf4NfCcCPH+DFv24HEDUAtyQxsIFt48GsB+YVZti252BCoxbC3TcKAjSet2CIhDY/DgCH28W2bXZ7c8fbHEj/+1Bnwsx/eeOODDR6HMQAdg8oFEXg1MDAwf8AvPwpGwSgYBSMeAADzsFFQ+VR2ZgAAAABJRU5ErkJggg==","orcid":"","institution":"Balıkesir University","correspondingAuthor":true,"prefix":"","firstName":"Serap","middleName":"","lastName":"DOGAN","suffix":""},{"id":340717906,"identity":"146b04d1-e97f-4c8e-be88-5d72bdf76292","order_by":2,"name":"Begumhan YILMAZ KARDAS","email":"","orcid":"","institution":"Balıkesir University","correspondingAuthor":false,"prefix":"","firstName":"Begumhan","middleName":"YILMAZ","lastName":"KARDAS","suffix":""},{"id":340717907,"identity":"8cf4e9a5-48d1-42ec-a3ef-de95ac5e52f3","order_by":3,"name":"Mehmet Emin DIKEN","email":"","orcid":"","institution":"Balıkesir University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"Emin","lastName":"DIKEN","suffix":""},{"id":340717910,"identity":"afca7e4e-a528-4bf4-9d45-24d072d0a6b9","order_by":4,"name":"Ömer Faruk KARASAKAL","email":"","orcid":"","institution":"Üsküdar University","correspondingAuthor":false,"prefix":"","firstName":"Ömer","middleName":"Faruk","lastName":"KARASAKAL","suffix":""},{"id":340717911,"identity":"ea6e8757-756c-4712-831b-7a8ce58a8332","order_by":5,"name":"Mikail ACAR","email":"","orcid":"","institution":"Munzur University","correspondingAuthor":false,"prefix":"","firstName":"Mikail","middleName":"","lastName":"ACAR","suffix":""}],"badges":[],"createdAt":"2024-07-22 08:51:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4780456/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4780456/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10722-024-02268-w","type":"published","date":"2024-11-25T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62892064,"identity":"1c83ca36-9f43-47b7-977e-0ab8695afb35","added_by":"auto","created_at":"2024-08-20 17:51:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113901,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity results ofPhlomis pungens shown as inhibition zones (mm ± SE).Inhibition zones observed with gentamicin were 21.97 ± 0.28, 22.60 ± 0.22, 22.68 ± 0.09, 24.52 ± 0.15 and 24.71 ± 0.21 mm for Aeromonas sp., E. coli, E. aerogenes, P. aeruginosa and S. aureus, respectively. \u003csup\u003e* \u003c/sup\u003eis statistically significant according to gentamicin.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780456/v1/5e75b20dc48fdd9d411a0b06.jpg"},{"id":62892063,"identity":"0dbcc59a-c550-4c21-9e7d-71c099c3876a","added_by":"auto","created_at":"2024-08-20 17:51:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71073,"visible":true,"origin":"","legend":"\u003cp\u003eMTS results of healthy human lymphocyte cells treated with \u003cstrong\u003e(a)\u003c/strong\u003e water, \u003cstrong\u003e(b)\u003c/strong\u003e methanol and \u003cstrong\u003e(c) \u003c/strong\u003eethanol extracts of \u003cem\u003ePhlomis pungens\u003c/em\u003e. * indicates significancy compared to negative control (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780456/v1/6cfe1b3836108c1dd0ad78ee.jpg"},{"id":62892062,"identity":"53c6e92d-6ffa-42b8-98a9-3cf518b10c41","added_by":"auto","created_at":"2024-08-20 17:51:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59170,"visible":true,"origin":"","legend":"\u003cp\u003e8-OHdG (ng/mL) levels observed in cells treated with \u003cstrong\u003e(a)\u003c/strong\u003e water, \u003cstrong\u003e(b)\u003c/strong\u003e methanol and \u003cstrong\u003e(c)\u003c/strong\u003eethanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e* indicates statistical significance (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4780456/v1/70de401a0a2efb88ab0aa520.jpg"},{"id":70382330,"identity":"b1d8f2fb-09b5-493a-8337-ca0d53bd166f","added_by":"auto","created_at":"2024-12-02 16:26:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1328648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4780456/v1/55c29e4f-91d2-4c70-9ca2-3c661ad23632.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIn vitro and in silico analyses of cytoprotective, antigenotoxic and antimutagenic potential of Phlomis pungens var. hirta extracts against oxidative damage\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eCellular integrity mainly depends on DNA stability that can be disrupted by genetic mutations and aberrations. Reactive oxygen species (ROS) induced oxidative stress scan threaten the DNA stability and this can lead vigorous results on biological pathways governed by DNA (Dumont \u0026amp; Monari \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). ROS (superoxide anion, hydroxyl radical, hydrogen peroxide) can induce both cytotoxic and mutagenic damages (Benhusein et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Ray et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) is known to act as a spreader by transporting itself to regions where the extremely toxic HO\u0026bull; radical can form in the presence of free iron(Lenzen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and HO\u0026bull; are showing their toxic effects in concert and dependent on each other in oxidative stress (Lenzen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In fact, the main reason of the cell death is the iron-mediated oxidative DNA damage caused by the hydroxyl radical in eukaryotes/ prokaryotes(Perron et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).The reactive molecules can cause destruction of the bases and sugar-phosphate backbones of DNA, so base modifications (bulky like cyclo purine/etheno adducts and non-bulky like 8-oxoguanine/form amido pyrimidine), DNA crosslinks, a-basic sites, protein-DNA adducts and single-strand breaks can occur(Berquist \u0026amp;Wilson \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe oxidative DNA damages should be repaired before the replication or transcription processes in order to prevent mutagenesis. Otherwise, several pathologies like carcinogenesis and neurodegeneration can occur. In order to protect the DNA damage, cells have important defense and repair strategies against ROS (Berquist \u0026amp;Wilson \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Homologous recombination, single strand annealing and non-homologous/microhomology mediated end joining pathways are the repair mechanisms for double strand DNA breaks while mismatch repair, base and nucleotide excision repairs (BER/NER) are the repair mechanisms for single strand DNA breaks (Kaur \u0026amp;Mojumdar \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). NER and BER mechanisms are also responsible for the protection of DNA from the detrimental effects of base modifications like 8-oxo-7,8-dihydroguanine caused by oxidative stress such as ROS, ultraviolet light or genotoxic agents. 8-oxo-7,8-dihydroguanine is an oxidized form of guanine and it can cause transversion mutation such as Guanine-Thymine or Gunanine-Adenine bonding(Ock et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, the 8-hydroxydeoxyguanosine (8-OHdG), a nucleoside form of 8-oxo-7,8-dihydroguanine, is considered as a well-known biomarker of the oxidative damage (Emam et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).8-OHdG accumulation was detected in various carcinoma cells and mutated genes(Kanazawa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Extensive DNA damages can also lead to the production of acentric chromatid/chromosome fragments which in turn cause tiny extra-nuclear bodies called micronuclei (MN) and the identification of the MN is frequently used to determine genomic instability and genotoxic exposure levels in cells(Luzhna et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Xu et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).Moreover, somatic mutation recombination test (SMART) is able to identify point mutations, deletions, rarely seen mitotic recombinations (a consequence of DNA damage repair process resulting in genome instabilities because of the heterozygosity loss), chromosomal aberrations and gene conversions(Lafave \u0026amp;Sekelsky \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, MN, 8-OHdG test and SMART are frequently used in literature for the detection of mutagens (Ceker et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012a\u003c/span\u003e, Demir et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Dirican et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Haverić et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Marković et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Okada \u0026amp;Okada \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Patenkovic et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to their usage for the nutrition, herbaceous plants have been consumed as medicine by people to take advantage of its useful features like protection against DNA damage and carcinogenesis (Lin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). On the other hand, plants are known to have several phytochemicals or secondary metabolites identified as toxins which help plants to protect themselves against bacteria, fungi, insects or predators (Duan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, detailed toxicology analyzes should be done so that toxic constituents of the plant extracts are not ignored while investigating the beneficial effects. \u003cem\u003ePhlomis pungens\u003c/em\u003e var. \u003cem\u003ehirta\u003c/em\u003e (Lamiaceae) is a member of \u003cem\u003ePhlomis\u003c/em\u003e L. genus whose members are known to have medicinal properties like ulcerogenic, anti-diabetic / microbial / inflammatory / nociceptive / mutagenic and immunosuppressive (Ulukanli \u0026amp;Akkaya \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Although \u003cem\u003eP. pungens\u003c/em\u003e is known to be used by local people, there are limited number of studies in literature about it (\u0026Ouml;zcelik et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u0026Ouml;zkan et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Ulukanli \u0026amp;Akkaya \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In one of those studies, researchers investigated the antibacterial activity of hexane, methanol and acetone extracts of \u003cem\u003eP. pungens\u003c/em\u003e and found that hexane extract of the plant was antibacterial (Ulukanli \u0026amp;Akkaya \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In another studies, it was shown that petroleum ether and methanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e had antibacterial and antifungal effects (\u0026Ouml;zcelik et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u0026Ouml;zkan et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although it has a therapeutic importance, the cytotoxicities and antigenotoxic/mutagenic properties of this medicinal plant have never been tested. The cytotoxicity and genotoxicity of \u003cem\u003eP. pungens\u003c/em\u003e were aimed to be evaluated in this study by MTS assay, micronucleus assay, 8-OHdG ELISA test and SMART. Other biochemical characterization techniques like liquid chromatography (LC), antibacterial and antioxidant activity tests of \u003cem\u003eP. pungens\u003c/em\u003e extracts were also revealed here.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eStandards used for liquid chromatography and cell growth media were purchased from Merck-Sigma Aldrich. MTS assay reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-inner-salt) was obtained from the Promega Company. Bacterial growth medium was obtained from Biomark. Bacteria (kwik-stik) were obtained from Microbiologics of France. \u003cem\u003eP. pungens\u003c/em\u003e (the aerial parts) were collected when they are in flowering period between Lice and Kulp, Diyarbakir (Turkey) districts at 1100 m (altitude of ca.) in June 2020. In addition, a voucher specimen coded as MA 2019is preserved in the Munzur University (Turkey).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of the plant extracts\u003c/h2\u003e \u003cp\u003ePlant extraction was performed using standard methods found in literature using water, methanol and ethanol as the solvents (Dirican et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Biochemical Characterization Techniques\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. LC analysis of the plant extracts\u003c/h2\u003e \u003cp\u003eLC system (1260 Series, Agilent, USA) with UV-Visible detector was used for the analysis of phenolic compounds according to previous studies (De Villiers et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Antioxidant activities of plant extracts\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of the plant extracts were determined by three methods. Firstly, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test was performed according to the literature(Re et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The plant extract (0- 4000 mg/L) and ABTS\u003csup\u003e+\u003c/sup\u003e solution were combined (1.66% v/v) and the IC\u003csub\u003e50\u003c/sub\u003e values were calculated according to the absorbances obtained at 734 nm. Secondly, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) test was performed using standard methods (Alothman et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Aminjafari et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The plant extracts (0- 4000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the control group were then mixed with 10% DPPH reagent (v/v). The mixture was kept in dark for 1 hour and the absorbances were measured at 517 nm. IC\u003csub\u003e50\u003c/sub\u003e values were calculated, accordingly. Thirdly, ferric reducing/antioxidant power (FRAP) assay was performed according to the literature(Benzie \u0026amp;Strain \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The absorbances were taken at 593 nm after the interaction of the plant extracts with the oxidant solution because it is known that the Fe(III)-2,4,6-tripyridyl s-triazine complex was reduced to the Fe(II) form at pH:3.6. With the help of the calibration curve obtained by Trolox (hydrophilic vitamin E analogue) the \u0026ldquo;\u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Trolox\u0026rdquo; values were calculated (Melilli et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Antibacterial activities\u003c/h2\u003e \u003cp\u003eStandard disc diffusion method was used with \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC-8739), \u003cem\u003eAeromonas sp.\u003c/em\u003e (ATCC-51107), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC-9027), \u003cem\u003eEnterobacter aerogenes\u003c/em\u003e (ATCC-13048) and \u003cem\u003eStaphylacoccus aureus\u003c/em\u003e (ATCC-6538)(Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 6 mm diameter discs were soaked in 4 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of plant extracts or gentamicin and they were placed onto the agar (Muller Hinton) inoculated with the bacteria (10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). After incubation at 37\u0026deg;C for 24h, area of the inhibitions were measured (Basile et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cytotoxicity and Genotoxicity Tests\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Lymphocyte isolation and cell culture conditions\u003c/h2\u003e \u003cp\u003eThe lymphocyte cells are important model systems for \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity and genotoxicity tests (Amani et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Bailon-Moscoso et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the lymphocyte cells were used in this study and they were isolated from healthy human blood samples (obtained in heparinized tubes) by Ficoll-Paque technique (Jaatinen \u0026amp; Laine \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The cells were dissolved in a complete cell culture medium containing RPMI 1640 (500 mL), 2.5 mL of Penicillin-Streptomycin, fetal bovine serum (50 mL) and 1 g/L phytohemagglutinin. 37\u0026deg; Cis used for the cell growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. MTS assay\u003c/h2\u003e \u003cp\u003eMTS test was performed to test cytotoxicity with healthy human lymphocytes. 24 h after the isolation step, the plant extracts prepared with cell culture media (0-100 mg/L final concentrations, no treatment for negative control) were added into the lymphocyte cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003ecells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Then, the treated cells were kept in the incubator further for 24 and 48 h. MTS was added into the cells (1:5) at the end of the periods. After 4 h, the absorbances were measured at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Micronucleus assay\u003c/h2\u003e \u003cp\u003eFor the genotoxicity analysis the micronucleus assay was used with healthy human lymphocyte cells(Ari et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Celikler Kasimogullari et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The plant extracts were added into the lymphocyte cell cultures at the 24th hour of the culture (50 mg/L final concentrations, no treatment for negative control, 6.5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for positive control). The micronuclei were identified from the microscopic images (Fenech et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The NDI (nuclear division index)was calculted using the Eq.\u0026nbsp;(1) and micronuclei \u0026permil; observed among the two nuclei cells was calculated (Ari et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{N}\\text{D}\\text{I}=\\frac{(\\text{O}\\text{n}\\text{e}\\:\\text{n}\\text{u}\\text{c}\\text{l}\\text{e}\\text{u}\\text{s}\\:\\text{c}\\text{e}\\text{l}\\text{l}+(2\\text{*}\\text{T}\\text{w}\\text{o}\\:\\text{n}\\text{u}\\text{c}\\text{l}\\text{e}\\text{i}\\:\\text{c}\\text{e}\\text{l}\\text{l})+\\left(3\\text{*}\\:\\text{T}\\text{h}\\text{r}\\text{e}\\text{e}\\:\\text{n}\\text{u}\\text{c}\\text{l}\\text{e}\\text{i}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\right)\\:+(4\\text{*}\\:\\text{F}\\text{o}\\text{u}\\text{r}\\:\\text{n}\\text{u}\\text{c}\\text{l}\\text{e}\\text{i}\\:\\text{c}\\text{e}\\text{l}\\text{l})}{200}\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. 8-hydroxydeoxyguanosine assay (8-OHdG) Assay\u003c/h2\u003e \u003cp\u003eThe oxidative DNA damage levels in the lymphocyte cells treated with 50 mg/Lplant extracts and 6.5 \u0026micro;g/mL H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h was analyzed with an oxidative DNA damage ELISA kit (Oxiselect TM, Cell Biolabs STA-320, USA). No treatment was applied for negative control. Genomic DNA was isolated from the cells using the DNA extraction kit (Monarch Genomic DNA Purification Kit, New England Biolabs) and the concentration of nucleic acids was determined by measuring the absorbance at 260 nm against a blank. The 8-OHdG assay was performed according to the supplier's instructions by coating the plate with 8-OHdG conjugate, blocking with the assay diluent, adding DNA samples and treating them with primary/secondary antibodies. At the end the substrate was added (100 \u0026micro;L) and after 2 minutes of incubations the absorbance was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5. \u003cb\u003eThe somatic mutation recombination test (SMART\u003c/b\u003e)\u003c/h2\u003e \u003cp\u003eSMART was performed using the standard protocols with flare-3, \u003cem\u003eflr\u003c/em\u003e\u003csup\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/ In (3LR) TM3, Bd\u003c/em\u003e\u003csup\u003e\u003cem\u003es\u003c/em\u003e\u003c/sup\u003e) \u003cem\u003eD. melanogaster\u003c/em\u003e strain and the multiple wing hairs strain with the genetic constitution \u003cem\u003ey; mwh j\u003c/em\u003e) (Graf et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Patenkovic et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). All flies were fed with standard growth medium and kept at 22 \u0026deg;C(Chung et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Yakovleva et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eflr3\u003c/em\u003e female flies (10 adults) were crossed with the \u003cem\u003emwh\u003c/em\u003e males (10 adults). Then, the trans heterozygous larvae (\u003cem\u003emwh+/+flr3\u003c/em\u003e) were collected after 72\u0026thinsp;\u0026plusmn;\u0026thinsp;4 h of incubation and washed with distilled water. Plant extracts (50 mg/L), solvent controls (5%, v/v) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (6.5 mg/L) were added into separate growth media with the larvae. The adult wings were examined under microscope(Sarikaya et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The wing spots were classified as small single spots ( \u003cem\u003eflr3\u003c/em\u003e or \u003cem\u003emwh\u003c/em\u003e phenotypes observed in \u0026le; 2 regions), large single spots (\u003cem\u003eflr3\u003c/em\u003e or \u003cem\u003emwh\u003c/em\u003e phenotypes observed in \u0026gt;\u0026thinsp;2 regions) or twin spots (both of the phenotypes observed). The spot frequencies (\u003cem\u003eFr.\u003c/em\u003e) were determined as the spots (n) per number of wings (N) (Graf et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Patenkovic et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Sarikaya et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The inhibition % observed in the cotreatment groups were calculated by Eq.\u0026nbsp;(2) (Abraham \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Mezzoug et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{I}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{\\%}=\\frac{(\\text{F}\\text{r}.\\:\\text{o}\\text{f}\\text{H}2\\text{O}2\\:\\text{a}\\text{l}\\text{o}\\text{n}\\text{e}-\\:\\text{F}\\text{r}.\\:\\text{o}\\text{f}\\:\\text{P}\\text{l}\\text{a}\\text{n}\\text{t}\\:\\text{e}\\text{x}\\text{t}\\text{r}\\text{a}\\text{c}\\text{t})}{\\text{F}\\text{r}.\\:\\text{o}\\text{f}\\:\\text{H}2\\text{O}2\\:\\text{a}\\text{l}\\text{o}\\text{n}\\text{e}}\\text{x}100\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(2)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistics\u003c/h2\u003e \u003cp\u003eAll of the experiments were performed in at least three biological repeats and standard errors (SE) were calculated according to them. Student\u0026rsquo;s (paired)\u003cem\u003et\u003c/em\u003e-test was applied using Excel software and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was determined as significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eIn silico\u003c/em\u003e analysis\u003c/h2\u003e \u003cp\u003ePrediction of Activity Spectra for Substance (PASS) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.way2drug.com/passonline/predict.php\u003c/span\u003e\u003cspan address=\"https://www.way2drug.com/passonline/predict.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) is a publicly available internet-based software tool that predicts the biological activity profiles of drug-like organic compounds, such as organic or phytochemicals. The results of the software tool are given as \"Pa; probably active\" and \"Pi; probably inactive\". If the Pa value is greater than 0.7, the compound is predicted to have a high probability of experimental, biological, and pharmacological activity. Ames toxicity, skin sensitisation, hepatotoxicity, etc. the pkCSM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biosig.lab.uq.edu.au/pkcsm/\u003c/span\u003e\u003cspan address=\"https://biosig.lab.uq.edu.au/pkcsm/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) software tool was used to estimate toxicity-related values. The \u003cem\u003ein silico\u003c/em\u003e analyses were done by adding SMILES formats from PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to these software tools.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 LC Results\u003c/h2\u003e\n \u003cp\u003ePlant phenolics are the most abundant secondary metabolites in plants and have important roles in defense against pathogens, UV radiation, parasites and predators (Dai \u0026amp;Mumper \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is also known that phenolic acids are the significant factors of odor and taste in plants and they are involved in the browning reactions, so it is important to identify those that occur in crops that have economic importance(Torres et al. \u003cspan class=\"CitationRef\"\u003e1987\u003c/span\u003e). The most significant property of the phenolic compounds is the protective role from the oxidative damage and related diseases like stroke, coronary heart disease and cancer. Thus, many analytical methods like LC\u0026ndash;DAD,LC\u0026ndash;MS and GC have been used for phenolic compound quantifications in plants(Matei et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, LC was performed to unveil the phenolic compounds of \u003cem\u003eP. pungens\u003c/em\u003e According to the findings (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), rutin hydrate (560.27 \u0026plusmn; 6.18\u0026micro;g/g) and quercetin (110.22 \u0026plusmn; 63.14 \u0026micro;g/g) were found in highest concentrations. Ferulic acid (7.03 \u0026plusmn; 0.78 \u0026micro;g/g), vanillic acid (1.61 \u0026plusmn; 0.36 \u0026micro;g/g) and \u003cem\u003ep\u003c/em\u003e-coumaric acid (0.45 \u0026plusmn; 0.05 \u0026micro;g/g) were also found in the plant. Although their concentrations differ, it can be clearly concluded that \u003cem\u003eP. pungens\u003c/em\u003e is very rich in phenolic compounds (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In many studies, these compounds were analyzed and their protective properties were revealed. For example, rutin hydrate was classified as an antioxidant flavonoid and its extraordinary ability to prevent neurotoxicity by inhibiting the endoplasmic reticulum stress was proven (Mostafa et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e, Noon et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e, Ojha et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e, Sana et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Rutin is a glycoside of quercetin. Because of its various protective biological properties, quercetin has been the focus of attention in many genotoxicity/carcinogenicity studies (Ozyurt et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). It was shown that quercetin has anti-inflammatory, antihypertensive, vasodilatory, antihypercholesterolemic, anti-atherosclerotic, antioxidant and anti-cancer properties (Lagunas-Rangel \u0026amp;Bermudez-Cruz \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Quercetin is known to protect DNA from oxidative damage and enhance DNA repair by modulating the DNA repair enzyme expressions (Min \u0026amp;Ebeler \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is known that quercetin and rutin are able to interact with the hydrated electron at close to diffusion control rate as efficient scavengers (Cai et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e, Zhao et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). Thus, quercetin and rutin can protect DNA from hydrated electron attack in addition to their capacity to repair DNA damage caused by radiation (Zhao et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). Moreover, ferulic acid is a well-known antioxidant molecule and it is known to suppress homologous recombination during DNA repair by inhibiting RAD51 production in breast cancer cells (Choi \u0026amp;Park \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, Rahman et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Oxidative damage protection capacities of vanillic acid and \u003cem\u003ep\u003c/em\u003e-coumaric acid were also reported previously (Sevgi et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, Shen et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). To conclude, it was determined by the LC results that\u0026nbsp;\u003cem\u003eP. pungens\u003c/em\u003e has an excellent phenolic content and the protective effect of this plant on living systems may be due to this rich content.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLC results of \u003cem\u003ePhlomis pungens\u003c/em\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmounts (\u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaffeic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL.O.D.*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGallic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL.O.D.*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVanillic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRutin hydrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e560.27\u0026thinsp;\u0026plusmn;\u0026thinsp;6.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-coumaric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFerulic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003e*\u003c/strong\u003eL.O.D. indicates the values below the limits of detection.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 \u003cem\u003eIn silico\u003c/em\u003e Results\u003c/h2\u003e\n \u003cp\u003eBiological activity profiles and toxicity analysis of rutin hydrate and quercetin, which are the major phenolic ingredients detected in \u003cem\u003eP. pungens\u003c/em\u003e, were determined \u003cem\u003ein silico\u003c/em\u003e (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In particular, the results of the PASS software tool used to estimate the biological activity profile show that the two components have high effects in terms of antioxidant, free radical scavenger, lipid peroxidase inhibitor, hepatoprotectant, hemostatic, cardioprotectant, anticarcinogenic, apoptosis agonist, vasoprotector, antineoplastic, and chemopreventive (Filimonov et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e, DE et al.\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePrediction of biological activity and toxicity based on major phenolic compounds in \u003cem\u003eP. pungens\u003c/em\u003e via \u003cem\u003ein silico\u003c/em\u003e analysis.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e analyses\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRutin hydrate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"11\"\u003e\n \u003cp\u003ePrediction of biological\u003c/p\u003e\n \u003cp\u003eactivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntioxidant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,923 Pi: 0,003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,872 Pi: 0,003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFree radical scavenger\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,988 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,811 Pi: 0,003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLipid peroxidase inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,987 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,788 Pi: 0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHepatoprotectant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,986 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,706 Pi: 0,007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHemostatic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,993 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,771 Pi: 0,003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCardioprotectant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,988 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,833 Pi: 0,003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnticarcinogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,984 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,757 Pi: 0,007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eApoptosis agonist\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,747 Pi: 0,011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,887 Pi: 0,005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVasoprotector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,980 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,824 Pi: 0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntineoplastic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,849 Pi: 0,007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,797 Pi: 0,012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChemopreventive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,968 Pi: 0,001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePa: 0,717 Pi: 0,006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"10\"\u003e\n \u003cp\u003ePrediction of toxicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMutagenic (AMES toxicity)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMax. tolerated dose (human)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.451 (log mg/kg/day)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.499 (log mg/kg/day)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehERG I inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehERG II inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOral Rat Acute Toxicity (LD50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.491 (mol/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.471 (mol/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOral Rat Chronic Toxicity (LOAEL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.672 (log mg/kg_bw/day)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.612 (log mg/kg_bw/day)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHepatotoxicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSkin Sensitisation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT. \u003cem\u003ePyriformis\u003c/em\u003e toxicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.285 (log ug/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.288 (log ug/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMinnow toxicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.893 (log mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.721 (log mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ePa\u0026thinsp;=\u0026thinsp;Probably active (\u0026gt;\u0026thinsp;0,70), Pi\u0026thinsp;=\u0026thinsp;Probably inactive, ERG: human Ether-a-go-go-Related Gene;\u003c/p\u003e\n \u003cp\u003eLD50: lethal dose of 50%; LOAEL: lowest observed adverse effect level\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Antioxidant Activity Results\u003c/h2\u003e\n \u003cp\u003eAntioxidants constitute the molecules that are able to inhibit or quench the free radical reactions which lead to delay or prevention of cell damages. Every species has its own antioxidant mechanism and plants are known to be extremely rich in compounds with antioxidative activities (Dumanovic et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The antioxidant capacities of water, methanol and ethanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e were investigated in this study and the data obtained as a result of the analyzes are given in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. According to the IC\u003csub\u003e50\u003c/sub\u003e values obtained in the results of both DPPH and ABTS tests, the methanol extract of the plant showed higher antioxidant capacities (DPPH: 0.18\u0026plusmn; 0.001 \u0026micro;g/\u0026micro;L, ABTS: 0.45\u0026plusmn; 0.004 \u0026micro;g/\u0026micro;L) than other extracts (Ethanol extract:0.28\u0026plusmn; 0.002 \u0026micro;g/\u0026micro;L for DPPH and 0.68\u0026plusmn; 0.009 \u0026micro;g/\u0026micro;L for ABTS; Water extract: 0.77\u0026plusmn; 0.009 \u0026micro;g/\u0026micro;L for DPPH and 1.03\u0026plusmn; 0.031 \u0026micro;g/\u0026micro;L for ABTS; Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) and these differences were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). FRAP data also showed similar results (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Methanol extract of \u003cem\u003eP. pungens\u003c/em\u003e showed the highest metal chelating capacity (92.58\u0026plusmn; 0.005 \u0026micro;g/g), while ethanol extract (79.74\u0026plusmn; 0.007 \u0026micro;g/g) and water extract (30.71\u0026plusmn; 0.007 \u0026micro;g/g) showed lower chelating capacities (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When the previous studies with different species belong to the \u003cem\u003ePhlomis\u003c/em\u003e genus are examined, it can be seen that similar antioxidant activity results were obtained. For example, Firuzi et al. used DPPH and FRAP methods to determine the antioxidant and metal chelation capacity of methanol extracts of \u003cem\u003ePhlomis elliptica\u003c/em\u003e, \u003cem\u003ePhlomis olivieri\u003c/em\u003e, \u003cem\u003ePhlomis persica\u003c/em\u003e and \u003cem\u003ePhlomis bruguieri\u003c/em\u003e species and found that \u003cem\u003ePhlomis olivieri\u003c/em\u003e had the highest radical scavenging activity (0.42 \u0026micro;g/\u0026micro;L), while \u003cem\u003ePhlomis persica\u003c/em\u003e showed the lowest radical scavenging activity (1.19 \u0026micro;g/\u0026micro;L)(Firuzi et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). The researchers also determined that \u003cem\u003ePhlomis elliptica\u003c/em\u003e had the highest metal chelating capacity (23.1 \u0026micro;M/g DW), while \u003cem\u003ePhlomis bruguieri\u003c/em\u003e had the lowest (11.0 \u0026micro;M/g DW) according to the FRAP results (Firuzi et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). In another study, it was observed that ethanol extract of \u003cem\u003eP. pungens\u003c/em\u003e L. started to scavenge DPPH radical at the lowest concentration studied (10 \u0026micro;g/L)(Isik et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Taskin et al. also used DPPH, ABTS and FRAP tests to investigate the antioxidant activities of the methanol extract of \u003cem\u003eP. pungens\u003c/em\u003e and IC\u003csub\u003e50\u003c/sub\u003e values were observed as 0.064\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 mg/mL, 23.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 mM trolox/mg and 8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 mM Fe\u003csup\u003e2+\u003c/sup\u003e/mg, respectively (Taşkın et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Researchers also revealed the IC\u003csub\u003e50\u003c/sub\u003e values of \u003cem\u003eP. pungens\u003c/em\u003e determined by DPPH and ABTS tests as 2.41mg/mL and 3.32mg/mL, respectively (Okur et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). These results showed that our plant \u003cem\u003eP. pungens\u003c/em\u003e, showed better antioxidant activity than the common form of the plant and other \u003cem\u003ePhlomis\u003c/em\u003e species. In addition, \u003cem\u003eP. pungens\u003c/em\u003e has very high antioxidant capacity similar to the famous antioxidant plant species like sage (\u003cem\u003eSalvia\u003c/em\u003e sp.), lemon balm (\u003cem\u003eMelissa officinalis\u003c/em\u003e), peppermint (\u003cem\u003eMenttha piperita\u003c/em\u003e) and thyme (\u003cem\u003eThymus serpyllum\u003c/em\u003e, \u003cem\u003eOriganum vulgare\u003c/em\u003e) reported before (Dogan et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e, Mekinic et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Considering the fact that flavonoids act as antioxidants, free radical scavengers and radio protectors, it is not surprising that the antioxidant activity of \u003cem\u003eP. pungens\u003c/em\u003e was very high because of the generous concentrations of quercetin (a well-known flavonoid) observed in this plant (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Quercetin is known to function as a free radical quencher to prevent lipid peroxidation because of its diffuse ability into the membranes and scavenging the oxyradicals in lipid bilayer or it can act as a metal ion chelator by orthodihydroxyphenolic structure so it scavenges alkoxyl/peroxyl radicals (Muthukumaran et al.\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntioxidant activities of \u003cem\u003eP. pungens\u003c/em\u003e extracts (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) .\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eABTS (\u0026micro;g/\u0026micro;L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDPPH (\u0026micro;g/\u0026micro;L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFRAP (\u0026micro;g/g Trolox)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWater Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031\u003csup\u003ea,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003ea,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003ea,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethanol Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEthanol Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGallic Acid (Positive Control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001885\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000004\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000005\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrolox (Positive Control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to positive control (gallic acid for ABTS and DPPH tests, trolox for FRAP test), \u003csup\u003eb\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to water extract, \u003csup\u003ec\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to ethanol extract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Antibacterial Activity Results\u003c/h2\u003e\n \u003cp\u003eDrug resistance has been accelerated because of the extensive use of antibiotics and it is possible to discover new drugs with potential antibacterial activities using plants extracts which are known to have various secondary metabolites(Bhatia et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, the antibacterial activity of \u003cem\u003eP. pungens\u003c/em\u003e was investigated (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The largest inhibition zones were observed against \u003cem\u003eAeromonas\u003c/em\u003e sp. (12.67 \u0026plusmn; 0.32 mm) and \u003cem\u003eE. aerogenes\u003c/em\u003e (12.26 \u0026plusmn; 0.47 mm). The plant also caused inhibition zones against \u003cem\u003eE. coli, P. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e as 10.97\u0026plusmn; 0.12, 11.14 \u0026plusmn; 0.33 and 11.70\u0026plusmn; 0.38 mm, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). There are similar results in previous studies. For example, Alpay et al. investigated the antibacterial activities of the ethanol extract of \u003cem\u003ePhlomis russliana\u003c/em\u003e species against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e and the inhibition zones were 10 mm, 11.2 mm and 12.4 mm, respectively (Alpay et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The antibacterial activities of hexane, acetone and methanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e were also studied before and it was determined that acetone and methanol extracts of the plant did not show antibacterial activity, but hexane extract (1000 \u0026micro;g per disc) showed the highest antibacterial activity against meat-isolated \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e ATTC 6538 and \u003cem\u003eS. epidermidis\u003c/em\u003e strains (diameter of inhibition: 10 mm) (Ulukanli \u0026amp;Akkaya \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). Ozcelik et al. investigated the antimicrobial activity of petroleum ether and methanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e and found that the extracts had antibacterial and antifungal activities but they didn\u0026rsquo;t show any antiviral activity (\u0026Ouml;zcelik et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). In another study, methanol extract of \u003cem\u003eP. pungens\u003c/em\u003e was shown to have an antibacterial effect against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e (\u0026Ouml;zkan et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). The emergence and spread of multidrug-resistant bacterial strains has become a significant public health threat due to the availability of fewer or sometimes no effective antimicrobial agents for infection by pathogenic bacteria. Therefore, the discovery of new antimicrobial agents is of great importance. Many plants offer an effective alternative in the treatment of these problematic bacterial infections, as they can be natural antimicrobial compounds (Manandhar et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). One of these plants, \u003cem\u003eP. pungens\u003c/em\u003e, has proven to be an important candidate for drug studies or biotechnological applications with its high antibacterial activity capacity.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e Antibacterial activity results of\u003cem\u003ePhlomis pungens\u003c/em\u003e shown as inhibition zones (mm\u0026thinsp;\u0026plusmn;\u0026thinsp;SE).Inhibition zones observed with gentamicin were 21.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, 22.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22, 22.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, 24.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 and 24.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mm for \u003cem\u003eAeromonas\u003c/em\u003e sp., \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eE. aerogenes\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, respectively. \u003csup\u003e*\u003c/sup\u003e is statistically significant according to gentamicin.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e MTS results of healthy human lymphocyte cells treated with \u003cstrong\u003e(a)\u003c/strong\u003e water, \u003cstrong\u003e(b)\u003c/strong\u003e methanol and \u003cstrong\u003e(c)\u003c/strong\u003e ethanol extracts of \u003cem\u003ePhlomis pungens\u003c/em\u003e. * indicates significancy compared to negative control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 MTS Assay Results\u003c/h2\u003e\n \u003cp\u003eCytotoxicity studies are known to be a useful first step to determine the potential toxicity of a test substance like plant extract and minimal toxicity is needed for the development of a pharmaceutical or cosmetic product (McGaw et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e).In order to test the cytotoxicity of \u003cem\u003eP. pungens\u003c/em\u003e extracts, MTS assay was used in this study and the results are given in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. After 24 h of incubation, none of the plant extracts caused any cytotoxicity and there were statistically significant increases in ethanol and methanol groups (50 and 100 mg/L, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). When the cells were treated with the samples for 48 h, the absorbances started to decrease and these decreases were statistically significant for water extracts (50 and 100 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), methanol extract (100 mg/ L) and ethanol extract (100 mg/ L) but the 50 mg/L doses of methanol and ethanol extracts didn\u0026rsquo;t cause any significant decrease (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). It is seen that the growth of the lymphocyte cells were supported in all of the extract groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), but the increased incubation time for the samples treated with the higher dose of the extracts (100 mg/ L) negatively affected the viability of the cells which is an expected situation as primary cells have limited life spans (Sultan \u0026amp;Haagsman \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). There isn\u0026rsquo;t any study in literature showing the cytotoxicity of \u003cem\u003eP. pungens\u003c/em\u003e but there are some cytotoxicity studies performed with other \u003cem\u003ePhlomis\u003c/em\u003e species. Mamadalieva \u003cem\u003eet al\u003c/em\u003e. investigated the cytotoxic effect of \u003cem\u003eP. bucharica\u003c/em\u003e on HeLa and HL-60 leukemia cell lines using the MTT method and it was observed that methanol, hexane, chloroform and water extracts of \u003cem\u003eP. bucharica\u003c/em\u003e showed cytotoxic effects on cells due to increased concentrations of the extracts (Mamadalieva et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). It was also shown that methanol, hexane and water extracts of \u003cem\u003eP. bucharica\u003c/em\u003e showed lower cytotoxic activity against HL-60 and HeLa cells than the chloroform extract (Mamadalieva et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Sarkhail et al. investigated the cytotoxic effects of the methanol extracts of \u003cem\u003eP. anisodontea\u003c/em\u003e, \u003cem\u003eP. bruguieri\u003c/em\u003e, \u003cem\u003eP. caucasica\u003c/em\u003e, \u003cem\u003eP. olivieri\u003c/em\u003e, \u003cem\u003eP. persica\u003c/em\u003e and \u003cem\u003eP. kurdica\u003c/em\u003e against tumor and normal cell lines using the MTT test and all of the plants were shown to have cytotoxic activity at the concentrations above 309 .15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Sarkhail et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thoppil et al. also showed that \u003cem\u003eP. platystedia\u003c/em\u003e was cytotoxic on human hepatocellular carcinoma cells after a 48-hour incubation period (Thoppil et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Plant extract and phytochemical usage is very important in therapeutic treatments but medicinal plants in nature are known to synthesize toxic substances as a defense mechanism against insects, herbivores and infections which affects all of the organisms eating them. Therefore, evaluation of the cytotoxicity is necessary for safe use of the medicinal plants(Varalakshmi et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). In conclusion, \u003cem\u003eP. pungens\u003c/em\u003e supported the healthy human cells in a dose dependent manner and can be considered as a potential candidate for therapeutic biomedical applications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Micronucleus Assay Results\u003c/h2\u003e\n \u003cp\u003eIn order to analyze chromosomal damage resulted from the exposure of mutagens, the MN assay is a common technique and it detects micronuclei structures which are the chromosomal fragments separated during mitosis (Cik \u0026amp; Jurzak \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e).At the same time, the MN assay performed with peripheral blood lymphocytes is considered as a standard method to monitor chromosome damage in human populations (Bonassi et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). In this study, MN test with the healthy human lymphocyte cells treated with \u003cem\u003eP. pungens\u003c/em\u003e extracts was used and the results are given in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. According to the results, none of the extracts showed a significant change compared to the negative control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). On the contrary, when MN \u0026permil; of the extracts were compared with the cells treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (6.5 \u0026micro;g/mL), all of the extracts showed significant decreases (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The water extract of \u003cem\u003eP. pungens\u003c/em\u003e caused approximately 6 fold lower MN \u0026permil; (5.18 \u0026plusmn; 0.33) than the one that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e caused (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but it was similar to the one observed with the water solvent control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In addition, methanol and ethanol extracts of the plant caused 2.15 and 2.58 fold lower MN \u0026permil; than the one that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e caused (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The MN \u0026permil; of the methanol and ethanol solvent controls were also similar to the \u003cem\u003eP. pungens\u003c/em\u003e methanol extract and \u003cem\u003eP. pungens\u003c/em\u003e ethanol extract samples(p\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These results showed that none of the extracts was genotoxic against healthy human cells and this effect can\u0026rsquo;t be attributed to the solvents used for the extractions. In addition, the nuclear division index (NDI) values were determined in this study (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). NDI value is known to increase or decrease abnormally as it shows the cellular proliferative capability (Ipek et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). According to the results, there wasn\u0026rsquo;t any significant difference in NDI values between samples showing the fact that the micronucleus test in this study was conducted using the cells dividing in similar rhythm (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). To our best knowledge, there isn\u0026rsquo;t any study in literature about the genotoxicity of \u003cem\u003eP. pungens\u003c/em\u003e. However, there are some studies about other members of the Lamiaceae family. For example, Dirican et al. investigated the antigenotoxic effects of water, methanol and ethanol extracts of \u003cem\u003eThymbra spicata\u003c/em\u003e L. species belonging to Lamiaceae plant family on mercury-induced human lymphocyte cells by micronucleus method and they found that \u003cem\u003eT. spicata\u003c/em\u003e extracts were able to protect the mercury-induced micronuclei formation (Dirican et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). In another study, the antigenotoxic activity of the essential oil isolated from \u003cem\u003eOriganum vulgare\u003c/em\u003e L. (Lamiaceae) against the genotoxic effect of aflatoxin B1 on human lymphocyte cells was investigated by micronucleus test and the essential oils in 1 \u0026micro;L, 1.5 \u0026micro;L and 2.0 \u0026micro;L concentrations showed the highest antigenotoxic effects against aflatoxin B1 (Ceker et al.\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e2012b\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMicronucleus assay results of \u003cem\u003eP. pungens\u003c/em\u003e extracts.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMN \u0026permil; \u0026plusmn; SE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNDI\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (6.5 \u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater Solvent Control (5% v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol Solvent Control ( 5% v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanol Solvent Control ( 5% v/v)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Water Extract (50 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Methanol Extract (50 mg/ L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Ethanol Extract (50 mg/ L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to negative control, \u003csup\u003eb\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (6.5 \u0026micro;g/ mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 8-OHdG Assay Results\u003c/h2\u003e\n \u003cp\u003eROS are known to have an ability to attack DNA and as a result of this attack, the guanine (G) base is modified into 8-OHdG which lead to mutations during DNA replication as polymerases can\u0026rsquo;t identify it as guanine (Emam et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, 8-OHdG was used as an indicator of oxidative DNA damage in this study and the results of 8-OHdG ELISA test are given in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. When the 8-OHdG amounts (ng/mL) of the samples treated with plant extracts were compared to the ones treated with an oxidative damage source, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, there were dramatic decreases (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Although the water extract of \u003cem\u003eP. pungens\u003c/em\u003e showed only 17% decrease, the methanol and ethanol extracts showed 41 and 50% decreases, respectively and all of those decreases were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When the solvent controls were examined, the water solvent control (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) caused the similar amount of 8-OHdG compared to negative control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) but it was lower than the one observed in the cells treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, the methanol and ethanol solvent controls (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb \u0026amp;Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) caused statistically significant increases in 8-OHdG level compared to negative control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the values were similar to the one observed with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results showed that methanol and ethanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e were able to decrease the oxidative DNA damage (41 and 50%, respectively) and this effect can\u0026rsquo;t be attributed to the solvents used for the extractions which couldn\u0026rsquo;t decrease the 8-OHdG levels when applied alone. Although there isn\u0026rsquo;t any study in literature showing the preventive effect of \u003cem\u003eP. pungens\u003c/em\u003e against 8-OHdG but various studies with different \u003cem\u003ePhlomis\u003c/em\u003e species showed that this genus generally has antigenotoxic and antimutagenic properties. For example, Uysal et al. showed that methanol, ethyl acetate and water extracts of \u003cem\u003eP. nissoli\u003c/em\u003e, \u003cem\u003eP. pungens\u003c/em\u003e var. \u003cem\u003epungens\u003c/em\u003e and \u003cem\u003eP. armeniaca\u003c/em\u003e had significant antigenotoxic effects according to the Ames test results(Uysal et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Dellai et al. also demonstrated the antigenotoxic effects of ethyl acetate, chloroform and methanol extracts of \u003cem\u003eP. crinita\u003c/em\u003e Cav. by Ames test (Dellai et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). In another study, researchers used Ames test to show that methanol and ethyl acetate extracts of \u003cem\u003eP. mauritanica\u003c/em\u003e had the capacity to inhibit mutations (Limem et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is known that unrepaired DNA lesions can inhibit replication and transcription processes which potentially lead to mutations and/or cell death (Knezevic-Vukcevic et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, the DNA damage prevention capacity of \u003cem\u003eP. pungens\u003c/em\u003e shown in this study revealed significant potential for its use in the development of therapeutic products and applications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8 SMART Results\u003c/h2\u003e\n \u003cp\u003eSMART is an assay used for the detection of mutagenic and recombinogenic activities in somatic cells induced by xenobiotics (Yilmaz Cetinkaya \u0026amp;Yurtsever \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is based on the genetic damage induction in dividing wing disc cells of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e larvae which results in heterozygosity loss during development and this can be observed on the adult wings as mutant wing spots(Pitchakarn et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). SMART is considered as a very sensitive genotoxicity test because the imaginal disc cells are known to duplicate every 10 hour during larval development so the genotoxins have a bigger chance of interacting with the larval genome(Pitchakarn et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, in this study SMART was used for the detection of \u003cem\u003ein vivo\u003c/em\u003e antigenotoxic effects of \u003cem\u003eP. pungens\u003c/em\u003e extracts and the results are given in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.As a genotoxic agent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e caused significant increases in small single, large single and twin spots (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).On the other hand, water, ethanol and methanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e showed similar small single spot frequencies like negative control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and there weren\u0026rsquo;t any large single or twin spots. Therefore, it can be concluded that the \u003cem\u003eP. pungens\u003c/em\u003e extracts weren\u0026rsquo;t genotoxic. In order to evaluate the influence of the solvents used to prepare the extracts, solvent control groups were also investigated (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The results showed that, ethanol and methanol solvents caused higher frequencies of large single spots compared to negative control and the small single spot frequencies were similar to the ones observed with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, while the water solvent control was non-genotoxic. Thus, the solvents are not responsible for the antigenotoxic effects of the \u003cem\u003eP. pungens\u003c/em\u003e extracts. When the extracts were coadministered with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, all of the extracts caused inhibitions in frequencies of different spot types (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). In fact, ethanol and methanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e caused more than 74% of inhibitions. Therefore, \u003cem\u003eP. pungens\u003c/em\u003e extracts can be considered as the antigenotoxic substances with an ability to suppress the genotoxic effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. There isn\u0026rsquo;t any SMART data observed with \u003cem\u003eP. pungens\u003c/em\u003e in literature. However, there are many studies showing the protective characteristics of the members of Lamiaceae family against the genotoxic agents shown by SMART (Guterres et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e, Magombe et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e, Yilmaz Kardas et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).To sum up, it can be said that mutations and mitotic recombination, which is known to be stimulated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induced DNA breaks, were inhibited by the \u003cem\u003eP. pungens\u003c/em\u003e extracts used in this study(Qi et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e is one of the medicinal plants that has been used by local people to treat diabetes, ulcer and infections. However, there are limited number of studies in literature about this plant and cyto/genotoxicity analyses have not been done before. Therefore, this study aimed to find out cell protective activity, DNA damage prevention capacity and antioxidant/antibacterial properties of the water, methanol and ethanol extracts of \u003cem\u003eP. pungens.\u003c/em\u003e According to the LC results of this study, rutin hydrate (560.27 \u0026plusmn; 6.18 \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and quercetin (110.22 \u0026plusmn; 63.14 \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were found in highest concentrations. Antioxidant activity results showed that the methanol extract of the plant showed higher antioxidant capacities than other extracts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The plant was found as antibacterial against all of the strains tested but the largest inhibition zones were observed against \u003cem\u003eAeromonas\u003c/em\u003e sp. (12.67 \u0026plusmn; 0.32 mm) and \u003cem\u003eEnterobacter aerogenes\u003c/em\u003e (12.26 \u0026plusmn; 0.47 mm). Although the increased incubation time for the samples treated with the higher dose of the extracts negatively affected the viability of the cells, it was clearly seen that the growth of healthy human lymphocyte cells were supported by all of the extracts. Micronucleus assay showed that none of the extracts was genotoxic against healthy human cells. 8-OHdG ELISA test also showed that methanol and ethanol extracts of \u003cem\u003eP. pungens\u003c/em\u003e were able to decrease the oxidative DNA damage (41 and 50%, respectively). According to the SMART results, \u003cem\u003eP. pungens\u003c/em\u003e extracts were shown as the antigenotoxic substances with an ability to suppress the genotoxic effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. As a result of the in vitro analyses, the biological activity profiles and toxicity of routine hydrate and quercetin, which discovered to be the main constituents of the plant, were investigated in silico analyses, and the obtained prediction results support our experimental findings. To conclude, this is the first study which demonstrated the \u003cem\u003ein vitro\u003c/em\u003e cell protective activity and DNA damage prevention capacity of \u003cem\u003eP. pungens\u003c/em\u003e which should be regarded as an important medicinal plant that can be used for nutritional supplements and therapeutic-products.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of the somatic mutation recombination test (SMART).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"12\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAverage Number of wings (N)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eSmall single spots\u003c/p\u003e\n \u003cp\u003e(S)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLarge single spots (L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eTwin spots\u003c/p\u003e\n \u003cp\u003e(T)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInhibition %\u003c/p\u003e\n \u003cp\u003e(S,L,T, Total)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFr.\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFr.\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFr.\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.069 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.23 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater Solvent Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.014 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanol Solvent Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.067 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol Solvent Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.141 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Ethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Methanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Water\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.7, 100, 100, 91.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Ethanol\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.009 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.7, 86.7, 100, 93.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. pungens\u003c/em\u003e Methanol\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.009 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.2, 86.5, 100, 93.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"12\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), according to negative control, \u003csup\u003eb\u003c/sup\u003e statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), according to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (6.5 mg/L), \u003cem\u003eFr.\u003c/em\u003e: Frequency, n: Average spot\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is produced from a part of F. J. Wild Korkmaz\u0026rsquo;s MSc Thesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from Balikesir University Faculty of Medicine Clinical Research Ethics Committee (2021/132).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved to participate in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the final manuscript and submitted it to this journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Scientific Research Projects Commission of Balikesir University (BAP 2021/110)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that, there is no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbraham SK. 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Biochem Pharmacol 65: 1967\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Phlomis pungens, cytotoxicity, genotoxicity, 8-OhdG, in silico","lastPublishedDoi":"10.21203/rs.3.rs-4780456/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4780456/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCellular integrity depends mainly on the stability of DNA, which can be disrupted by genetic mutations and aberrations. Here, we show that the cell-protective activity and DNA damage prevention ability of \u003cem\u003ePhlomis pungens\u003c/em\u003e var. \u003cem\u003ehirta\u003c/em\u003e have been investigated. We found cell protective activity, healthy cell proliferation promoter activity and DNA damage preventing capacity in the controlled in vitro assays. Additionally, \u003cem\u003eP. pungens\u003c/em\u003e was shown to possess the ability to suppress the genotoxicity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as an antigenotoxic agent. Rutin hydrate and quercetin are the major flavonoids in \u003cem\u003eP. pungens\u003c/em\u003e, which exhibit a wide range of biological activities, including antioxidant, neuro- and hepatoprotective and Aβ-oligomer reducing activities. \u003cem\u003eP. pungens\u003c/em\u003e has shown impressive activity against Gram-positive and Gram-negative bacteria as well as strong scavenging activity against various radicals. Subsequently, \u003cem\u003ein silico\u003c/em\u003e software tools PubChem, pkCSM and PASS Online were used for biological activity profiling and toxicity predictions of the major compounds in \u003cem\u003eP. pungens\u003c/em\u003e. All evaluations of \u003cem\u003eP. pungens\u003c/em\u003e could be suggested as a potential source for dietary supplements and therapeutic products.\u003c/p\u003e","manuscriptTitle":"In vitro and in silico analyses of cytoprotective, antigenotoxic and antimutagenic potential of Phlomis pungens var. hirta extracts against oxidative damage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-20 17:51:41","doi":"10.21203/rs.3.rs-4780456/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-09T23:43:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-15T13:38:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-31T10:41:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263600838168475632327904864369499253561","date":"2024-07-25T00:32:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254005897665474096370997412236919013886","date":"2024-07-24T08:31:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-24T07:20:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-24T05:51:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-24T05:50:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genetic Resources and Crop Evolution","date":"2024-07-22T08:50:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e9d5ff7e-dac4-48f2-a267-9ac2f602cc12","owner":[],"postedDate":"August 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T16:00:55+00:00","versionOfRecord":{"articleIdentity":"rs-4780456","link":"https://doi.org/10.1007/s10722-024-02268-w","journal":{"identity":"genetic-resources-and-crop-evolution","isVorOnly":false,"title":"Genetic Resources and Crop Evolution"},"publishedOn":"2024-11-25 15:57:18","publishedOnDateReadable":"November 25th, 2024"},"versionCreatedAt":"2024-08-20 17:51:41","video":"","vorDoi":"10.1007/s10722-024-02268-w","vorDoiUrl":"https://doi.org/10.1007/s10722-024-02268-w","workflowStages":[]},"version":"v1","identity":"rs-4780456","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4780456","identity":"rs-4780456","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}