After performing PSM, there were no statistically significant differences between the groups, except for creatinine clearance (79.9 vs. 96.8 mL/min); p = 0.01. There were also no differences regarding the −455 G/A βfibrinogen polymorphism distribution between the two groups. After constructing the LRM, we found no performance enhancement for the CHA2DS2-Vasc score by adding the fibrinogen concentration or creatinine clearance alone, but when all three variables were put together, there was a significant improvement in LAT prediction (AUC 0.64 vs. 0.72), p = 0.026. Conclusion: Our study found no evidence of elevated levels of circulating fibrinogen in patients with LAT or a connection between those levels and the A/A and A positive polymorphism. When used together with renal function markers such as creatinine clearance, plasma fibrinogen concentrations can provide additional power to the CHA2DS2-Vasc score for predicting LAT.Background: As has been shown previously, patients with atrial fibrillation (AF) who have left atrial thrombus (LAT) also have elevated plasma concentrations of fibrinogen. In this study, we tried to determine if this is the consequence of a genetic trait and whether elevated concentrations of fibrinogen could be used to predict LAT in patients with AF. Methods: We recruited 181 consecutive patients scheduled for pulmonary vein isolation (PVI) or direct current cardioversion. The primary endpoint was the presence of LAT on transesophageal echocardiography (TOE). We recorded routine clinical and biochemical data as well as the polymorphism type of the fibrinogen gene for the β chain. To control potentially interfering variables, we performed propensity score matching (PSM). Multivariable and univariable logistic regression models (LRM) were computed using the CHA2DS2-Vasc score, the fibrinogen concentration and creatinine clearance as estimated by the Cockcroft–Gault equation. Results: 60 of 181 patients had LAT as detected by TOE. As expected, patients with LAT had significantly higher concentrations of fibrinogen (3.9 vs. 3.6 g/L);  AF is the most common arrythmia in human adults. It is linked to significant mortality and morbidity—primarily due to thromboembolism (mostly stroke)—by promoting left atrial appendage (LAA) thrombus formation . The LAAWe included 181 patients who were scheduled for PVI or direct current cardioversion between January 2018 and December 2019 in our university hospital center and who needed a transesophageal echocardiography exam (TOE) prior to the procedure. All patients had a transthoracic echocardiography exam (TTE) performed as well. We divided patients into two groups, one with thrombus in LAA detected on TOE, and the other without thrombus in LAA. Excluding criteria were any prior prosthetic valve surgeries, patients with documented thrombophilia, severe illness, or severe acute infection or active malignant disease. All patients have signed an informed consent form prior to recruitment in the study. The research protocol and the review of EMRs were approved by the ethics committees of the Medical University of Zagreb and the University Hospital Centre Zagreb.We included demographic data from electronical medical records , relevant medical histories , stroke/transient ischemic attack (TIA)/thromboembolism, vascular disease , and congestive heart failure, liver disease, smoking habits (yes/no) and current anticoagulation status). The CHA2DS2-Vasc score was calculated from the relevant variables . GeneticAll patients scheduled for direct current cardioversion for AF or for PVI, who had indications for TOE, were included in the study. Together with TOE, TTE was performed routinely prior to the procedure. TOE examinations are usually conducted within a few hours prior to the scheduled procedure (at most within 24 h before the procedure). All TOE and TTE studies were performed by certified echocardiographers (accreditation in echocardiography by the Section of Echocardiography of the Croatian Cardiac Society), using a VIVID 9 Ultrasound Machine . In cases where the study’s findings were in doubt, they were reviewed by two echocardiographers in order to arrive at a conclusive diagnosis that was the most trustworthy, to enable safe referral for cardioversion or ablation.All patients had peripheral venous blood samples taken, which were all kept in deep freezers. For DNA extraction, 200 μL of dissolved blood samples were centrifuged at 3000 bpm with a solvent solution for 5 min. For this extraction, we used a commercially available reagent for DNA extraction . The next step was amplification and digestion of the multiplied fragments of the gene promotor-encoding β chain of fibrinogen, which was carried out with the commercially available reagent AMPLI-β FIBRINOGEN −455 G/A .DNA samples were amplified and biotinylated in multiplex polymerase chain reactions (PCR). The amplification conditions were as follows: an initial denaturing step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min and extension at 72 °C for 2 min, and a final extension step of 72 °C for 10 min. After this, DNA amplification detection of the −455 G/A polymorphism was performed with the specific restrictive enzyme Hae III. The finished product’s genotypes were examined using a UV transilluminator after electrophoresis on a 1.5% agarose gel.The primary endpoint was the presence of a left atrial thrombus on TOE. As mentioned above, this is frequently employed as a surrogate marker for thromboembolic events, as following hard endpoint outcomes  would require substantially larger sample sizes and longer periods of time.2 test. Continuous values are presented as medians and interquartile ranges, and differences were analyzed using the Mann–Whitney U test. As the study was designed to be observational, we expected significant differences across several variables of interest. In order to counteract those differences, we implemented propensity score matching (PSM) in both groups, using a multivariable logistic regression model with LAT as the outcome, and matched individuals based on their risk score. By compiling a sample of LAT patients that is comparable to a sample of control group patients based on all the observed factors, PSM aims to decrease assignment bias and imitate randomization. After PSM, we were left with two new (matched) groups that could be compared in a case–control type analysis. Then, we analyzed the matched groups using the McNemar test for categorical variables and the Wilcoxon signed ranks test for continuous variables. Lastly, we generated logistic regression models for CHA2DS2-VASc, CHA2DS2-VASc and fibrinogen concentrations;CHA2DS2-VASc and eGFR; and CHA2DS2-VASc, fibrinogen concentration and eGFR. ROC curves were drawn and AUCs calculated and compared using the DeLong method. For all tests, a p-value of less than 0.05 was considered significant. All tests were two-tailed. All calculations were performed using MedCalc version 19.2.6  and Office Excel 2007 .First, we analyzed the unadjusted data in both groups. Categorical and nominal values are presented through corresponding frequencies and shares, and differences between individual groups were analyzed using the χA total of 181 patients were included, of whom 60 (33.1%) had LAT—predominantly thrombosis of the LAA. The group differences in the unadjusted analysis are described in p = 0.0027). Patients in the thrombus group had significantly more vascular disease, heart failure and diabetes mellitus , compared to the control group, where on the other hand, there were significantly more patients with paroxysmal atrial fibrillation (p = 0.0115). The thrombus group had higher fibrinogen levels that were statistically significant compared to the control group, as well as creatinine clearance, which was statistically significantly lower in the thrombus group .The demographic factors that were noted did not differ significantly, except in the control group (without thrombus), where there were significantly more men than women  compared to the control group, where only 65.3% patients were of high risk. The LAT and control groups differed significantly from each other in terms of thromboembolic risk; The use of anticoagulant therapy was generally high (over 90% of patients) and did not significantly differ between the two groups. Anticoagulation was mostly achieved by NOACs.There was no meaningful difference between the two groups in terms of LA size. The global systolic function of the left ventricle (measured as ejection fraction) also did not differ significantly between the two groups. There were no differences regarding the prevalence of the homozygous AA genotype between the two groups. When analyzing together with the heterozygous GA genotype , we also failed to detect any significant difference between the groups.A multivariable logistic regression model was computed to perform propensity score matching for the two groups (thrombus and control). Individually obtained risk scores were listed and matched between the two groups. After PSM, we got two new (matched) groups that could be compared in a case–control type analysis that was now adjusted for confounding variables. The adjusted results of the comparison of the 120 patients (60 pairs) are described in p = 0.01). There were also no differences regarding polymorphism distribution between the two groups. No statistically significant changes between the groups were found following PSM, except for creatinine clearance  had an area under the receiver operating characteristic curve (AUC) of 0.642, with a standard error of 0.0408 and a 95% confidence interval of 0.567 to 0.712.p = 0.318 and p = 0.054 for pairwise comparison with the reference model, respectively), as seen in The combined models of only fibrinogen and CHA2DS2-Vascand only creatinine clearance and CHA2DS2-Vasc yielded no significant improvements , which is shown in After constructing the LRM containing CHA2DS2-Vasc, fibrinogen and creatinine clearance, there was a significant improvement in prediction , while the CHA2DS2VASC score showed poor sensitivity and specificity (AUC = 0.642), as in previous studies. [The CHA2DS2VASC + FIB + CC score demonstrated good sensitivity and specificity in patients with LAA thrombus  and not ischemic stroke. However, LAA thrombus formation is thought to be the main cause of thromboembolic events in AF patients, making it appropriate to be utilized in risk assessments of AF. Secondly, our study was observational, with a possible selection bias which limits its generalizability, as our database included only hospitalized patients in one hospital center. Additionally, as this was an observational study and we were only able to use the factors that were sampled, additional possible predictors may have gone overlooked. Given that we used PSM, this is especially significant; it balances treatment and control groups on a number of covariates without utilizing a large number of observations and employs a linear combination of factors to obtain a single score (average treatment effect estimate). PSM has the drawback of not accounting for latent traits, but only for observed (and observable) factors. The matching technique cannot take into consideration factors that influence treatment assignment and outcome that cannot be seen. Any concealed bias resulting from latent factors may still be present after matching because the process merely adjusts for observed variables. However, the study’s objective was to identify risk factors that are frequently assessed in clinical practice. There are several studies that have shown seasonal variations in fibrinogen concentration, as well as the fact that fibrinogen concentrations are influenced by many environmental factors  ,26,27,28In the light of already published data, our study provides further evidence of the thrombogenic nature of inflammation markers and renal dysfunction. Two variables not included in the CHA2DS2VASC score  proved strong predictors of LAT on TOE, and therefore might improve thromboembolic risk stratification in patients with AF. Nevertheless, the proposed CHA2DS2-VASC-FIB-CC score requires validation in further studies.


Ptf1aCre/+;Vglut2flox/flox mutant mouse to define fundamental phenotypes and measures that are valuable for testing the cellular, circuit, and behavioral mechanisms that drive dystonia. In this model, excitatory neurotransmission from climbing fibers is genetically eliminated and, as a consequence, Purkinje cell and cerebellar nuclei firing are altered in vivo, with a prominent and lasting irregular burst pattern of spike activity in cerebellar nuclei neurons. The resulting impact on behavior is that the mice have developmental abnormalities, including twisting of the limbs and torso. These behaviors continue into adulthood along with a tremor, which can be measured with a tremor monitor or EMG. Importantly, expression of dystonic behavior is reduced upon cerebellar-targeted deep brain stimulation. The presence of specific combinations of disease-like features and therapeutic responses could reveal the causative mechanisms of different types of dystonia and related conditions. Ultimately, an emerging theme places cerebellar dysfunction at the center of a broader dystonia brain network.There is now a substantial amount of compelling evidence demonstrating that the cerebellum may be a central locus in dystonia pathogenesis. Studies using spontaneous genetic mutations in rats and mice, engineered genetic alleles in mice, shRNA knockdown in mice, and conditional genetic silencing of fast neurotransmission in mice have all uncovered a common set of behavioral and electrophysiological defects that point to cerebellar cortical and cerebellar nuclei dysfunction as a source of dystonic phenotypes. Here, we revisit the  Dystonia is a neurological disease that causes muscles to work against, rather than with one another . DystoniTOR1A, GNAL, SGCE, and THAP1 [Tor1a in mice, have paved the way for testing the circuits [Tor1a in the cerebellum [Great efforts have been made to determine the relationship between possible dystonia etiologies and the spectrum of known clinical characteristics of the disease. Many cases of isolated dystonia, where dystonia is not comorbid with other movement disorders, are thought to be genetic in origin. Among the most common genes with mutations that cause dystonia are nd THAP1 , 18. Whind THAP1 . Still, circuits , 20 and circuits . The cercircuits . These scircuits . More recircuits . These scircuits  and shRNcircuits . Similarrebellum . MechaniAtcayji−hes mouse model of dystonia [lamb1t [Ptf1aCre/+;Vglut2flox/flox mice [Ptf1aCre/+;Vglut2flox/flox mutant mice [A key issue that has been under scrutiny is the exact defects in the cerebellum that are responsible for initiating the dystonia. Much attention has been placed on two primary cell groups, the Purkinje cells in the cerebellar cortex and the neurons in the cerebellar nuclei. Normally, both types of cells fire at relatively high rates, often exceeding ~50 Hz. In addition, although their in vivo firing pattern is not tonic, both cell types fire with robust and continuous activity with minimal pausing in healthy awake animals . In dysdystonia , the ouadystonia , and the [lamb1t . To bettlox mice . These mant mice .Ptf1aCre allele [LoxP-flanked glutamatergic vesicular transporter 2 allele, Vglut2flox (JAX #012898) [Vglut2flox. The resulting Ptf1aCre/+;Vglut2flox/flox offspring had a conditional deletion of the Vglut2 allele that is specific for the Ptf1a lineage (achieved by driving the Cre). As a consequence, the loading of glutamate into presynaptic vesicles during fast neurotransmission is genetically blocked, which results in the precise elimination of neurotransmission at glutamatergic synapses only within the Ptf1a-expressing neurons [Ptf1a lineage are inhibitory and therefore are not affected by the removal of Vglut2. However, the inferior olivary neurons in the brain do express both Ptf1a and Vglut2 and, ultimately, the olivary climbing fiber projections that terminate upon the dendrites of Purkinje cells in the molecular layer of the cerebellar cortex no longer express the VGLUT2 protein at their synapses.To generate the mice, we crossed male mice that were heterozygous carriers of the e allele  and homo#012898) , to fema neurons . ImportaHere, we describe the application of these mice to behavior, electrophysiology, and deep brain stimulation and argue that mouse models of cerebellar function hold critical insights for understanding dystonia pathogenesis and treatment options. The specificity of the approach not only allows for the dissection of cerebellar mechanisms in dystonia, but also provides a framework for disentangling how the cerebellum might contribute to a number of motor disorders, including ataxia, tremor, and seizures –36. We dThe experimental approaches and analyses presented here were described in detail in White and Sillitoe, 2017 . In addiPtf1aCre/+;Vglut2flox/flox mutant has proven to be especially useful for studying these severe dystonic symptoms. These include twisting of the torso . We implant EMG recording electrodes into the tibialis anterior (TA) and the gastrocnemius muscles (GC)  Fig. . During GC) Fig. . Consistice Fig. . Cross-cype Fig. . TherefoPtf1aCre/+;Vglut2flox/flox mutants, which lack inferior olive to Purkinje cell communication, an ~10 Hz tremor accompanies the severe dystonia [Thap1 mice [It is estimated that upwards of 50% of individuals with dystonia also have tremors . At the dystonia . In animdystonia , 45 , and dentate nuclei. In regards to dystonia, the interposed is an interesting area because of its role in limb movements and ongoing motion –54. Accochanisms . Deep brlox mice   The developmental onset of the dystonia in this mouse indicates that the causative defects may derive from a dynamic set of structural and functional interactions long before the mature circuit is established. This idea is supported by the phenotype of a different mouse model in which granule cell neurogenesis is obstructed [Ptf1aCre/+;Vglut2flox/flox mutant mice also exhibit a dramatic burst firing phenotype [Ptf1aCre/+;Vglut2flox/flox mice, stimulation of the interposed nucleus significantly improves mobility [Car8wdl/wdl mice, which have dystonia, ataxia, and tremor, chlorzoxazone recovers movement and corrects Purkinje cell firing by eliminating burst activity [The structed . (2) Dysstructed . In our structed  and ataxstructed . (3) Worstructed . Similarstructed , 28. Cerhenotype . (4) Deehenotype , 66. In mobility  Fig. 5)Ptf1aCre/mobility , in our mobility . In the activity . We postPtf1aCre/+;Vglut2flox/flox model. First, even though the dystonia is reliable , robust, and easily quantifiable, the phenotype is not reversible. It would be remarkable to have an equivalent model in which the dystonia, and the associated cellular defects, could be eliminated on demand and at different time points during the animal’s life. One could then examine what aspects of the circuit maintain enough function for recovery, therefore unveiling additional therapeutic targets; whether specific circuits exhibit plasticity-related changes during dystonia, and an experimenter could carefully examine how circuits adapt during the onset and removal of dystonia. Second, even though the loss of climbing fiber to Purkinje cell function could be a feature in human dystonia , it remTSPOAP1; . RegardlPtf1aCre/+;Vglut2flox/flox mouse provided an ideal model to test the role of the interposed nucleus as a target for stimulation, it remains possible that the fastigial and dentate nuclei could also offer similar or even complimentary benefits. A patient with hemidystonia showed improved motor function after deep brain stimulation of the dentate nucleus [The design of strategies to target the cerebellum with noninvasive and invasive methods of brain stimulation has recently gained wide appreciation in the field . Althoug nucleus . More br nucleus  and opto nucleus  in rats  nucleus , 74, not nucleus  and exis nucleus –78 highl


Camellia sinensis L.) extraction,carried out usingwater and hydroalcoholic solvents, for the subsequent synthesis ofsilver nanoparticles (AgNPs). The value ranges for independent variables,including pH, time, and temperature, were selected based on single-factorexperiments and used for extraction in the order presented by theBox–Behnken design. Three-dimensional response surface graphswere used to visually present the optimization results and determinethe optimal extraction conditions: pH = 7, 30 min, 80 °C forwater and pH = 5.5, 50 min, and 80 °C for water–ethanol.Our findings indicate that the water–ethanol mixture extractedmore polyphenols. We compared the physicochemical properties of AgNPsobtained using both types of extractants via DLS and TEM analysis.We proposed a predicted mechanism for the reduction and stabilizationof AgNPs based on the Fourier transform infrared data. The hydroethanolicextract leads to significant nanoparticle aggregation, which can beexplained by the nucleation theory and agglomeration of nanoparticlesin the presence of excess macromolecular organic substances (flocculation).In this paper, we present the optimization of green tealeaf ( Among this vastarray of bioactive substances, polyphenols stand out as potent naturalantioxidants.9 Green tea leaves (Camellia sinensis L.) are a popular plant sourceof polyphenolic compounds.10 The place,cultivation method, climatic conditions, and harvesting time significantlyinfluence the chemical composition. Green tea leaves contain variousbioactive substances, such as amino acids, proteins, carbohydrates,minerals, and pigments. However, the primary chemical component comprisingup to 30% of the mass of dry leaves are polyphenols, particularlyfour catechins: epicatechin, epicatechin gallate, epigallocatechin,and epigallocatechin gallate.14 A significant advantage of extracting polyphenolsfrom plant material is the absence of the need for a sophisticatedmethod to aid in facilitating the extraction process. Extracting polyphenoliccompounds from plant leaves is an example of a solid–liquidseparation. Optimizing its crucial parameters has become necessaryto transfer this process to a pilot scale. Mathematical and statisticalmodels, including surface response techniques, are commonly employedto determine optimal conditions for obtaining extraction productsefficiently, quickly, and economically. Several parameters are usuallyconsidered, such as the solid–liquid ratio, temperature, processduration, pH, and extractant type. In cases where ultrasound-assistedextraction is utilized, optimization also includes verifying the durationand power of ultrasound.17 Various leaves of plants havebeen extracted, and the process parameters have been optimized. Examplesof these plants include Hibiscus cannabinus L,15Lobelia rucotianifolia,17Azadirachta indica (neem),20Eucalyptus globulus,21Murraya koenigii (Linn),22Myrtus communis L,23Rhamnus alaternus,24Stevia redaudiana25 and pure heart plant, and olive leaves.27 Green tea, known for its easy commercial availability and richnessin polyphenols, is the most widely used type of tea for extraction.12 The procedures employing microwaves (MAE),30 ultrasound (UAE)31 and other green conditions32 are popular in the extraction process of theseplant leaves beyond conventional methods. The literature has examplesof extracting polyphenols from green tea leaves with the assumptionof using statistical models.37 So far, Box–Behnken design (BBD),36 central composite design (CCD),37 and orthogonal assay design29 have been used. Additionally, the prepared extracts are tested toevaluate antioxidant activity. It is thought that the extractant andgrain size of the green tea matrix often have a significant impacton the efficiency of the extraction process.38 Menezes and Bindes proved that a specific grain size (0.15–0.74mm) optimized based on the statistical Turkey test is important formaximizing the extraction efficiency of polyphenols using water asan extractant.33 Kim and his group examinedthe influence of three important parameters affecting the extractionof polyphenols, including ethanol concentration (0–100%), temperature(10–70 °C), and time (3–15 min) as input valuesfor the BBD model combination matrix.35 The highest antioxidant activity above 85% was observed for thehighest values of time and temperature and also for an ethanol concentrationof 57.7%. On the other hand, the model was applied to extraction inwater, choosing biomass/solvent ratio (1:10–1:50), temperature(40–50 °C), and extraction duration (15–45 min)as independent variables.36 Based on CCD,Lee et al. optimized a conventional green tea extraction method usingtemperature, time, and ethanol concentration as initial parameters.14 They compared the yield and oxidative activityobtained under optimal conditions with those measured for ultrasound-assistedextraction. Thus, they proved that ultrasonication promotes the reductionof the extraction time and ethanol concentration without affectingthe antioxidant activity of polyphenols. More recently, Al-Hatim andcoauthors performed green tea extraction under microwave conditionsusing an orthogonal assay design.29 Statisticalanalysis included the following extraction conditions: microwave intensity,extraction time, biomass/water ratio, and also frequency of microwaveexposure. Interestingly, the time required for extraction under theindicated conditions was only 3 min. Less common tea leaves, suchas black39 and oolong,40 were also used in the extraction processes.Throughout history, numerousbiologically active compounds havebeen identified and extracted from various plant parts that are usedas valuable medicines, yet many more await discovery.43 The qualitative andquantitative composition of the tea extract is the primary factoraffecting the synthesis of silver nanoparticles (AgNPs). Therefore,tea fermentation processes have been optimized,44 and microwaves have been used during extraction.45 Tea leaves as a reach plant source of naturalreducing compounds have gained recognition among the interests ofresearchers not only in terms of extraction but also in the synthesisof nanoparticles of various metals.48 Of all types of tea,green tea deserved special attention. AgNPs obtained through its extractshave been examined for antibacterial and antifungal activity.55 In certain cases, even their catalytic activity in the degradationof dyes present in industrial wastewater like Reactive Yellow 160,56 Direct Fast Rose FR Red 227,57 Basic Brilliant Flavine Y-40,57 Reactive Red 81,58 Congo Red Direct,59 Orange E360 and CrystalViolet61 have been confirmed. Sun and hisco-workers assessed the effects of time, optimal dosage of extract,and temperature on the synthesis of AgNPs (20–90 nm).53 The most pleasing result of synthesis was obtainedafter 2 h at room temperature. They also examined the inhibitory propertiesof these AgNPs against Escherichia coli. On the other hand, Babu et al. proposed an efficient method tosynthesize these nanoparticles as a colorimetric sensor of cysteine.52 They employed different dilutions of the aqueousgreen tea extract under ambient temperature conditions. AgNPs possessedhigh stability for more than six months without the need for specialstorage conditions, such as low temperature or lack of light. Interestingly,it was also confirmed that the properties of AgNPs are significantlyinfluenced by the elevation of the green tea growing fields in Nepal.54 More recently, Widatalla and coauthors demonstratedthe high destructive activity of the produced AgNPs toward two bacteria: Klebsiella pneumoniae and Staphylococcusaureus.62 Onitsuka et al.63 compared the reducing properties of aqueousand methanolic extracts of green and black tea for the preparationof AgNPs. The stability of the AgNPs was comparable; however, a differencewas observed in their size. For the aqueous extract, AgNPs were smallerfor green tea, while for the methanolic extract using black tea leaves.Maintaining specified nanoparticle dimensions is necessary for commercializingAgNPs.64 Nucleation and agglomeration processesmainly influence the size of the synthesized nanoparticles. The modelformation of nanoparticles assumes a two-step process: nucleationand growth.66 The course of these two stagesis significantly influenced by process parameters such as pH value,temperature, ionic strength of the solution, and mixing intensity.Compounds in the reaction mixture, such as reducing and stabilizingagents, are equally important. It has been shown that trisodium citrateas a reducing agent and stabilizers such as sodium dodecyl sulfate(SDS) and Tween 80 influence the stability of AgNP suspension andprevent agglomeration.67 Biosynthesis usingplant extract leads to nucleation and nanoparticle growth processesaccompanied by numerous organic compounds whose effects are not yetfully understood.70Bothblack and green tea extracts have been previously proven effectivefor silver nanoparticle synthesis.Despite the very rich literature based on thepreparation of metallicnanoparticles using plant material as a source of potential reductants,there is still a discernible place to provide protocols related tooptimizing efficient, sustainable, as well as green procedures inthis research area. The ever-increasing industrial demand for nanomaterialsthat play an important role within the context of therapeutic, biotechnological,or technical applications is forcing the use of more friendly andinexpensive synthetic solutions based on the application of naturalresources and decreasing the ecological footprint. The lack of onespecific general extraction protocol tailored against all plants iscurrently a key problem facing the field of nanotechnology using plantextracts in the synthesis of nanomaterials. In this situation, scalingup nanosynthesis poses an additional challenge. This is due to thedifferent conditions or regions of plant cultivation, the diversityin the profile of the chemical compounds hidden in them, and alsothe great flexibility of the parameters that determine effective extraction.The hope to deal with this problem is noticeable in process optimization.As mentioned above, the selection of specific parameters will makeit possible to determine the conditions that ensure optimal effectivityby evaluating the relationship between them. Thus, it will be possibleto reduce the economic effort and time required to develop the procedure.Therefore, keeping in mind the requirements of the industry, the studyaimed to develop an efficient process for extracting polyphenoliccompounds from ground green tea leaves based on three independentparameters: pH value, time, and extraction temperature, utilizingthe combination prescribed by the statistical Box–Behnken responsesurface model. The total number of polyphenolic compounds (TPC) wasused as a model response for two solvent systems with low environmentaltoxicity: water and ethanol–water mixture. Furthermore, anapplication test was conducted using the obtained extract under optimalconditions as a source of natural reducing agents for the biosynthesisof AgNPs.22.1Figure S1).Commercial store-boughtgreen tea was used as the material for polyphenol extraction. Driedgreen tea leaves were ground using a domestic coffee grinder, thoroughlywashed, and dried to increase the extraction efficiency. Each grindingprocess lasted for 1 min using the maximum power of the grinder. Afterthe milling process, the particle size distribution of green tea wasmeasured. The mean particle size was determined to be 205 μmusing a Beckman Coulter LS 13 320 LS Particle Size Analyzer . Thefinal total polyphenol content was calculated using the equation shownbelow (The green tea leaves were mixed with an extraction solvent (0.2 g/25mL) and left at room temperature before being heated to a specifictemperature in a water bath. The biomass was then separated from theextract by filtration using a 0.22 μm filter (Sigma-Aldrich).The total amount of polyphenols in the extract was determined usingthe Folin–Ciocalteu method.wnbelow 11CTPC [mg GAE/mL]—total polyphenol concentrationcalculated using the standard curve and accounting for the extract’sdilution; V [mL]—the volume of solvent selectedfor the extraction experiment; and m—the weightof the dry biomass (green tea leaves).The elements of the equation representthe following: TPC [mg GAE/g db]—total polyphenol content,which is the response for statistical optimization; 2.3HPLC UV–vis analyses were conductedto confirm the presence of characteristic components of tea in theaqueous and hydroethanolic extracts. A 500 μL sample of theextract was taken and diluted with the appropriate solvent by addingthe same volume to the volumetric vial. The sample vial was placedin the measuring apparatus, an HPLC Dionex equipped with an AerisPEPTIDE XB-C18 column . The mobile phase comprised solvents A (water with 0.1% TFA)and B . The measurement was performedusing a gradient elution: 5–40% of solvent B for 30 min withUV detection at a wavelength of 280 nm. The injection volume was 20μL, and the flow rate was 1.0 mL/min.2.4–3 M). Then, 5 mL of the extractusing the previously optimized conditions and a defined pH value wereadded dropwise. The mixture was heated in the dark at 65 °C for45 min with constant stirring. The change in the color of the mixtureand the appearance of light yellow turbidity indicated the reductionof Ag+ ions from the precursor to the Ag(0) nanoform. Oncethe reaction time had elapsed, the mixture was left to cool in thedark. The suspension of AgNPs was then transferred from the reactionvessel to an amber glass bottle and stored in a refrigerator at 4°C for further characterization. A similar procedure for thesynthesis of AgNPs using green tea extract and the temperature of65 °C indicated as optimal, but with different proportions ofthe volume of the extract and the amount of AgNO3, waspreviously used by Riaz et al.74Aflask equipped with a magnetic stirrer introduced an aqueous solutionof silver nitrate  at a temperature of 25 °C. TEM measurements were takenusing an FEI Tecnai G2 20 X-TWIN high-resolution transmission electronmicroscope with a LaB6 cathode, an FEI Eagle 2K CCD camera, an EDSdetector, and a STEM detector. The appropriate functional groups identifyingthe biomolecules present in the aqueous green tea extract and thoseresulting from distinct interactions between the polyphenolic compoundsof the extract and the surface of the AgNPs formed with their participationas reducing agents were analyzed using Fourier transform infrared(FTIR) spectra. According to the standard pellet KBr method, FTIRmeasurements were performed for freshly prepared dried tea leaf extractas well as green tea extract-mediated AgNPs in the range of 4000 to400 cm–1 using a Bruker Vertex 70 FTIR spectrometer.50The synthesized AgNPs were characterized using several techniques,including UV–vis spectra, DLS analysis, potential zeta, andTEM measurements.2.678 The experimental design and levelsfor water and water–ethanol solvents are presented in The impact of three selectedparameters, solvent pH, temperature (°C), and time (min), onthe extracted total phenolic content was assessed using the Box–Behnkenfactorial design with three levels: (−1), (0), and (1). Itshould be emphasized here that temperature and time are the most importantparameters factored in statistical designs in terms of optimizingthe extraction of polyphenols from plant material, including tea leaves.xi—is the mean dimensionless value of the process parameters, Xi—is the uncoded valueof the three test variables, X0—isthe value of each uncoded variable at the central point, and Δx—is the mean step change of the value of the actualvariable (Xi) correspondingto a variation of the coded value of process variables.Based on the impact of different factor values onthe extractionefficiency, which was expressed as TPC [mg GAE/g db], the levels ofindependent variables were determined and marked as (−1), (0),and (1) for statistical optimization using the Box–Behnkenmethod. The coded values of the independent variables were calculatedfrom their uncoded values using the following equation 2, takingAccordingto this design, 17 experiments were necessary to optimizeand construct a model to generate the extraction efficiency expressedas the total polyphenol content (TPC) response. This included fivereplicated experiments under the same conditions where pH, temperature,and time were at level 0, as shown in Y—is the predictedresponse , β0, βi, and βii, βij—areregression coefficients, which represent the variable correlationresponse: βi—is the linearcoefficients, βii—is thequadratic coefficients, and βij—isthe coefficients of cross-interactions between two independent variables, k—is the number of analyzed independent variables, Xi and Xj—are the values of the independentvariables.The second-order polynomial equation, which is theimage of theresponse function obtained by regression analysis, is presented by eq p-value, R2 and Radj2 coefficients,and obtaining the response surface curvature.The statistical calculation software Statistica 13.3was utilizedto perform the regression analysis and obtain the coefficients ofthe polynomial equation presented above. The ANOVA statistical testprovided by the program allowed for the correction of the regressionequation by assessing the model’s usefulness using parameterssuch as lack of fit (LoF) and 3The green synthesisof AgNPs using plant extracts is a common methodfor obtaining metallic nanoparticles while meeting the principlesof green chemistry. Many reports in the literature, developed overmore than a hundred years, have concluded that several factors affectthe efficiency and course of the extraction process of bioactive compoundsfrom plant structural elements. Carrying out experiments to determinethe optimal value for each factor is an impractical point of view.Therefore, an essential step in developing an effective green technologicalextraction or AgNP formation process is to select a few parametersand conduct preliminary experiments to determine their most effectivevalues. Based on literature data, the parameters selected for optimizingthe polyphenol extraction process from Green Tea L. are pH, extractionprocess duration, and temperature 1 and 2.3.1Aninitial step in the work was to study the effect of individual parameterson the extraction process of polyphenols from Green Tea L. in water1. Figure79 This explains why the extraction efficiencydecreases when the water’s pH is beyond the neutral value.The analysis of extraction yields (TPC) at different time intervalsshowed that initially, the extraction efficiency increased and reachedits maximal value after 30 min  1A. The rr 30 min 1B. Howevto 80 °C 1C. The r81 However, there was aslight decrease in extraction efficiency when the temperature wasincreased to 80 °C, possibly due to the formation of insolubleforms of polyphenolic compounds as a result of their detachment fromthe cell wall or decomposition processes of the entire plant cell.83 The final values of the independentvariables pH , time , and temperature used for statistical modeling for water extraction,selected based on the described single-factor experiments, are summarizedin The highest TPCvalue was recorded for the temperature at 70 °C,reaching 39.89 [mg GAE/g db]. This finding is consistent with thegeneral statement that traditional methods of polyphenol extractionachieve the highest level of isolation at temperatures between 60and 80 °C.3.2Optimizing the polyphenol extraction process wasbased using the Box–Behnken statistical model and analyzedusing the response surface methodology (RSM). Three independent variables,including pH value, time, and temperature, were assessed accordingto the planned experiment. The Box–Behnken model matrix, withimposed connections between the extracted parameters’ codedand uncoded values, was used to design 17 experiments, as shown in The experimental datafor the extraction of Green Tea L. showed that the yields (TPC) ofthis process ranged from 37.39 to 39.89 [mg of GAE/g db]. The highestTPC value was obtained by performing the extraction at 70 °Cfor 30 min while maintaining a neutral pH of water as the solvent.The lowest yield was obtained when the sample was heated at the sametemperature for 20 min in a solvent with an acidic pH value of 5.5.Based on all results, the average extraction efficiency of polyphenoliccompounds in water was 38.14 [mg of GAE/g db].x1, x2, and x3 denotethe coded independent variables: pH value, time, and temperature,respectively. The model and, thus, its final equation were verifiedby statistical analysis, which was the ANOVA test, and its resultsare presented in The predictionmodel for the extraction of polyphenols from driedGreen Tea L. in an aqueous environment is described by the followingequation 44where xFigure S4). The probabilityvalue  is used to estimate the significanceof the model. Typically, the response variable is considered significantif the p-value is less than 0.05.Examination of the parameters obtained through the ANOVAstatisticaltest allowed for assessing the significance of the generated mathematicalmodel for extracting polyphenols from Green Tea L. and identifyingthe factor that had the greatest impact on the course of the process(x1), time (x2), and temperature (x3) werefound to be significant, as well as the quadratic coefficients relatedto time (x22) and temperature(x32) variables, and the interactioncoefficients between the pH value and time (x1x2) and time and temperature ofextraction (x2x3). However, the square coefficient of pH (x12) and the interaction between pH and temperature(x1x3) wereinsignificant. After the insignificant terms are removed from themodel, the final equation describing the investigated response ofthe process is presented in the form of eq In the testedmodel for the effective extraction of polyphenolsfrom green tea leaves, all the generated linear coefficients for thevariables pH (R2), adjusted correlationcoefficient (Radj2), predictedcoefficient (Rpred2), F-valueof the model and LoF significance,adequate precision, and coefficient of variation (C.V.) are crucialfor validating the obtained model. The prepared regression analysisyielded an R2 correlation coefficientof 0.9804, indicating a good fit between the experimental and model-predictedextraction yields expressed as TPC. The linear fit is shown in Figure S5 (see Supporting Information). The equally high value of the adjusted R2, which is 0.9553, provides information aboutthe model’s ability to explain 95.53% of all variances. Thepredicted R2 (0.9807) is close to Radj2, which is desirable, as theyshould agree and the difference between them should be less than 0.2.The model’s usefulness is further demonstrated by the insignificantvalue of the LoF (>0.05) and the significant p-valuefor the F-value of the model, which is less than0.0001. Furthermore, the C.V. parameter value of 2.2%, which is lowerthan 10, confirms the model’s accuracy. The estimated valueof the signal-to-noise ratio, equal to 23.2733, also called adequateprecision, exceeds the value of 4, which additionally indicates thatthe obtained model is precise in determining the optimal conditionsbased on the analysis of the surface response in the form of three-dimensional(3D) graphs . Laboratory experiments confirmed the compatibilitybetween predicted and experimental TPC values under optimal conditions,with an average value of 40.34 [mg GAE/g db] in three replicates.Based on the 3D plots of the surface response, theoptimal conditionsfor the extraction of polyphenols from Green Tea L. were identified,with the highest yield of 40 [mg GAE/g db] achieved at pH = 7, 30min and 80 °C .Significant effects observed include linear correlations of time (x2) and temperature (x3), quadratic correlations described by coefficients of pH (x12) and temperature (x32), and coefficients describing the interactionbetween pH and extraction temperature (x1x3) as well as time and temperature (x2x3). Conversely,coefficients calculated for the linear pH effect (x1), the quadratic time coefficient (x22), and the coefficient calculated from theinteraction between pH and time (x1x2) were insignificant. Thus, they should beomitted in the final extraction model equation, as represented by eq This factor strongly influences, as confirmed by thePareto graph. The R2, Radj2, and Rpred2 coefficients are satisfactory,exceeding 0.9 and equaling 0.9654, 0.9210, and 0.9654, respectively.This indicates that the model effectively describes 92.10% of allvariances represented by the model, and the data values predictedby the model closely match the experimental data concerning TPC obtainedin the hydroethanolic extraction process as observed in the aqueoussystem. The highly linear relationship between the data was furtherconfirmed by the comparison shown in Figure S7 (see Supporting Information). The insignificanceof the LoF parameter of 0.0776 and the highly significant p-value parameter for the entire model (less than 0.0001)allow the model to be accepted as useful for determining optimal conditionsfor extracting polyphenols from green tea leaves using an ethanol/watermixture. For this model, the adequate precision and coefficient ofvariation values were determined to be 15.4279 and 4.2, respectively,falling within the range established by statistical rules. This ledto the full characterization of the model and the possibility of usingresponse surface analysis to determine the optimal extraction conditions.According to the three-dimensional graphs obtainedby modeling4A–C,whiThis combination of results appears in the Box–Behnkenmodelmatrix and represents the highest extraction process efficiency, 121.82[mg of GAE/g db]. The TPC value predicted by the model is 122.43 mgof GAE/g of db, indicating that both values are very close. Basedon this result, it can be concluded that the proposed model is a goodfit and can be used to determine the TPC value for extracting theselected plant research material.3.5The firstevidence for the biosynthesis of AgNPs using an aqueous or hydroalcoholicextract of Green Tea L. was a visible change in the color of the reactionmixture from colorless to a yellow-green or light brown suspension,depending on the extract used. Additionally, spectrophotometric studiesconducted in the subsequent step confirmed this fact by identifyingthe maximum surface plasmon resonance absorbance found in the typicalwavelength range for AgNPs, between 420 and 500 nm 5.Notably, the AgNPs obtained using both proposedplant extractsas natural reducing agents exhibit differences in morphology primarilydue to their size 6.AgNPs formed during the green synthesis, wherethe hydroethanolicextract was employed, showed a tendency to exist in large aggregates,with an average size of nanoparticles equal to 139.8 nm C–E, whilTable S3). The composition of the main polyphenols found inthe aqueous and hydroethanolic extracts indicates that a higher amountof them is achieved using an ethanol/water mixture for the extractionprocess. This is in agreement with the results of ethanol extractionreported in the literature.89The reason for their ability to agglomerate shouldbe sought inthe qualitative and quantitative composition of both extracts usedfor the synthesis  mixturethat replaced the plant extract as reducing agent failed under thesame reaction conditions. The peak confirming the formation of AgNPsdid not appear in the UV–vis spectra either immediately afterthe reduction process or after 24 h of storage of the sample, whichwas protected from the light. It can therefore be assumed that theratio of the water/ethanol mixture used in the extract increases theefficiency of polyphenol extraction compared to water but has no directeffect on the synthesis of AgNPs. Probably, the concentration of ethanoland its amount may be insufficient to observe the symptoms of reductionin the conditions of synthesis proposed by us. It provides informationthat only biomolecules present in the extract are responsible forthe reduction process.The reducing propertiesof ethanol in the synthesis of metal nanoparticleshave been described in the literature.Additionally, it should be emphasizedthat (−)epigallocatechingallate (EGCG) and (−)epicatechin-3-gallate (ECG), which werefound in higher concentrations in the hydroethanolic extract comparedto the aqueous extract, may have the most significant impact on thereduction of the silver precursor to nanoparticles.FTIR measurementswere taken to further confirm these predictions.Interpretation of the FTIR analyses consisted of comparing the presenceand location of bands corresponding to specific functional groupsbelonging to various bioactive compounds contained in the green teaextract with those observed in the sample measured after AgNP synthesis8. This a–1 probably came from thestretching vibration of the O–H bond in free OH groups foundin the structure of catechins, phenols, and alcohols, but it can alsobe attributed to the stretching vibration of the N–H bond inamine. At 2927 cm–1, a band corresponding to thevibration of the C–H bond was observed. The strong marked bandat 1645 cm–1 is assigned to the stretching vibrationof the C=O or C–N bonds present in compounds like gallicacid, catechin, or theanine. The presence of a peak in the positionof 1042 cm–1 informed us about the vibrations ofthe C–OH bonds belonging to alcohols or proteins. In the FTIRspectrum of green tea extract, bands at 820 and 610 cm–1 were also found, corresponding to out-of-plane vibrations attributedto aromatic C–H bonds and bending vibrations of the C=Cbonds in alkenes, respectively.The FTIR spectrum for the aqueous green tea leafextract was representedby a blue line 8. The sh–1. Other representative bands wereobserved at 2360 cm–1 and attributed to a stretchingvibration from a bond in the COOH group and also at 1629 cm–1, which corresponds to the vibration of bonds found in carboxylicacids. A particularly important peak is the one present at 1384 cm–1, which confirms the presence of bending vibrationsassigned to phenolic C=O groups formed as a result of the simultaneousoxidation of hydroxyl groups and reduction of Ag+ ionsto the Ag0 form. On the other hand, a small peak locatedat a wavelength of 386 cm–1 on the FTIR spectrumof the sample after nanoparticle synthesis is considered to identifythe formation of silver metal. This is in line with previously recordedFTIR spectra for nanoparticles synthesized using tea extracts by otherauthors.76The FTIR spectrum measured forthe green tea-mediated AgNPs sampleis marked with the red line 8. Detail+ ions confirmed by FTIR analyses can play a doublerole: act as reducing agents in the synthesis of AgNPs and also stabilizethem.Additionally, it should be underlined thatthe obtained FTIR resultsare in agreement with our earlier predictions and suggest that polyphenoliccompounds determined by HPLC: EGCG and ECG and their interactionswith AgThe general scheme below 9 include95A predicted scenario of changes in the functionalgroups of thetwo main components determined in the green tea leaf extract due toa specific mechanism of reaction with silver ions is proposed in + ions to nanosilver. The presence of silver ions adjacent to polyphenolsconsidered to be important reducing agents: EGCG and ECG leads tobreaking the existing O–H bonds in hydroxyl groups attachedto the aromatic ring belonging to the catechol fragment of biomolecules.As a consequence, an enol intermediate is formed, and then silverions can be bound. Due to the low stability of this compound, thebond formed can be easily broken, which will occur with the simultaneoustransfer of an electron to the metal ion, which is associated withits reduction to the Ag(0) form. This is evidenced by the presenceof nanoparticles observed with TEM images. The reduction of metalions also entails the formation of keto groups due to the oxidationof hydroxyl groups.It can be seen that the mechanism presented aboveassumes the existenceof two main stages, leading finally to the reduction of AgAs mentioned before, the biomolecules presentin the extract alsohave the function of controlling the stability of nanoparticles throughinteractions occurring on their surface. It was confirmed using FTIRspectra. A predicted schematic image of such stabilization is shownin The stabilization process is important from thepoint of view ofavoiding the formation of aggregates as a result of covering the surfaceof nanoparticles with enol forms of biomolecules established in placeof forms containing initially hydroxyl functional groups and, as aconsequence, the appearance of suitable interactions between themand active sites on the surface of AgNPs.4C. sinensis) using two types of extractants:aqueous and hydroethanolic (ethanol/water ratio = 1:1). The optimizationof the extraction based on the Box–Behnken statistical model’smatrix was successfully carried out in both cases using three factorsas independent variables: pH, time, and temperature. The ranges forthese levels were selected through single-factor experiments.In this paper, we focusedon comparing the optimization of theextraction process of polyphenolic compounds from green tea leaves(+ ions to Ag(0) form, enabled us to propose a scheme for themechanism of reduction and show the predicted course of nanoparticlestabilization. In conclusion, an ethanol/water mixture is a bettersolvent system for polyphenol extraction than water alone. However,for AgNP synthesis, it is preferable to use an aqueous extract, which,despite its lower concentration of polyphenols, does not lead to agglomeration.The aggregation of AgNPs can be attributed to the increased amountof organic macromolecules in the water–ethanol extract andchanges in the reaction mixture’s solution parameters.The total polyphenol content (TPC) was used as a surface responseand determined in milligrams of gallic acid equivalent (GAE) per gramof dried biomass. TPC values obtained during extraction in the water/ethanolsystem were higher than those in water. As a result of optimization,two models with high experimental data fit to the model predictionswere obtained. This allowed us to determine the optimal extractionconditions based on 3D response surface plots with different valuesfor both extraction systems. The optimal conditions for water extractionwere determined as follows: a temperature at 80 °C, a pH of 7.0,and a processing time of 30 min. The optimal parameters for the ethanol/watermixture extraction were the temperature at 80 °C, a pH of 5.5,and a duration time of 30 min. Both extracts were employed for synthesizingAgNPs; however, their physicochemical analysis revealed a tendencyfor partial nanoparticle aggregation in the hydroethanolic extract.It is important to note that this phenomenon does not occur when anaqueous extract is used. The differences in the bands observed throughFTIR spectra of the extract and AgNP suspension samples allowed usto identify modifications occurring in the hydroxyl functional groupsof polyphenols found in green tea leaf extract. Their final oxidationto ketone groups, being the result of the observed reduction of Ag5The main challengefor nanobiotechnologists and nanotechnologistsis the development of a laboratory and, consequently, a pilot protocolfor the synthesis of stable, durable, and nontoxic nanomaterials throughbiosynthesis procedures, including the extraction of plant componentsand then biosynthesis of NPs. Biological methods for the synthesisof NPs are now a key approach to replacing current chemical reductionmethods, which are not environmentally or human health beneficial.Due to the fact that NPs, as the main building block of nanomaterialsobtained with the assistance of plant material, often show brief stabilityin relation to shape and size. In addition, there is often a tendencyto form aggregates during storage. Therefore, it would be appropriateto focus on finding nontoxic, affordable, and green stabilizing agents.The structure of the stabilizer should be designed based on the anticipateduse of the nanomaterial and, preferably, where possible, use componentsthat occur in nature as renewable resources. Here, interest shouldbe turned toward ionic liquids, whose range of properties is wide.A major challenge is also posed by the absolutely unpredictable compositionof biomolecules contained in plants, a large number of which havenot yet been identified. Owing to the fact that the mechanism of reductionshould be studied on each plant separately, with the goal of determiningthe biomolecules responsible for the reduction and stabilization ofNPs. In order to maintain the low economic and time investment envisionedfor a process, it is planned for implementation on an industrial productionscale. It is important to direct further research toward optimizingprocesses based on statistical models. In our opinion, this trendis gaining popularity at the moment. It represents an opportunityto obtain the final reaction conditions at which performance is highestby dispensing with the need for a tedious series of experiments againsteach individual factor in favor of a few specific ones based on themost important parameters, as we mentioned in our article.Therefore,the continuation of the research presented in this publicationshould be seen in attempts to increase the stability of nanoparticlesby using suitable green stabilizers in the form of selected ionicliquids for both aqueous and ethanolic extracts, where the tendencyto aggregation is evident. It is also possible to consider an optimizationapproach for the biosynthesis of nanoparticles in the presence ofa stabilizer. Then the development of such a procedure from the pointof view of application research will provide a starting point forresearch for antimicrobial, medical, and even catalytic applications.It is important to fulfill the conditions relating to the principlesof Green Chemistry and sustainable development simultaneously.


Thymes kotschyanus, Allium hooshidaryae, and Cerasus microcarpa plants and to evaluate their antibacterial properties. The healthy samples were collected, dissected and surface-sterilized before cultured on four different selection media at 28 °C. Nine EA were isolated and identified based on morphological and molecular properties, and scanning electron micrograph analyses. Based on phylogenetic analysis, they were taxonomically grouped into four families Streptomycetaceae, Nocardiaceae, Micromonosporaceae, and Pseudonocardiaceae. Their branched aerial mycelia produced chains of cylindrical or cube or oval shaped spores with smooth or rough surfaces. Four strains; IKBG03, IKBG05, IKBG13, and IKBG17 had less than 98.65% sequence similarity to their closely related strains, which constitute them as novel species/strains. Besides, three strains; IKBG05, IKBG13, and IKBG18 were reported as endophytes for the first time. Preliminary antibacterial activity conducted on the all isolates revealed potent antibacterial effects against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. All isolates strongly inhibited the growth of at least one of the tested pathogens. Our results reveals that the test plants are novel sources for isolating a diverse group of rare and common actinobacteria that could produce a wide range of novel biologically active natural products with antibacterial activity which have a great potential in pharmaceutical and biotechnological applications.The arbitrary and overuses of antibiotics have resulted in the emergence of multidrug resistance bacteria which encounters human to a serious public health problem. Thus, there is an ever-increasing demand for discovery of novel effective antibiotics with new modes of function against resistant pathogens. Endophytic actinobacteria (EA) have currently been considered as one of the most prospective group of microorganisms for discovery of therapeutic agents. This study aimed to isolate EA from  This, in addition to genomic mutations, translocations, and genetic alterations, has led to the development of microbial resistance to existing antibiotics. As a result, millions of people around the world, mostly children and the elderly, die each year due to increased bacterial resistance2. Due to this fact, it is indispensable to discover and develop novel and more powerful bioactive compounds and antibiotics from unexplored environments towards drugs resistant bacteria. Despite the efforts, we are still facing an increasing demand for new effective antibiotics, since microbial warfare seems to be inevitable in the near future3. Therefore, scientists have sought to identify and introduce more effective antibiotics over the past three decades4. In recent years, untapped drug sources have attracted a great deal of attention among researchers. Hence, many methods and technologies have been studied and conducted to discover new drugs5.Antibiotics are among the most widely used drugs in the worldStreptomyces, still is the biggest producer of secondary metabolites and bioactive compounds6, accounting for over 70% of commercially useful antibiotics7. In addition, many bioactive substances with anti-tumor, anti-fungal, anti-inflammatory, and anti-cancer properties have been reported from them, which introduces these microorganisms as one of the promising sources for the production of new drugs10. Microbes commonly coexist in diverse communities in nature. Endophytic actinobacteria that coexist with medicinal plants residing within their robust tissues have very beneficial and important effects on the survival and life of their hosts, which are mostly unknown12. Plants harbor novel and diverse range of actinobacteria, and have always been considered as one of the new untapped sources for isolation of EA. Isolation of actinobacteria strains from different environments will probably lead to the identification of new species with high ability to produce bioactive compounds13. Accordingly, the isolation and identification of actinobacteria have recently become a productive area of research that has consequently led to the identification of novel Actinomycete species that need to be exploited to unveil possible biosynthetic pathways and discover new bioactive natural metabolites.One of these attractive sources is microorganisms from unknown environmental origin. Natural products have been used for millennia to treat different infections. They provide us a great opportunity for discovery of therapeutic agents. The natural products of microorganisms are relatively cost-effective, affordable and justified for a variety of economic and non-economic reasons including non-toxic to host and active in low concentration, diversity in their environments, genetic potential, and cheap industrial production. Search for microorganisms in lesser-known environments such as mountainous, marine, and desert environments as well as those which coexist with medicinal plants which have been less studied due to difficulties in sampling, transport, storage, etc., can be a great reservoir for discovery new therapeutic compounds. Within microorganisms, the family of actinomycetes, especially the genus 17. Most of them are free-living microorganisms which use the remains of other organisms as saprophytes and also spend most of their lives as spores, especially in harsh environmental conditions19. Actinobacteria are prolific producers of bioactive metabolites with a diverse range of chemically structure complexity20. The actinobacteria phylum currently includes the most prospective group of microorganisms for the discovery of biologically active compounds including peptides, polyketides, macrolides, quinolones21. Actinobacteria alone produce approximately two-thirds of all know antibiotics in the market with a diverse range of biological activities namely cytotoxic, antiparasitic, antibacterial, anticancer or antiproliferative, antifungal, antimicrobial, insecticidal and immunosuppressive activities6. Actinobacteria also play an important role in the recycling and degradation organic substances from dead organisms to a depth of two meters in the soil23. They can indirectly inhibit the growth of plant pathogens through biological control or antagonistic activities, especially the production of extracellular enzymes or antibiotics24, or produce compounds, which directly promote plant growth and facilitate the absorption of soil nutrients, such as nitrogen fixation, phytohormones and siderophores production, dissolution of soil minerals, etc.25. Due to the climatic and geographical diversity of Iran, a broad spectrum of plants and subsequently endophytic microorganisms are available.Actinobacteria as one of the largest ubiquitous bacterial phylum are generally Gram-positive, having high percentage of G + C bases in their genome, mycelial growth, and spore production. They are widely distributed in both normal and extreme aquatic and terrestrial ecosystemsAllium hooshidaryae, Cerasus microcarpa, and Thymes kotschyanus Boiss & Hohen plants have generally been consumed as traditional medicine for many years due to their medicinal properties. Modern pharmacology of Allium hooshidaryae, Cerasus microcarpa, and Thymes kotschyanus indicated that the essential oils of these plants have anticancer, anti-anti-inflammatory properties. Allium hooshidaryae is a native wild plant in northwestern Iran, traditionally used as folk medicines. However, so far, no study has been published on their microbial diversity and endophytic populations. Therefore, the aims of this study were to isolate and phylogenetically identify EA from abovementioned plants and to evaluate the antibacterial effects of their extracts against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853.Having over 8,000 plants species, the Iranian ecosystem has a very high potential to become a source of unknown endophytic actinobacteria (EA) with unique metabolic profile and properties which can be widely used for discovery and development of new drugs and even genetic engineering of useful strains to improve their properties. To date, however, very little is known about the EA of the native plants in Iran. Therefore, the exploration of potential actinobacteria from untapped habitats is a step-wise approach for discovery of novel antibiotics to meet the current needs to cope with the life-threatening infections. Surface sterilization is a crucial step for the isolation of plant endophytes. The final rinse water of surface-sterilized was cultured on ISP2 medium, and no microbial colony was observed after two weeks of incubation at 28 °C which highlights the effectiveness and success of the surface-sterilization protocol in suppressing the growth of unwanted phyllospheric microorganisms.Thymes kotschyanus, Cerasus microcarpa, and Allium hooshidaryae on the media after 4–8 weeks of incubation at 28 ºC. The morphological isolation and purification of colonies were conducted onto ISP2 in order to identify the isolates. ISP2 was also used as a master plate to store colonies for further analyses. The isolates were then characterized based on their molecular properties. Most isolates had different pigmentation as demonstrated in Fig. To isolate as many actinobacteria as possible, the samples were plated simultaneously on four different media including TWYE, YECD, PDA and HVA. A total of nine different isolates recovered from different tissues of Differential morphological and cultural characteristics include spore arrangement, spore surface ornamentation, growth conditions and the colors of the aerial and substrate mycelia on ISP2 medium are summarized in Table Streptomycetaceae, Nocardiaceae, Micromonosporaceae, and Pseudonocardiaceae within class actinobacteria , followed by Cerasus microcarpa (two strains), and Allium hooshidaryae (two strains). Among nine isolates, four were isolated from TWYE, two isolates from YECD and three from HVA culture media. The result indicated that TWYE was the suitable medium for the isolation, and yielded the highest number of isolates which all belonged to Streptomyces genera. Over 50% of isolates were found to be a member of rare actinobacteria. It was documented that the incubation time seems to be more influential than the ingredients of isolation media in yielding rare genera of actinobacteria27.The isolates were distributed into four families ia Table , which iCerasus microcarpa were of stem samples, whereas the highest number of isolates from Thymes kotschyanus was from roots. The isolates of Allium hooshidaryae were from stems and leaves. All isolates showed a good sporulation after 10 days of incubation on ISP2, except IKBG18 which had poor sporulation even after 14 days  was prominent genus. Phylogenetic analysis revealed that the IKBG13 and IKBG14 formed a distinct clade with some strains of the genus Nocardia. However, both strains demonstrated distinct clade against KBG07, IKBG19 and IKBG20 strains that were most closely associated with the genus Streptomyces, isolated from the roots of Thymes kotschyanus. The strains IKBG07, IKBG17, IKBG19 and IKBG20 were found to form a unique clade that were different from other closely related strains, while IKBG03 and IKBG05 were placed in another distinct clade. The results of phylogenetic analysis also indicated that strain IKBG19 is associated with Streptomyces cyaneofuscatus under a bootstrap supported value of %100.As the Fig. Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Preliminary data revealed that all nine isolates had strong inhibitory activity against at least one pathogenic bacterium. Some of the isolates exhibited a wide spectrum of inhibitory activity against target bacteria ranging from two to all of the test organisms. IKBG19 had inhibitory effects against all target strains, while IKBG05 exhibited the weakest antibacterial activity, which was effective only against one pathogen. The inhibitory effect of isolates against P. aeruginosa was the most frequent detected antimicrobial activity. The rare identified actinobacteria  also showed antagonist activity against test bacteria. In some cases, the activity of tested extracts was comparatively greater than positive control.The antibacterial properties of isolates were screened against three different strains namely P. aeruginosa. Generally, the methanolic extracts of the isolates displayed stronger antibacterial properties against S. aureus than their ethyl acetate extracts. IKBG03, IKBG7, IKBG13, IKBG14, IKBG18, IKBG17, IKBG19, and IKBG20 had antibacterial activity against S. aureus. Most of strains had no effect on E. coli, except methanolic extracts of IKBG14 and IKBG19 showed a moderate activity in which IKBG14 had the greatest effect on E. coli. However, the ethyl acetate extracts of all isolates had no activity towards E. coli. All strains except IKBG14 exhibited antibacterial activity against P. aeruginosa. Table 28, Taechowisan et al.29. The results revealed that the isolates possess strong antibacterial activities against Gram-positive and Gram-negative bacteria, which is consistence with the findings of previous studies31. The results indicated that bacteria under the family of Streptomycetaceae presented a remarkable antagonistic activity as compared to other isolates against the test pathogens.Overall, crude methanolic extracts had greater potential antibacterial activities against the test pathogens than ethyl acetate extracts. The highest antibacterial effect of both extracts was on Thymes kotschyanus, Allium hooshidaryae, and Cerasus microcarpa. The majority of EA were isolated form the roots and stems. Primary identification was based on morphological characteristics  color system.33. Among the media used for isolation, TWYE seems to be the best medium for isolation of actinobacteria, which was in line with findings of Mahdi et al.17 who isolated three strains using the same medium. There are many reports highlighting TWYE and HVA, which are low nutrient-media, as the best media for isolating actinobacteria35 in which corroborated our funding23. This is the first study to investigate the population of EA in the target plants.The properties of mycelia and spores were also examined using light and electron scanning microscopes, and then the coloration of the colonies was determined using the ISCC-NBS systemThe strains were observed to form abundant straight or spiral branched aerial mycelium, occasionally twisted at the end, which differentiated into straight or spiral spore chains. Scanning electron micrograph results revealed that spores of isolates were detected to be large cylindrical (IKBG03) or large sausage (IKBG05) or cylindrical (IKBG07) or small oval (IKBG13 and IKBG20) or cubed (IKBG14 and IKBG19) or small cubed (IKBG17)-shaped, with indent smooth surfaces, whereas IKBG18 had a chain of cylindrical- shaped spores with indent coarse surfaces.Streptomycetaceae, Nocardiaceae, Micromonosporaceae, and Pseudonocardiaceae in which Streptomycetaceae were predominant which is in accordance with the previous reports36. Over 50% of isolates were found to be a member of rare actinobacteria. The strains IKBG03, IKBG05, IKBG13, IKBG14 and IKBG18 belonged to rare actinobacteria. They included Pseudonocardia spp., Nocardia spp., and Micromonospora spp., of these, Pseudonocardia spp., and Nocardia spp., were the dominant groups. In corroboration with our results, previous studies also recorded these strains as endophytes from medicinal plants38. They have also been reported as main Actinomycetes genera in cave ecosystems39. All identified families are important for the production of antibiotics41. Numerous rare actinomycetes belonging to different genera, have been reported from cave samples as a source of antibacterial compounds during 1999 and 201842. In agreement with our study, Biranvand et al. isolated and identified endophytic bacteria in a number of medicinal plants43. Of the 23 endophytic species isolated, seven belonged to the actinobacteria class, with families Streptomycetaceae, Nocardiaceae, Thermomonosporaceae, Dietziaceae, and Micrococcaceae. To the best our knowledge, to date, there is no publication on the population, and antibacterial activity of EA from Thymes kotschyanus, Allium hooshidaryae, and Cerasus microcarpa, and this is the first report. In a study of Thymes roseus in China, researchers have isolated a total of 126 actinobacterial species belonging to 24 genera using three methods of sample preparation of which the majority are Streptomyces which is consistent with previous studies44.The 16S rRNA gene sequence analyses revealed that the isolates belong to four families 45. In the current study, the highest diversity of genera belonged to the family Streptomycetaceae. This family is the largest family of actinobacteria and has been the most frequently reported genus of Streptomyces in the numerous literatures38.According to our results, there is no significant difference among the genera recovered from different segments of target plants and different genera have the same distribution. Similar to previous studies, most isolates were obtained from roots (4 isolates), followed by stems (3 isolates) and leaves (2 isolates), respectively27. It seems that it is possible to enhance the number of isolation strains by media optimization, the proper time of incubation, increasing the scale of cultured samples, and further improved isolation media as well as using molecular and non-culture dependent techniques27.Despite of using different strategies to maximize the isolation of endophytic actinomycetes, including diverse culture media, selection of native plants, and prolonged cultivation, up to 16 weeks, the number of isolated strains was less than expected. The reason which we have recovered a relatively lower number of isolates as compared to other studies, it might likely be due to the intrinsic feature of plant species which are herbaceous, not woody plants and are more vulnerable to the surface sterilization reagents and thus suggested that these chemicals might have penetrated to the samples and destroyed the EA or they might be unculturable on the selected media. There was also contamination of fast-growing bacteria and fungi, probably endophytic fungi, which might prevent the growth of actinobacteria and this was in line with previous publication datatuberculous effects of YIM65484 strain, having close relationship to the IKBG19 isolate, which has high similarity to Streptomyces cyaneofuscatus NBRC 13062T46. Four antibacterial compounds were reported from YIM65484, isolated as an endophyte from Tripterygium wilfordii, which showed IC50 values of 18–64 µg/ml against M. tuberculosis. Based on phylogenetic analysis, the closest relative strain to IKBG07 isolate was S. californicus strain, which had 99.01% similarity in sequence. S. californicus strain ADR1 strain was isolated as an endophyte from Datura metel47. Similar to our findings, secondary metabolites of this strain exhibited substantial antibacterial activity against pathogenic bacteria such as S. aureus. Based on their closely related strains, IKBG03, IKBG07, IKBG20, IKBG14, and IKBG17 strains had previously been reported as endophytes51. While the IKBG05, IKBG13, and IKBG18 strains were isolated and identified as endophytes for the first time. On the other hand, IKBG03, IKBG05, IKBG13, and IKBG17, in terms of 16S rDNA, had less than 98.65% sequence similarity with their closest relative strains, for which they could be considered as new species52. Therefore, it is necessary to perform whole genome analyses, along with new methods such as ANI, dDDH, and MLSA to define new strains53. In addition, along with genomic characteristics, morphological, chemotaxonomic, and phenotypic analyses are necessary for accurate evaluation and confirmation of new species. Furthermore, the intact nature of the Iranian ecosystem along with very limited studies in this area can increase the odds of species diversity which in turn might result in discovery of new medicines. Therefore, more efforts are needed to isolate new strains with the ability to produce secondary metabolites and to study the demographic diversity of these areas.Zhou et al. studied anti-S. aureus, E. coli and P. aeruginosa. Both methanolic and ethyl acetate extracts of isolates exhibited a wide range of antagonistic activities against the target pathogenic bacteria, which is in agreement with the antibacterial finding reported by Gebreyohannes et al.54.The preliminary antibacterial activity of isolates was tested against three pathogenic bacteria namely S. cyaneofuscatus, extracts exhibited a wide spectrum of antibacterial activity in comparison to other isolates against the test pathogenic bacteria. Except IKBG05, all isolates had antibacterial activity against S.aureus. Meanwhile, methanolic extracts of IKBG07, IKBG14, and IKBG19 isolates showed significant activity against S. aureus, as compared to positive control which is similar to a previous report55. IKBG14 and IKBG19 were also the only strains active against E. coli, while other isolates had no significant effect against this strain. Most of isolates exhibited a strong antibacterial activity against P. aeruginosa from which IKBG18 strain showed the highest activity. In this study, rare actinobacteria rather IKBG-14 exhibited antagonist activity against test bacteria which was consistence with finding reported by Qin et al.56. However, the majority of isolates showed antagonistic activities against two test pathogens. S. aureus and P. aeruginosa were affected by most of isolates. IKBG03, IKBG13, IKBG18, IKBG19, and IKBG20 were shown a strong growth inhibitory to P. aeruginosa. IKBG05, IKBG07 and IKBG17 also had moderate activity against this pathogen strain. The results of current study and other similar studies uncover that EA are still the main source of bioactive compounds that may help to discover new drugs58. Further research on identification of all secondary metabolites produced by these strains could lead to discovery of new pharmaceutical and biotechnological leads59. The high inhibitory activity and wide antimicrobial array of tested strains suggested that the isolated EA are potential candidates for novel antimicrobial agents.Among all isolates, IKBG19, closely related strain to E. coli and P. aeruginosa, both Gram negative bacterial strains. The results clearly indicated that P. aeruginosa was highly susceptible to the tested crude extracts as compared to E. coli. The discrepancy in sensitivity could be attributed to morphological differences between these two bacteria. For example, outer membrane of E coli having lipopolysaccharide which makes the cell wall impermeable to lipophilic extracts, Lipid Transfer Proteins (LTPs) and the structure of pores and efflux pumps or it might be likely due to functionally difference in the PhoP-PhoQ systems of E. coli and P. aeruginosa. Two-component systems (TCSs) assist bacteria to rapidly recognize and adapt to changes in their environment. A large number of TCSs exist in these pathogens, which are associated in regulation of gene expression in response to environmental signals such as antibiotic exposure60. The other example of autoregulation within TCSs is the PhoQ/PhoP system in E. coli. They also likely differ in the expression of periplasmic space enzymes, degrading the antibiotics. The expression of periplasmic enzymes in the intact cell relies on the presence of hydrophilic channels in the outer membrane. The outer membrane in gram-negative bacteria makes a significant role in pathogenicity of bacteria. The features of these porins are crucial for the intrinsic level of antibiotic resistance in gram-negative bacteria. The major outer membrane porin of P. aeruginosa (OprF) carries solutes at least two times slower than that of bacteria, such as E. coli61. E. coli, was generally less susceptible to antibacterial extracts which might likely be due to their outer membrane lipopolysaccharide.The bioactivity of the isolates varied between All organic solvents were purchased from Merck  except when the supplier was mentioned, and were either of HPLC grade or the highest degree of purity. All bacterial culture media were purchased from Merck-Millipore company , Fluka , Fischer , or Sigma Aldrich  unless otherwise stated. All chemicals used for DNA extraction were purchased from Sigma-Aldrich  and SinaClon.Thymes kotschyanus, and Allium hooshidaryae were collected from mountainous areas bordering Iran and Iraq in Piranshahr city, West Azerbaijan province, Iran, ((36° 47′ 44′′ N 44° 58′ 47′′ E), and Cerasus microcarpa was collected off mountainous region of Goldareh, Nojubaran, Kermanshah province, Iran  during the period of April 2019 to May 2020. All the procedure related to plants were complied with relevant institutional, national, and international guidelines and legislations. The collection of specimens was done randomly from the wild under the Research Center of Agriculture and Natural Resources of Kermanshah (RANK) permit, provided by the governments. The plant specimens were then identified and authenticated by experts at the RANK and Research Center of Agricultural and Natural Resources of Kurdistan Sanandj (HKS) according to the Flora of Iran and Flora Iranica. Thymes kotschyanus and Allium hooshidaryae were identified by a botanist (Mr. Hiva Ghaderi), and their voucher specimens 6639 and 8858 were then deposited in the herbarium of HKS, Iran, respectively. Cerasus microcarpa was identified by Dr Nastaran Jalilian, a botanist at Kermanshah Agricultural and Natural Resources Research and Education Centre, Kermanshah, Iran, and a voucher number  was deposited at the herbarium of RANK. All plant samples were packed in sterilized polythene bags and transferred to the laboratory for the surface sterilization and isolation of EA within 24 h. The plants were dissected to stems, leaves, roots, and bulbs, surface-sterilized and cultured within 48 h of sampling.The healthy plant specimens of 31 and Mahdi et al17. Briefly, the samples were first washed several times with running tap water to remove all contaminants including soil, sand, epiphytic and rhizospheric microorganisms. They were then cut into small pieces of one to two centimeters with a sterile scalpel and subjected to surface sterilization and validation as reported by27. First, the samples were immersed in 1% Tween-20 solution for 5 min, then in 70% ethanol for 5 min, followed by 5 min washing in 6% NaOCl and then washed 5 times with sterile distilled water to eliminate the chemicals. Finally, the tissues were immersed in sterile 10% NaHCO3 for 10 min to delay the growth of endophytic fungi and washed three times with sterile double distilled water. Upon sterilization, the samples were air-dried in a laminar air flow.The surface sterilization of plant samples was conducted according to the methods described by Ali et al37. The pH of all media was adjusted to 7.2. The surface-sterilized samples were then aseptically dissected into 1–2 cm and placed directly on the culture media in triplicate and the petri-plates incubated at 28 ± 2 °C, and the growth of bacterial samples was monitored over 16 weeks62. The isolation media were supplemented with nalidixic acid (10 µg/ml) and benomyl (25 µg/ml) to inhibit the growth of gram-negative bacteria and fungi, respectively58. The growth of actinobacteria colonies was regularly tracked by their morphological traits6. By emerging of each colony, the putative isolate with the typical morphology of actinobacteria colonies was picked out from isolation media, and transferred and inoculated onto ISP2 medium and purified. Pure cultures were obtained after two to three successive sub-culturing rounds and transferred to the fresh isolation media. Colonies were purified through repeated streaking onto half-strength potato dextrose agar  plates. The pure isolated strains were then maintained on ISP2 cultures and used as master plates to establish stock cultures and the isolates stored in Tryptic Soybean Broth medium (TSB) containing 20% glycerol at − 20°C for further analysis.The sterilized tissues were imprinted on standard culture media namely TWYE, YECD, HVA, and PDA to isolate EA Table 37. The p37.To ensure the efficacy of the surface sterilization, 100 μL of the last double distilled water was cultured onto ISP-2 media and incubated at 28 ± 2 °C for one week. No growth of any bacteria or fungi was considered as effective surface sterilization67.The isolates were sub-cultured onto ISP2 for 10–14 days at 28 °C, and monitored daily. The color of aerial and matrix hyphae and spores was ascertained as compared with the color chart from the ISCC-NBS. Morphological characteristics of strains were studied. The colonies were classified based on their macroscopic, morphological and cultural characteristics including mycelia, the color of culture and diffusible pigments. Microscopic monitoring of stained (Gram staining) and unstained (wet mount) bacteria was further applied for identification. Morphological differences of isolates were recorded according to color of aerial and substrate mycelia, the presence or absence of spores, spore shape, spore chain morphology, pigment or melanin production of colony and mycelium color and growth characteristics after 10 days of incubation onto ISP-2 medium, following the general guidelines of the International Streptomyces Project17. The isolates were cultured onto ISP2, the aerial mycelia and spores were obtained on a coverslip. Briefly, sterile coverslips were inserted into the ISP2 solidified medium plate at an inclination of 45° with the agar surface. The isolates were inoculated along the surface of the medium which meets the surface of the buried coverslip36, and incubated at 28 °C for ten days. The coverslips were then removed using sterile forceps and specimens were then splutter-coated with a film of gold about 150–200 A° thickness under vacuum, and then viewed on the SEM at an accelerating voltage of 25.000–30.000 V. The mycelium and spore surface structures, spore chain morphology and ornamentation of the potential strains were analyzed.The isolates were then studied by scanning electron microscopy (SEM)  according to the methods described by Mahdi et al.31. Biomass for molecular analysis was obtained by cultivation of the strains in TSB. Briefly, pure isolates, scrapped from ISP2 culture, were inoculated into TSB medium and incubated at 140 rpm in a rotary shaker  for 7 days at 28 ± 2 °C. After growth, the mycelium was centrifugated using Hettich 320R, Germany  to separate the supernatant. The biomass were then used to extract DNA or study their antimicrobial activities The Genomic DNA was extracted according to Ali et al.34. For each isolate, 70–100 mg of bacterial pellet was weighed and washed twice with 500 μL of Tris–EDTA  by vortex and centrifuging . After that, 500 µL TE buffer was added to each sample and then 0.2 mg ml−1 of Lysozyme enzyme added to each microtube and incubated for 60 min at 37 °C. Subsequently, 10 μL of 1% (w/v) proteinase K enzyme and 10 μL of 10% sodium dodecyl sulfate was added, and the mixed solution was incubated at 55 °C for 60 min. Afterwards, 100 µL 5M NaCl and 65 µL CTAB (0.27 M) /NaCl (0.7 M) were added to the microtubes and incubated for 10 min at 55 ℃, and after a short vortex spined down. The sample was then extracted with an equal volume of chloroform-isoamyl alcohol (24:1) (600 µL) and incubated for 30 min at room temperature followed by centrifugation using Hermle Z216M, Germany . The aqueous phase was carefully transferred to a new microtube and washed with 500 µL chloroform. After 15 min of incubation at room temperature, the lysate was centrifuged  and the aqueous phase transferred to a new microtube followed by adding 0.4 mg mL−1 of RNase and incubation at 37 ℃ for 60 min. Washing with chloroform was repeated. The mixture was then treated with 1X volume of 3M sodium acetate and 3X volume of 99% cold ethanol and kept in a freezer at – 20 ℃ overnight. After centrifugation at 16,000 RCF for 15 min, the supernatant was discarded and the DNA precipitated, the pellet was then washed twice with 70% ethanol. The dried pellet was then resuspended in 50 µl of sterile H2O.The quality and quantity of extracted genomic DNA were checked at A260nm/280 nm and A260 nm/230 nm ratios by Nanodrop 2000 spectrophotometer , and on 1% agarose gel and stored at −20 ℃ until further use.Genomic DNA extraction and PCR amplification of the 16S rDNA of strains were conducted based on Mahdi et al. and Ali et al.68. Polymerase chain reaction (PCR) was carried out in a total reaction volume of 25 μL as described by Coombs and Franco34 consisting of 1 μL of DNA template (50–100 ng), 0.3 μL of each primer (10 pmol/μL), 1 μL of dNTPs (2.5 mM each), 2.5 μL of 1 × PCR buffer, 0.75 μL MgCl2 (1.5 mM), and 0.125 μL of Taq DNA polymerase (5 U/μL), and 19.025 distillated water (D.W). PCR conditions included an initial denaturation at 94 °C for two min, followed by 30 cycles of denaturation at 94 °C for one min, 50 °C for one min, and 72 °C for two min and a final extension at 72 °C for 10 min and then cooled to 4 °C. The PCR products were then separated and confirmed by gel electrophoresis and UV-transilluminator. The 1.5 Kb amplified 16S rDNA fragments was monitored on 1% agarose gel electrophoresis and visualized using a Quantum-ST4, GelDoc (France). After determining the appropriate concentration of DNA using , the PCR products were sequenced by Macrogen Inc., on an Abi3130 DNA sequencer .The isolates were subjected to 16S rDNA gene sequence analysis. The 16S rDNA was amplified using Mastercycler® nexus, Eppendorf. The 16s RNA gene of isolates was amplified using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGGGTACCTTGTTACGACTT), synthesized by Metabion, Germany. The 16S rDNA gene sequences were achieved by the BLASTn searches program and then compared with existing sequences of bacteria available in the National Center for Biotechnology Information (NCBI) and EzBioCloud databases to determine similarity percentages70. Multiple DNA sequence alignment of selected 16S rDNA was performed using the ClustalW (version 1.83) algorithm in the MEGA X software71. The phylogenetic tree was constructed by MEGA X using maximum- likelihood method based on the Kimura 2-parameter model72. Pairwise distances for the neighbor-joining algorithm were calculated based on the Kimura two-parameter model (Kimura 1980). Strength and reliability (topologies of the neighbor- joining) of the resultant tree was assessed after a 1000 bootstrap-replicate analysis73. Escherichia coli was used as an outgroup strain for tree constructions74. The 16S rDNA gene sequences of the isolates were then deposited in GenBank and their accession numbers acquired. The phylogenetic tree was constructed using the neighbor-joining algorithm/method (based on 1000 bootstrap iterations) of nucleotides sequence of 16S rDNA gene.The closely related strains to isolates were identified through phylogenetic analyses. The taxonomy characterization of isolates was according to 16S rDNA analysisThe pure isolates were grown on TSA medium . The full plate containing the culture and the agar medium was then cut into small pieces with a sterile scalpel, and placed into 250 ml Erlenmeyer flasks containing 50 mL of ethyl acetate, and put on a shaker for 24h (200 RPM) at room temperature. The ethyl acetate solution was filtered and the supernatant collected. Then, 50 mL of methanol was added into the flasks, allowing the device to operate for another 24 h under the same conditions, the extract was separated. Both extracts were concentrated under reduced pressure using a rotary evaporator  to yield a dry extract. Then, the extracts were stored at – 20 °C until used for evaluation of antimicrobial activity.Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, using a typical agar diffusion assay75. The test organisms were grown in tryptone soy broth  and incubated at 37 °C for 18–24 h. The growth of the cultures was evaluated by measuring the optical density (OD) using a Spekol 1500, spectrophotometer at 600 nm (OD600 nm), and OD was adjusted to 0.2. The antibiotic agar medium No.1 (AAM- MERCK) was seeded with the test culture (1% V/V) and dispensed into petri dish plates, and 6 mm diameter wells were prepared using a sterile cork borer at regular intervals to make 10 wells. Each well was then filled with 50 μL of extracts, allowing the wells to dry fully, and the plates were incubated at 37 °C for 24h. Vancomycin (25 µg/ml) and Imipenem (30 µg/ml P. aeruginosa ATCC 27853 and 1.5 µg/ml E. coli ATCC 25922) were used as positive controls for Gram-positive and Gram-negative bacteria, respectively, with ethyl acetate and methanol as negative controls. Each test was conducted in duplicates and repeated twice. The diameters of the zone of inhibition around each well were then compared with the negative and positive controls.The antibacterial potential of the extracts was evaluated against Thymes kotschyanus, Allium hooshidaryae, and Cerasus microcarpa were selected to identify the population of EA. A total of nine EA was isolated and identified from different tissues of these plants. Most isolates gave different pigmentation. The well-developed, branched substrate hyphae and aerial mycelia differentiate into straight or spiral spore chains composed of cylindrical or cube or oval spores with smooth or indented or rough surfaces. While Streptomyces was the most prevalent genus among isolates and have a cosmopolitan distribution, several isolates that show bioactivity belonged to rare actinomycetes. The data corroborated the results reported by44 who isolated a number of Streptomyces from Thymus roseus. Based on 16S rDNA sequence and phylogenetic analyses, the IKBG03, IKBG05 IKBG13, and IKBG17 isolates could be considered as potentially novel species; therefore, more genomic studies and biosynthetic gene clusters analyses of the active strains and polyphasic study into novel strains are needed. Antibiotic resistance and nosocomial infections are a global health problem and one of the major causes of death among hospitalized patients. Therefore; to fight against the bacterial resistance, it is a clear need to develop new antibiotics, particularly against Gram-negative bacteria which have some superior features such as a second polar outer membrane and numerous efflux pumps compared to Gram-positive bacteria that render these bacteria less susceptible to antibiotic treatments. Natural products have generally had crucial roles in improving human health and wellbeing, namely their application in microbial remedies and cancer prevention and treatment regimens. Our study revealed that plants are rich sources for the isolation and identification of EA with a potential to produce a vast array of bioactive metabolites which have a wide range of pharmaceutical and biotechnological applications. This study, for the first time, the plants To increase the number of identities, it seems that media with low levels of nutrients are more suitable to isolate actinobacteria and result in the isolation of a large number of actinobacteria or it might use the unculturable methods such as molecular techniques for identification of isolates to increase the number.All strains exhibited substantial antibacterial effects against at least one of the test pathogens, indicating the potential of these strains to produce secondary metabolites. This study provides a valuable snapshot of the antibacterial potentials of EA from untapped native plants in Iran, having activity against both Gram-positive and Gram-negative bacteria. To the best of our knowledge, this is the first report on antibacterial property of novel strains from these plants. The IKBG18 and IKBG20 strains showed a potent antibacterial activity. 75. Further research is necessary to evaluate the novelty of molecules responsible for the preliminary bioactivity. Further chemical study is also required to purify and elucidate the structure of bioactive compounds from extracts with antibacterial activity in order to develop putative drugs against resistant bacteria. Previous reports have insinuated that the derived actinomycetes bioactive compounds are of alkaloids, macrolides, phenolic, diketopiperazine (DKP), peptides, polyketides, xiamycins, dilactones, salinosporamides, indole alkaloids, 2-pyranones, sesquiterpenes, and phenazines76. Therefore, it is vital to perform bioassay-guided fractionation and purification researches on the extracts to explore the structure of pure compounds. Besides, the mechanism of action of bioactive compounds must be scrutinized before they can be used as therapeutic agents. This research topic expands our knowledge on the actinobacteria- derived bioactive compounds and their biological potential applications. We may need to use different production media to induce silent cluster genes as previous studies highlighted the importance of media for production of bioactive compoundsSupplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.