Enterococcus thailandicus, Pseudomonas putida, Marinobacter hydrocarbonoclasticus, and P. geniculata for Copper (Cu), Iron (Fe), Cobalt (Co) and Zinc (Zn) Nanoparticles (NPs) production sequentially. Nitrate reductase enzyme was assayed for bacterial isolates which demonstrated that P. putida, and M. hydrocarbonoclasticus have the maximum enzyme production. The produced NPs were characterized by using XRD, TEM, UV–VIS spectroscopy. Magnetic properties for all selected metals NPs were measured using Vibrating Sample Magnetometer (VSM) and demonstrated that FeNPs recorded the highest magnetization value. The antibacterial activity of selected metals NPs was tested against some phytopathogenic bacteria causing the following diseases: soft rot , blackleg (Pectobacterium atrosepticum and Dickeya solani), brown rot , fire blight (Erwinia amylovora) and crown gall (Agrobacterium tumefaciens). All metals NPs showed an antagonistic effect against the tested isolates, particularly, FeNPs showed the highest antibacterial activity followed by CuNPs, and ZnNPs. Due to the small size, high reactivity, and large surface area of biologically synthesized NPs, they are used as a good disinfector, and can be considered as a new and alternative approach to traditional disease management methods.Bacterial metal reducers were isolated from water samples collected from harsh condition locations in Egypt. Four selected isolates were identified as  There are many ways to control bacterial plant diseases such as antibiotics; due to the natural development of bacterial resistance2 often antibiotics are ineffective in disease management3. Using chemical pesticides cause a hazardous effect on the environment, animals, and human health4. Nanotechnology as a new approach was applied to the development of novel antibacterial agents for the management of phytopathogenic bacteria affecting crops. The reduction of macro materials into Nanoscale particles (1–100 nm) gives hope to new characteristics and the material behaves differently5.Plants are exposed to infections by a large spectrum of over 200 pathogenic bacterial species that cause diseases that can lead to damage and death6, physical7, and biological9 processes are currently widely used to synthesize metallic nanoparticles (NPs) for use as antibacterial agents. Many biological systems, can convert inorganic metal ions into metal NPs via the reductive capacities of the bioproduct of these organisms. The low cost of cultivation, low energy requirements, short production time, safety, and the ability to up production volumes and eco-compatibility make biological synthesis an attractive platform for nanoparticle synthesis alternative to physical and chemical preparation10.Different chemicals11, X-ray diffractometry12, Raman scattering14, Energy-Dispersive X-Ray15, Transmission electron microscopy16, vibrating sample magnetometer17, Particle size analysis18, and inductively coupled plasma-optical emission spectrometry19.The physicochemical properties of NPs are important for their behaviour, bio-distribution, safety, and efficacy. Therefore, the characterization of NPs is important to evaluate the functional aspects of the synthesized particles. Characterization is performed using a variety of analytical techniques, including UV- VIS spectroscopyThe objectives of this study were a biological synthesis of different types of metallic NPs using eco-friendly bacterial isolates obtained from the Egyptian ecosystem, production, and characterization of these metals NPs from bacterial isolates, as well as, studying their effects on some phytopathogenic bacteria in vitro.3+, Zn2+, Cu2+, and highly toxic heavy metals like cadmium (Cd2+) and Pb2+ as well. Sample A1 contained 19 mg/L lead (Pb2+) and 25 mg/L Cd2+, sample A2 contained 5.1 mg/L Cu2+ and 3.4 mg/L Fe3+, and also sample A5, A6, A7, and A8 contained traces of Pb2+ and Cd2+. Sample A5 and A6 showed the highest pH values 8.6 and 8.1 respectively. Besides, sample A1 recorded the highest nitrate and salinity concentration 87 mg/L, 95.1 psu.The experimental data on the chemical parameters of the collected water samples are presented in Table 3+, Cu2+, Co2+or Zn2+). After 6 days incubation, results indicated that samples A1, A4, A7, and A8 contained metal-reducing bacteria, as shown in Fig. Table 3+, Cu2+, Co2+, and Zn2+ nitrate respectively. Figure Nitrate reductase enzyme was produced by selected isolates T65, E12, M69, and G80 grown on FePseudomonas, Enterococcus, and Marinobacter spp.Data on morphological, physiological, and biochemical characterization of selected bacterial isolates T65, E12, M69, and G80 (S1) showed that isolates belonged to www.ncbi.nlm.nih.gov.The resulting partial DNA sequences of the 4 bacterial isolates were analyzed using the BLAST tool in NCBI  P. putida, E. thailandicus, M. hydrocarbonoclasticus, and P. geniculata. The bacterial isolates were illustrated with the Gene Bank accession numbers in S2.Obtained data analyses revealed that inferred 16S rRNA sequences of isolates showed similarity to E. thailandicus KY968665.1, isolate M69 showed the highest similarity (99.0%) with M. hydrocarbonoclasticus JQ045804.1, as well isolate T65 showed the highest similarity (98.0%) with P. putida KU860101.1, also, the isolate G80 showed the highest similarity (99.0%) with P. geniculata KU550146. The phylogenetic trees of selected isolates were related to isolates from the gene bank  with ank Fig. .Figure 33.The reduction of metals ions to metals NPs could be optically appreciated after incubation of the bacterial culture by color changes of the medium from yellow to dark brown/black as illustrated in SThe UV–VIS spectroscopic analysis of synthesized metals NPs was shown in Fig. . UV–VIS P. putida , followed by P. putida that can accumulate all amount of Fe3+ from the culture medium within 4 days (S4A), while isolates E. thailandicus and M. hydrocarbonoclasticus can accumulate Cu2+ (S4B) and Co2+ (S4C) within 5 and 6 days sequentially.The bacterial bio-sorption and reduction of metals capability was assayed by ICP-OES. Determination of Residual Metal was measured in cell-free supernatant compared with the control medium supplemented with metals. The ability of the selected bacterial isolates to accumulate metals intracellularly is shown in S (4). Bacterial isolate Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, and Dickeya solani respectively, while Ralstonia solanacearum showed the smallest means of IZ 26.55 mm. FeNPs at the concentration of 300 µg/mL had the highest mean value of IZ (42.93 mm) followed by 200 µg/mL (39.05 mm) comparing with other tested concentrations.The antibacterial effect of metallic NPs was determined by the well diffusion method. The mean value of IZ is reported in Table P. carotovorum subsp. carotovorum, D. solani and Erwinia amylovora were 36. 38, 34.13, and 32.69 mm sequentially, followed by R. solanacearum (30.36 mm), while the smallest mean of IZ (24.58 mm) was against A. tumefaciens. CuNPS concentration of 500 µg/mL gave the highest mean of IZ (38.70 mm) followed by 300 µg/mL (36.96 mm).IZ mean values recorded for CuNPs against D. solani, and the smallest means of IZ  against E. amylovora and Pectobacterium atrosepticum. CoNPs at the concentration 500 µg/mL gave the highest mean of IZ (33.50 mm) followed by 300 µg/mL (30.19 mm).CoNPs and ZnNPs exhibited a lower antibacterial effect than FeNPs and CuNPs. CoNPs showed the highest IZ mean, 26.76 mm, against D. solani showed the highest mean of IZ (18.71 mm), while the lowest mean of IZ (16.85 mm) was the one against P. atrosepticum. ZnNPs at the concentration of 1000 µg/mL gave the highest mean of IZ (23.41 mm), followed by the concentration of 700 µg/mL (20.13 mm).ZnNPs versus P. carotovorum subsp. carotovorum, E. colacae, R. solanacearum, E. amylovora at 200 µg/mL and the growth of P. atrosepticum, and A. tumefaciens at 400 µg/mL. MBC values of 50, 100, 200 µg/mL were recorded for FeNPs, CuNPs, CoNPs against D. solani, P. carotovorum subsp. carotovorum, and R. solanacearum. ZnNPs MBC values were 200 µg/mL against P. carotovorum subsp. carotovorum, D. solani, and E. cloacae, 400 µg/mL against R. solanacearum and E. amylovora and 800 µg/mL against P. atrosepticum and A. tumefaciens.To investigate the growth inhibition effect of NPs quantitatively against various plant bacterial pathogens, the MIC and MBC were measured and results are reported in Table 3+, Zn2+, Cu2+, and highly toxic heavy metals like Cd2+ and Pb2+. Nano-metals forming bacteria that could be adapted to detoxify these heavy metals were isolated from collected samples. Different studies reported that microorganisms have developed the capabilities to protect themselves from heavy metal toxicity by various mechanisms such as adsorption, uptake, methylation, oxidation, and reduction20.Water samples were collected from different locations in Egypt, industrial wastewater, seawater, wastewater, and lake water. The chemical analysis demonstrated that samples contained inorganic pollutants such as Fe.20 reported that the changes observed in the colony colors were due to chemical modification of metals when interacting with the bacteria, and not to the induction of real pigmentation. Zaki et al.21 reported that color change in Stenotrophomonas rhizophila grown at silver nitrate indicates to reduction reaction of AgNo3 and formation of AgNPs. The present study concluded that all selected bacterial isolates could be able to produce nitrate reductase enzyme. The nitrate reductase was reported to be responsible for nanoparticles (NPs) production especially AgNPs23.Metal-reducing bacteria were isolated with dark brown colony or dark zone around bacterial growth. Lima de Silva et alPseudomonas spp. and the other two isolates belonged to Enterococcus sp. and Marinobacter sp. Molecular approaches, i.e. 16S rRNA sequencing and phylogenetic analysis of selected isolates, revealed similarity to P. putida, E. thailandicus, M. hydrocarbonoclasticus, and P. geniculata. The literature reported the use of P. putida for bioremediation, due to its ability to degrade organic solvents such as toluene24, and for silver and selenium NPs synthesis25. Tang et al.26 reported that P. geniculata was capable of efficiently degrading nicotine. E. thailandicus was first isolated from fermented sausage in Thailand in 2008 and can produce L-lactic acid27. On the other hand, M. hydrocarbonoclasticus which use nitrate (NO3−) or nitrite (NO2−) as the terminal electron acceptor to form gas product such as N2O, was first isolated near a petroleum refinery in the Mediterranean Sea.The morphological, biochemical, and physiological behavior of 4 tested isolates confirmed that two isolates belonged to 28. Present data showed the absorption maximum peak of FeNPs, CoNPs, CuNPs, and ZnNPs in agreement with the results of Zhang and Lan29, Yuvakkumar et al.30, Annapurna et al.31 and Devasenan et al.32.UV–Vis spectroscopy used for characterization of metal NPs, had proven to be a very useful technique for monitoring the signature of colloidal particles, especially for noble metal since they exhibit strong surface plasmon resonance absorption in the visible region and are highly sensitive to the surface modification33. The presence of only a single band at such wavelength ranges reflected the presence of small spherical particles according to Mie's theory34. The position and shape of the surface plasmon absorption of noble metal nanoclusters were strongly dependent on the particle size, dielectric medium and surface adsorbed species35.All UV absorption peaks in the range from 200 to 600 nm, characterized by the absence of long tailing on large wavelengths suggested the absence of aggregation between particlesP. putida was observed. Such results were in agreement with Varshney et al.36, who found that P. stutzeri synthesized silver NPs within its periplasmic space. Synthesis of CuNPs was shown in the cytoplasm space of E. thailandicus, also particles were aggregated in the center of cells by the action of proteins of cytoplasm which prevent their release to the surrounding media through the cell wall. CoNPs synthesis in the case of isolate M. hydrocarbonoclasticus appeared diffused in the cytoplasm. ZnNPs appeared in periplasmic and cytoplasmic space of P. geniculata. Iravani37 explained that the ability of bacteria to survive and grow in stressful situations might be due to specific mechanisms of resistance which include: efflux pumps, metal efflux systems, inactivation of metals, impermeability to metals, the lack of specific metal transport systems, alteration of solubility, toxicity by changes in the redox state of the metal ions, intracellular precipitation of metals, and volatilization of toxic metals by enzymatic reactions. On the other hand, Gomathy and Sabarinathan38 suggested that cellular mechanisms may be implicated in the resistance and tolerance of microorganisms to excessive concentrations of heavy metals in the environment. The strategy adopted include slow transport into the cell, detoxification or incorporation of specific metals into enzymes.Data obtained from TEM concluded that the selected isolates were able to detoxify and reduce metallic ions to nanoscale particles. FeNPs in the cytoplasm and periplasmic space of .39 reported the process that involves the reduction of metals ions. The first step involves the interaction between these metal ions with nitrate reductase present inside the cell (periplasmic and cytoplasmic membrane) and their bioreduction to metallic form. The accumulation of intracellular NPs was reported in different studies like the synthesis of silver NPs by Stenotrophomonas rhizophilia21, E. coli, palladium NPs by Desulfovibrio desulfuricans and Bacillus benzeovorans40, and AuNPs by Lactobacillus kimchicus41.Deepak et al, based on TEM agreed with previous studies for FeNPs in the range of 1–4 nm42, CuNPs in the range 8–14 nm43, CoNPs in the range 9–20 nm and ZnNPs in the range 4–13 nm44. Also, in agreement with previous studies the PSA analysis showed that the range size distribution of FeNPs was 1–3 nm45, CuNPs in range 8–18 nm46, CoNPs in range 8–22 nm47 and ZnNPs in range 4–16 nm48.Results of characterization of size and morphology of metals NPs49. The XRD patterns correspond to FeNPs diffractogram, in which the characteristic peak of highest intensity occurred at 45.50° correspond to (111), crystallographic planes of face-centered cubic iron zerovalent crystals50. Two main characteristic diffraction peaks for CuNPs were observed at around 2θ = 42°, 50° which correspond to the (111), (200) crystallographic planes of face-centered cubic Cu2+ phase (JCPDS No.04-0784)51. The XRD patterns in of CoNPs show the diffraction peaks at around 43.2°, 54.5°, and 77.9° could be assigned to the (111), (200), and (220) planes of the cubic Co nanocrystal52. ZnNPs showed that all the diffraction peaks could be well indexed to the characteristics of the cubic of ZnNPs. The sharp and narrow diffraction peaks appeared at about 2θ of 43, 56 and 74.83°, and were assigned to (111), (200), and (220) plane values of ZnNPs. This diffraction pattern has corresponded to pure Zn Nanopowder. A small peak is also observed at around 36° indicated that a small amount of zinc was oxidized and converted into zinc oxide32. The XRD pattern of all metallic NPs indicated that the metals NPs had a spherical structure with a small size. The obtained results illustrated that metal ions had indeed been reduced to Nano-form by reducing enzyme of bacterial isolates and prof the ability of selected bacterial isolates to produce metals NPs9.Patterns of XRD were analyzed to determine peak intensity, position, and width. The diffraction peaks of all NPs appeared sharp, and clearly distinguishable, which indicates the ultra-fine nature and small crystallite size. The XRD spectrum contained no other phase, indicating the purity of the sample−1. Raman region of interest in this investigation was around 200–1100 cm-1, and it was confirmed to Fe zerovalent Fe0 NPs peaks. These results were matched with Dong et al.50. Besides, results of Raman spectra of CuNPs with peak absorptions around 500 cm−1, 1250 cm−1, and 1500 cm−1 indicated that the sample contains Cu0 NPs53. Raman spectra showed absorptions peak around 500 cm−1 and 1000 cm−1 and it corresponded with the Raman shift pattern of the CoNPs52. In addition to Raman spectra with 2 strong peaks around 300–500 cm−1 and 1400 cm−1, these peaks characteristics were identical to peaks of ZnNPs54. All Raman spectra of samples indicated the high quality of metal NPs, as well as, these results were compatible with XRD analyses.Raman spectroscopy is especially useful if minerals are to be identified that are poorly defined or that cannot easily be distinguished using other methods such as XRD. In this study, Raman spectra of metal NPs showed that the 3 Raman spectra of FeNPs had mainly 3 bands observed around 200, 290, and 1100 cmP. putida isolate was 46%, the content of Cu in E. thailandicus was 47%, which was coherent with the results by Eltarahony et al.55. Content of Co in M. hydrocarbonoclasticus was 41%, and Zn in P. geniculata was 59%, in agreement with finding by Eltarahony et al.56. These results fixed that all bacterial isolates could accumulate and reduce the high amount of metal for NPs intracellular synthesis. The presence of other metals might be referred to some elements in bacterial cell structure and biomolecules such as DNA, ATP, RNA, and amino acids19.Analysis using EDX indicated that the content of Fe in 57, with no hysteresis. The measured saturation magnetization value around 36.9 emu/g. The magnetic characterization of CuNPs indicated low magnetic behavior with a saturation magnetization value of around 1.3 emu/g without any hysteresis58. Also, magnetization curves of CoNPs were very low around 0.2–0.3 emu/g, with a soft hysteresis loop59. In addition to the magnetization curve of ZnNPs with saturation magnetization had a value of around 5 emu/g60. From these results, we concluded that Fe, Cu, and Zn were paramagnetic NPs, and Co was ferro-magnetic NPs.The magnetic properties of the metal NPs have been investigated using a VSM. The plots of the FeNPs indicate a superparamagnetic behavior at room temperature which is compatible with Eivari et al.P. putida could accumulate all amount of Fe3+ from the culture medium within 4 days, as well P. geniculata could accumulate all amount of Zn2+ within 2 days, and it might be referring to the ability degree of isolates to detoxification of metals and the toxicity level of metals like Cu2+ and Co2+. Different studies reported the importance of Fe3+ and Zn2+ to bacterial cells and their role in different biological pathways. Ahmad et al.61 reported that Zn2+ plays a significant role in cellular division and protein synthesis contributes to carbohydrate, lipid, and nucleic acid metabolism. Fe3+ participates in a large number of cellular processes, the most important of which were oxygen transport, ATP generation, cell growth, proliferation, and detoxification. It was a coenzyme or enzyme activator of ribonucleotide reductase, a key enzyme for DNA synthesis, which catalyzes the conversion of ribonucleotides to deoxyribonucleotides and particularly of deoxyuridine to thymidine. ICP-OES results were compatible and confirm ability of selected isolates to reduce and accumulate metals19.Determination of residual metal concentration using ICP-OES demonstrated that selected bacterial isolates could detoxificate and accumulates metals intracellular. , CuNPs and CoNPs increase the antibacterial effect and this effect significantly decreased with high concentrations. Rout et al.62 declared that size of the inhibition zone increased significantly with decreasing the size of the NPs. It is reasonable to state that binding of the NPs to the bacteria depends on the surface available for interaction. They added that smaller and monodispersed particles having the larger surface area available for interaction would give more of a biocidal effect than the aggregated and larger particles. Moreover, present data revealed that the antibacterial potential increased with an increase in the concentration of ZnNPs.The antibacterial effect of metal NPs against some phytopathogenic bacteria noted that in case of low concentrations of FeNPs63 reported the inactivation of E. coli by zero-valent Fe, furthermore, NPs leads to the production of reactive oxygen species (ROS), resulting in the generation of hydroxyl radicals (OH−) from superoxide (O2−) and hydrogen peroxide (H2O2) in microbial cells. These radicals promote oxidative stress and cause cell membrane damage, which contributes to the outflow of intracellular contents and, finally, cell death due to the penetration of the small NPs (sizes ranging from 10–80 nm) into E. coli membranes64. Other NPs such as ZnO-NPs65, Aerogel-MgO-NPs66, and TiO2-NPs67 have been reported also to cause a loss of membrane integrity and leakage. Other studies on ZnO-NPs and MgO-NPs concluded that antibacterial activity increased with decreasing particle size68. Moreover, researches had demonstrated that the small size of NPs, which have characteristic dimensions < 100 nm, can contribute to the bactericidal effect. Their uniquely small size results in novel properties, like the greatest interaction with cells due to a larger surface area-to-mass ratio and multilateral and controllable application69. The antibacterial properties of CuO NPs have been investigated, which were found that able to cause protein oxidation, lipid peroxidation and DNA degradation in E. coli cells70. Khalil et al.71 reported that synthesized cobalt oxide NPs were studied for their antibacterial potential against 3 G-ve  and 3 G+ve bacterial strains . It was noted that the antibacterial potential increased with an increase in the concentration of the nanoparticles.Several studies reported the antibacterial effect of metals NPs and their antibacterial mechanisms. Lee et al., CuNPs, and CoNPs were found to be effective at 50–100 µg/mL against some phytopathogenic bacteria, likewise, 200–400 µg/mL of ZnNPs were effective. This effectiveness at lower concentrations might be referred to the small size of bacterial synthesized metal NPs. It was reported that ultrafine particle size causes its action at a lower concentration, whereas our study used NPs with a size range of 2–10 nm. A previous study by Niemirowicz et al.72 indicated that MIC and MBC of FeNPs against Staphylococcus aureus and P. aeruginosa was 64, 182 µg/mL and 128, 265 µg/mL on the relay, also Ruparelia et al.73 noted that MIC and MBC of CuNPs against E. coli was 140, 160 µg/mL. On the other hand, Aysa and Salman74 demonstrated that 3.7 µg/mL was the MIC and MBC value of ZnNPs against P. aeruginosa.In this study, FeNPsFrom this study, we concluded that biological synthesis of NPs using bacterial cells is eco-friendly, fast, and inexpensive. The toxicity of FeNPs, CuNPs, CoNPs, and ZnNPs to phytopathogenic bacteria in addition to their bactericidal effect, recommend future investigations on the toxicity and safety concentrations of metal NPs to animals and human cells. These future studies seem to be necessary to be performed, in order to precise for toxicity prevention and the applicability of metals NPs in bactericide industry.Various environmental water samples showing harsh conditions for microbial growth , containing organic and inorganic pollutants were collected from several diverse habitats , during April 2015 Table .Table 6S2−, NO3−, salinity, Fe3+, Zn, Cu2+, Pb2+, and Cd2+ were analyzed and performed according to APHA75.pH, NO76 and incubated at 30 °C for 24 h at 150 rpm in an orbital shaker. Samples were serially diluted in 0.8% saline then plated on Luria Bertani (LB) agar plates supplemented with 5 mM of either Fe (NO3)3·9H2O, Cu (NO3)2. 3H2O, Co (NO3)2·6H2O and Zn (NO3)2·6H2O (Sigma Aldrich) and incubated at 30 °C for 6 days. Plates were examined daily for dark colonies and/or with a dark zone on the agar. The obtained pure bacterial colonies, through a single colony isolation technique77, were maintained by mixing bacterial suspension in 20% glycerol and stored at − 80 °C for further studies.Each water sample (1 mL) was inoculated in nutrient broth (NB), and then taken in a series of 250 mL Erlenmeyer flasks containing 49 mL of NB. In the case of seawater samples, 1 mL of each sample was inoculated in Marine Broth (2216), isolates were grown on LB broth supplemented with 3.5 mM of Fe3+, Cu2+, Co2+, and Zn2+ nitrate separately, then cells were collected and disrupted by an Ultrasonic generator  for 5 min. Nitrate reductase activity was assayed according to Antonio et al.78.For crude enzyme determination79. For testing the ability of isolates to grow under anaerobic conditions, bacterial isolates were tested using AnaeroGen jar 2.5 liters80.Motility, Gram stain, spore formation were done for Morphological tests. Physiological tests for the effect of different pH values on growth, temperature, and NaCl tolerance as well as gelatin liquefaction was determined according to Bergey's Manual of Determinative Bacteriology81.Biochemical characterization tests were performed using diagnostics ENC 8 kit and GN 24 kit identification system for Gram-positive and Gram-negative bacteria, respectively. As well DNAase test was performed by streaking each bacterial culture on the DNAase agar plate (HIMEDIA M482) and incubated at 28 ± 2 °C for 3 days, then the plate was flooded with 0.1% toluidine blue. A clearance zone around the colonies was recorded as a positive reaction82. PCR amplification of the 16S rRNA gene was performed for 4 isolates on a "Biometra PCR Thermocycler" using one pair of primer 16S-F (27F) AGAGTTTGATCMTGGCTCAG and 16S-R(1492R) GATTACCTTGTTACGACTT according to Wang et al.83.For DNA extraction protocol from bacterial cells, an aliquot of 1 mL of AMSHAGE DNA extraction kit was added and followed the steps of Abd-El-HaleemTen μL of PCR product was loaded per gel slot. Electrophoresis was performed at 100 Volt with (0.5 x) Tris–Acetate—EDTA buffer (TAE)  as running buffer in 1.5% agarose gel cast in 0.5 × (TAE buffer). The gel was stained in (1 μL) ethidium bromide . Finally, the gel contained PCR product was visualized with a syngeneic gel documentation system.84. Partial DNA sequencing was performed for the PCR amplified 16S rRNA gene using an ABI PRISM dye terminator cycle sequencing kit with Ampli Taq DNA polymerase and an Applied Biosystems 373 DNA Sequencer .PCR products were purified using NEPRAS DNA purification kithttp://www.ncbi.nlm.nih.gov).85 Production and Extraction of metals nanoparticles (NPs) were performed according to Zaki et al.86 and Kamal et al.87 respectively.Bootstrap neighbor-joining tree was generated using MEGA version 6.1 from CLUSTALW alignment. Comparisons with sequences in the GenBank database were achieved in BLASTN searches at the National Center for Biotechnology Information (NCBI) site 89, Raman Spectroscopy90, Particle size distribution (PSA)45, Energy dispersive X-ray (EDX) spectroscopy91, Vibrating Sample Magnetometer (VSM)17 and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES)92 were carried out for characterization of metals NPs.Characterization was performed using a variety of analytical techniques. Ultraviolet–Visible spectroscopy (UV–Vis), X-ray diffraction (XRD)93. The minimum inhibitory concentration (MIC) was determined based on batch cultures containing varying concentrations of metals NPs with serial two-fold dilution in suspension (25–800 µg/mL). All the experiments were carried out in triplicate. Also, the determination of the minimum bactericidal concentration (MBC) was performed as suggested by Avadi et al.94. To test for bactericidal effect, one mL of each culture flask which was used in the MIC experiment was plated in duplicates on LB agar free of NPs and incubated at 30 °C for 48 h. NPs concentration causing the bactericidal effect was selected based on the absence of colonies on the agar plate95.The metals NPs were evaluated for antibacterial activity against 7 molecular identified phytopathogenic bacteria (Table Supplementary Information.


Berberis lycium Royle has a long history of medicinal uses to treat different diseases. It naturally grows on the mountains of Indian subcontinent. Its ethnobotanical and biochemical study from the state of Azad Jammu and Kashmir (AJ&K) was not previously explored. So, the objective of the current study was to explore the ethnobotanical and biochemical properties of the B. lycium Royle population of AJ&K. For this purpose, samples of B. lycium Royle were randomly collected from five districts of Azad Jammu and Kashmir, including thirty-five locations. Demographic features of informants such as plant part used, methods of preparation, modes of administration, conservation status, and ethnomedicinal uses were documented. It was used for treating different diseases such as diabetes, arthritis, joint pain, and stomach ulcer. This plant is very famous for providing medicinal roots, leaves, and fruits which are extensively used in many parts of the world. The biochemical analysis was conducted for total phenolic contents (TPC), chlorophyll contents, and antioxidant activity. The highest level of TPC found was 88.66 ± 1.07 µg/g of gallic acid equivalent phenolic (GAE) from leaves collected from Patikka , Muzaffarabad District, AJ&K. The highest total chlorophyll contents (3.75 ± 0.53 µg/ml) were found in samples collected from Sathrian, Neelum District. The highest antioxidant activity with lowest IC50 value (33.26 µg/ml) was obtained from the root of sample collected from Bakreyali, Muzaffarabad District, as compared with other districts. The concentration of berberine was found to be 4.76 percent in the root bark of B. lycium Royle, estimated by high-performance liquid chromatography (HPLC). In syrup composition, 0.95 mg/5 ml of berberine was used. Hence, it is concluded that amongst the five districts, the plant parts  collected from Muzaffarabad District, AJ&K, showed the highest medicinal potential due to its unique climatic conditions. Berberis lycium Royle is a high value medicinal plant with known history in folklore medicine and traditionally used to treat different human diseases  ∗ 100, where Ao is the absorbance of the control reaction and As is the absorbance of the sample itself. IC50 was calculated after plotting data and getting the linearity correlation equation.The antioxidant activities of the plant extract were measured by using the stable DPPH radical according to the method of . 0.25 mMB. lycium Royle was selected from Muzaffarabad District (Patkai) The sample was washed thoroughly to remove dirt and stain. The sample was placed in the shade for almost 20 days in order to get dry. After drying, the sample was grounded into powder form. An aqueous solution was made of the powder form. The patent was filed, Abbas Nutraceutical Product made from locally available medicinal plant B. lycium Royle.The root bark of B. lycium was carried out by using HPLC, Cat # PM 951505-0, column C-18 isocratic, 5 µm, 4.6 mm × 250 mm. The liquid chromatography used was DLC-20 by Star-Chrom Lite. The software used was also Star-Chrom. The single-beam UV-spectrophotometer by IRMECO Germany, Model #2020, was used for spectrum analysis and absorbance peak comparison. The mobile phase contains 50 mM potassium dehydrogenase phosphate and acetonitrile in 70 : 30, and pH of solution was adjusted to 3 with phosphoric acid. The flow rate was retained 1 ml/min, and the injection volume was 20 µl. The berberine quantification was done at 271 nm. The peak in the B. lycium Royle sample was observed by retention time. The analysis was conducted in triplicate, and berberine was found. The validated method for HPLC is given in [The quantification of berberine from the root extract of given in .http://www.graphpad.com.The data in this study were expressed as a mean of triplicate. Statistical comparison and one-way ANOVA followed by Dunnett's multiple comparisons test were made in Prism, version 6.00, GraphPad software for Windows, La Jolla, California, USA, B. lycium Royle in the state of AJ&K.There are 10 districts in Azad Jammu and Kashmir. Data were collected from five districts of AJ&K, i.e., Muzaffarabad, Hattian, Bagh, Poonch, and Neelum, as shown . These fB. lycium Royle. It was found that medicinal values of B. lycium Royle are difficult to maintain because of their potential uses; therefore, they are recommended for viable harvest, not being used as fire wood [In the current study, there were 35 informants  having age ranging from 22 to 75 years. Among them, 11 were housewives, 10 were teachers, 9 were shopkeepers, 3 were farmers, and 2 were lecturers. The information  was collected through questionnaire, interviews, and discussions with villagers. Our questionnaire allowed descriptive response on plant prescribed, plant part used, and detailed information on the mode of preparation . Root bark powder was used for treating different diseases in the form of paste, given orally or placed on a wound or cut directly . Ethnoboire wood . People ire wood .µg/g) and the lowest in Qadrabad (9.50 ± 0.95 µg/g). The phenolic contents of stem showed the highest value of 56.39 µg/g ± 2.01 in Medan Syedan and the lowest value of 8.18 ± 0.72 µg/g. The phenolic contents of leaves were the highest in Patikka  (88.66 ± 1.07 µg/g) and the lowest in Dhaman Jholi (8.24 ± 1.25 µg/g). The thorn showed the highest concentration of 61.22 ± 1.4 µg/g in Patikka , and 11.14 ± 1.7 µg/g was the value for Dhaman Jholi. Patikka  again showed the highest value of phenolic contents in fruits of 66.43 ± 1.09 µg/g, and Dhaman Jholi was the lowest with 10.36 ± 1.47 µg/g. TP contents varied among the plant samples collected from different locations. The variation of phenolic contents was due to environment, soil texture, and altitude differences. It could be said that phenolic content variations were the result of a plant's interaction to its environment. The change in phenols directly affects the medicinal quality of the plant [Berberis species [TP contents' amount was estimated by using Folin–Ciocâlteu's phenol reagent and is denoted in GAE . The highe plant . The mai species .µg/ml (µg/ml) in Bagh and lowest (0.24 ± 0.06 µg/ml) in Shahkot. The chlorophyll b was highest in Sathrian (2.66 µg/ml ± 0.41) and lowest in Rawalakot (0.15 ± 0.04 µg/ml). The total chlorophyll content was found highest in Sathrian (3.75 ± 0.53 µg/ml) and lowest in Sheesha Mali (0.53 ± 0.15 µg/ml). The chlorophyll content changed in different areas as well as within the same plant due to differences in leaves' reception of sunlight. It also varied in different areas due to shade and time of exposure to sunlight [Chlorophyll concentrations were calculated in different demographic regions. The values of chlorophyll a and chlorophyll b and the total amount of chlorophyll were expressed as µg/ml . The chlsunlight .µg/ml in Serli Scha and the lowest for Kaalis (36.63 µg/ml). The IC50 for root showed the highest value of 513.33 in Serli Scha and the lowest for Bakreyali (33.26 µg/ml). The IC50 for leaves showed the highest value of 481.23 µg/ml in Sarran Chatiyan and the lowest for Subhai Mali (40.82 µg/ml). Bakreyali showed 33.26 µg/ml. The IC50 for thorn showed the highest value of 474.14 µg/ml for Laala and the lowest for Bagna (8.69 µg/ml). The highest antioxidant activity was exhibited by the thorn of plants collected from Bagna, having the lowest IC50 of 8.69 µg/ml. Stem and fruits showed the highest antioxidant activity compared with other parts, while roots also exhibited good antioxidant potential. The root bark of B. lycium is the best antioxidant compared to ascorbic acid and many other plants [The DPPH method is used for the radical scavenging activity of antioxidants. DPPH is a stable organic free radical and presents the ability to accept hydrogen radical or an electron. Scavenging activity was calculated in percentage of inhibition . The IC5r plants . Antioxir plants . The antr plants .B. lycium Royle, having a concentration of 4.1% in the root bark of the plant [B. lycium Royle roots, fruits, leaves, and stem [Berberis species and is said to be a very active compound with various pharmacological properties [Berberine is a major alkaloid that is found in he plant . Berberihe plant , 16. Berand stem . Berberioperties . Berberioperties . There aoperties .B. lycium Royle, having a concentration of 4.76 percent in root bark as estimated by HPLC : vitamin A (56.75 microgram), vitamin C (1.94 mg), calcium (3.24 mg), sodium (1.68 mg), phosphorous (2.73 mg), potassium (13.1 mg), iron (0.17 mg), zinc (1.88 mg), Mn (0.0055 mg), and berberine (0.95 mg). Fruits and thorns may be used for the development of antioxidant compounds or combinations. Fruits could also be used in its raw form [B. lycium Royle such as Na, Cu, Ca, S, Fe, Zn, Mn, and Pb that is enough to prepare syrup [Syrup formation was done by adding these constituents, i.e., the root bark extract of raw form . A wide re syrup , 16. Nowre syrup .Berberis lycium Royle is under heavy pressure from human activities including overexploitation, overgrazing, deforestation, and unscientific ways of collection. An increase in human population leads to depletion of natural resources. Because of the low potential of natural regeneration, constant high use is posing a great risk to B. lycium Royle. Increased livestock is also destroying this plant as they graze its leaves and branches due to its good taste. Goats specifically like to feed on this plant. The increasing human population is destroying the habitat of the plant. Habitat degradation is also due to deforestation, overexploitation, livestock grazing, and unscientific ways of collection. Other factors contributing to this problem are fuel wood cutting, stall feeding, soil erosion, fires, lack of awareness, weak law enforcement, and smuggling. As root bark of B. lycium Royle is used for the treatment, local Hakeems are using every possible way to obtain roots, resulting damage to the population of B. lycium Royle. Because of lack of awareness, people use this plant for stall feeding their animals and fire fuel, so in recent years, it has been vanished from a big area. It is the need of the hour to take the immediate attention to conserve this species.B. lycium Royle, estimated by high-performance liquid chromatography (HPLC). In syrup composition, 0.95 mg/5 ml of berberine was used. B. lycium Royle possesses powerful antioxidant activity in its all parts, especially root, stem, and fruit. Fruits are already used as food in hilly areas of AJ&K. Another important finding from the present study is that change in the environment has a big impact on the chemical composition of the same species, so its medicinal properties get changed. Hence, it is concluded that amongst the five districts, the plant parts  collected from Muzaffarabad District, AJ&K, showed the highest medicinal potential due to its unique climatic conditions.The TPC was high in leaf samples collected from Chanjhal, Muzaffarabad District of AJ&K. The highest total chlorophyll contents were found in samples collected from Sathrian, Neelum District. The highest antioxidant activity with the lowest IC50 values was obtained from the root sample collected from Bakreyali, Muzaffarabad District, as compared with other districts. The concentration of berberine was found to be 4.76 percent in the root bark (sample collected from Muzaffarabad District) of


To compare the dosimetric, normal tissue complication probability (NTCP), secondary cancer complication probabilities (SCCP), and excess absolute risk (EAR) differences of volumetric modulated arc therapy (VMAT) and intensity-modulated radiation therapy (IMRT) for left-sided breast cancer after mastectomy.Thirty patients with left-sided breast cancer treated with post-mastectomy radiation therapy (PMRT) were randomly enrolled in this study. Both IMRT and VMAT treatment plans were created for each patient. Planning target volume (PTV) doses for the chest wall and internal mammary nodes, PTV1, and PTV of the supraclavicular nodes, PTV2, of 50 Gy were prescribed in 25 fractions. The plans were evaluated based on PTV1 and PTV2 coverage, homogeneity index (HI), conformity index, conformity number (CN), dose to organs at risk, NTCP, SCCP, EAR, number of monitors units, and beam delivery time.p < 0.001). The HI was 0.11 ± 0.01 for VMAT and 0.12 ± 0.02 for IMRT, respectively (p = 0.001). The VMAT plan had better conformity  in PTV compared with IMRT. As opposed to IMRT plans, VMAT delivered a lower mean dose to the ipsilateral lung (11.5 Gy vs 12.6 Gy) and heart (5.2 Gy vs 6.0 Gy) and significantly reduced the V5, V10, V20, V30, and V40 of the ipsilateral lung and heart; only the differences in V5 of the ipsilateral lung did not reach statistical significance (p = 0.409). Although the volume of the ipsilateral lung and heart encompassed by the 2.5 Gy isodose line (V2.5) was increased by 6.7% and 7.7% , the NTCP was decreased by 0.8% and 0.6%, and SCCP and EAR were decreased by 1.9% and 0.1% for the ipsilateral lung. No significant differences were observed in the contralateral lung/breast V2.5, V5, V10, V20, mean dose, SCCP, and EAR. Finally, VMAT reduced the number of monitor units by 31.5% and the treatment time by 71.4%, as compared with IMRT.VMAT resulted in more homogeneous chest wall coverage than did IMRT. The percent volume of PTV1 that received the prescribed dose of VMRT and IMRT was 95.9 ± 1.2% and 94.5 ± 1.6%, respectively (Compared with IMRT, VMAT is the optimal technique for PMRT patients with left-sided breast cancer due to better target coverage, a lower dose delivered, NTCP, SCCP, and EAR to the ipsilateral lung and heart, similar doses delivered to the contralateral lung and breast, fewer monitor units and a shorter delivery time. Breast cancer is the most common cancer among women. Although breast-conserving surgery for early breast cancer has become the standard treatment in European and American countries , 2, radiElectron beam or arc irradiation , 8 and tIn recent years, volumetric-modulated arc therapy (VMAT) has been applied in PMRT to improve treatment efficiency –26. AlmoIn this study, we used SmartArc optimization algorithms, Equivalent Uniform Dose (EUD) optimization parameters and virtual block to restrict the low-dose area and explored the balance between the dose to the target area and that to normal tissue in VMAT planning. We then discussed dosimetric characteristics of the targets of CW and IMN and the target of SCN, as well as the dose to the ipsilateral lung, heart, contralateral breast/lung in IMRT and VMAT plans of left-sided breast cancer PMRT. We further predicted treatment outcomes focused on the lower irradiation dose of OARs .Thirty left-sided breast cancer patients after radical post-mastectomy with a clinical-stage above T3 or N1-3  were randomly enrolled in this study. All patients were positioned supine on a commercially available breast tilt board to render the sternum parallel to the table, with both arms fully abducted (90° or greater) and externally rotated, and the head was secured. A planning CT scan at 5-mm intervals from the mastoid process to 3 cm below the right breast fold was obtained for each patient with a CT simulator .http://www.rtog.org/CoreLab/ContouringAtlases/BreastCancerAtlas.aspx). CTV1 was defined as the ipsilateral chest wall (CW) and IMN regions, and the SCN region was regarded as CTV2. PTVs were obtained from CTVs by expanding a 5-mm margin in three dimensions. An additional bolus was applied on PTV1 only, and PTV2 was 4 mm from the skin surface, excluding the build-up region. Mean volumes and standard deviation of PTV1, PTV2 and whole PTV were 367.19 ± 84.08 cm3 (range 238.19–537.88 cm3), 163.48 ± 31.09 cm3 (range 112.00–256.44 cm3) and 530.67 ± 98.02 cm3 (range 376.94–757.45 cm3), respectively. OARs, such as the ipsilateral lung, heart, contralateral breast and lung, liver, spinal cord and trachea, were outlined on the axial CT sections.The CTV  was delineated according to the breast cancer atlas for radiation therapy planning consensus definitions of the Radiation Therapy Oncology Group . A Varian TrueBeam linear accelerator with a 6-MV photon energy beam was used. SmartArc and DMPO optimization types were used to optimize the VMAT and IMRT plans, respectively, and dose calculation was performed with a collapsed cone convolution algorithm. To ensure sufficient skin dose coverage, a virtual 5-mm bolus was applied to the CW of each patient before optimization and dose calculation. VMAT treatment plans were generated by two partial arcs starting from 295° to 145° clockwise and inverse, with one control point every 4°. To reduce the low dose to the normal organs, EUD and an artificially drawn block were used to limit the lower dose during plan optimization. Seven beams were used in the IMRT plan. The prescription dose was 50 Gy in 25 fractions, and the 95% PTV received 50 Gy. The aim, starting objective and constraints of planning optimization for the VMAT and IMRT plans are specified in Table 2%, D98%, , Dmean (mean dose of PTV), V95%, V100%, V107% and V110% (percent volume receiving greater than 95% to 110% of the prescribed dose) and the dose homogeneity index (HI), conformity index (CI) and conformity number (CN) for PTV. The HI was calculated according to ICRU 83 [2%–D98%)/D50%. CI and CN were proposed by Paddick et al. [50/VPTV and CN = (VPTV50/VPTV)*(VPTV50/V50), where VPTV is the target volume, V50 is the volume of prescribed isodose value and VPTV50 is the volume of the target that is covered by the prescribed isodose value. Smaller CI and larger CN represent better dose conformity. (2) For OARs, the mean doses and a set of appropriate Vx(Gy) values to the ipsilateral lung, heart, contralateral breast and lung and normal tissue were analyzed.For dosimetric analysis, the following indices extracted from dose-volume histograms (DVHs) were used: (1) D ICRU 83 : HI =  + γa) [Lung = 7.5, βBreast = 9.2), the age modifying factors γe and γa  were taken from Schneider et al. [. The agex is the patient’s age at the time of radiation therapy, and the agea is the attained age. An attained age of 75 yrs was used for cancer risk assessments. OED is organ equivalent dose and was calculated using the linear model for the contralateral breast, ipsilateral and contralateral lungs, and whole lungs [Vt is the total organ volume, and Vi is the volume receiving dose Di. SCCP was calculated using the product of the OED and the organ-specific absolute cancer incidence rate in percent per gray (Inorg) [Inorg for the lungs and contralateral breast were 1.68% Gy−1 and 0.78% Gy−1 from a previous publication [The NTCP for radiation-induced pneumonitis was computed for the lung using the Lyman-Kutcher-Berman model using the following values: D50 = 30.80 Gy, n = 0.98 and m = 0.37 . The EAR0) + γa) , where βr et al. . The agele lungs  Vt is th (Inorg) , and theP-value ≤ 0.05 was considered the threshold for statistical significance.Statistical analyses were performed to compare the two different techniques using a paired t-test, a 107%. The average percent volume of PTV1 receiving the prescribed dose for IMRT and VMAT was 94.5 ± 1.6% and 95.9 ± 1.2% (p < 0.001), that receiving 110% of the prescription dose was 0.3 ± 0.2% for IMRT and 0.2 ± 0.1% for VMAT (p = 0.018), the near-maximum dose and minimum dose were 54.5 ± 0.3 Gy and 48.3 ± 0.6 Gy for IMRT and 54.3 ± 0.2 Gy and 48.7 ± 0.3 Gy for VMAT  and the HI was 0.12 ± 0.02 for IMRT and 0.11 ± 0.01 for VMAT (p = 0.001). For the whole PTV, VMAT plans showed superiority over IMRT with respect to conformity, in which the average CI and CN were 1.10 and 0.84 for the VMAT plans and 1.17 and 0.78 for the IMRT plans, respectively.Figure 5, V10, V20, V30 and V40 of the ipsilateral lung and heart. Only the differences in V5 of the ipsilateral lung did not reach statistical significance (p = 0.409). Ipsilateral lungs that received 10, 20, 30 and 40 Gy were reduced by 2.8%, 3.3%, 3.4% and 3.0% in VMAT plans. Hearts that received more than 5, 10, 20, 30, and 40 Gy were reduced by 7.0%, 3.1%, 2.8%, 2.2% and 1.3% in VMAT plans, respectively. The average mean doses to the ipsilateral lung and heart were reduced by 1.1 and 0.8 Gy, respectively. Whereas VMAT would result in larger volumes of the ipsilateral lung and heart receiving low doses, the average volume percentage receiving the 2.5 Gy dose increased by 6.7% and 7.7%, respectively . Moreover, the V5, V20 and Dmean of body-PTV decreased in VMAT plans. For the contralateral lung and breast, the differences between VMAT and IMRT plans did not reach statistical significance.As shown in Fig. The SCCP and EARs of the ipsilateral lung, whole lung, contralateral lung and breast, and the NTCP of the ipsilateral lung and heart for the investigated breast radiotherapy techniques are shown in Table Table 95% and V100% were higher in the VMAT plans than in the IMRT plans: 48.7 ± 0.3 Gy, as compared with 48.5 ± 2.2 Gy from a study by Zhang [IMRT and VMAT can shape the dose to the concave target in the CW and IMN in breast cancer radiotherapy. In this study, we systematically compared the dosimetric parameters of two techniques, IMRT and VMAT, at our institute for 30 cases of post-mastectomy left-sided breast cancer patients. The results of our study indicated that both IMRT and VMAT provided good coverage of the target, while VMAT showed, with statistical significance, more conformity and more dose homogeneity in the target area of CW and IMN, compared with those of IMRT, by avoiding areas of under-dose, and at the same time eliminating areas of relative overdose. Our VMAT significantly reduced the near-maximum dose of the PTV of CW and IMN, which was 54.3 ± 0.2 Gy, as compared with 55.4 ± 1.7 Gy from a study by Zhang , and 56.by Zhang , and 48.by Zhang  and 47.7by Zhang . The valby Zhang –21, 26. by Zhang . We alsoby Zhang .2.5. In our study, the volume of heart received more than 2.5 Gy (lower dose) was increased by 8.80%, whereas the value in VMAT was also clinically acceptable. In addition, our NCTP values based on the relative seriality model further verified that the VMAT plans provide better protection for the heart (0.34% vs 0.86%). This result differs from those of Wang et al. [5, V10, V20, and V30 of the heart are the highest for VMAT out of these techniques. For the ipsilateral lung and whole-lung, both mean dose and volume received more than 5 Gy, 10 Gy, 20 Gy and 30 Gy were lower in VMAT plans; only the differences in the V5 were not statistically significant. Although the V2.5 was increased in VMAT plans, the risks of pulmonary toxicity, SCCP and EAR were not increased. Compared with previous studies of VMAT plans for PMRT patients [5 of VMAT plans were also lower, 46.1% and 20.9%. These values were 83.0% and 70.2% according to Popescu [In the optimization of VMAT and IMRT, the heart, ipsilateral lung and contralateral breast were considered the three most important OARs due to their large volumes. These OARs were protected by adjusting the priority values to reduce the maximum percent dose and scatter dose. Compared with our IMRT plans and other studies of VMAT plans, the mean dose to the heart was comparably lower in our VMAT plans, 5.2 ± 0.9 Gy, while it was 13.5 ± 5.0 Gy according to Zhang , 7.2 ± 2patients –24, the  Popescu , 61.1% a Popescu , 53.91%  Popescu , 70.36%  Popescu  48.9% an Popescu  and. 43. Popescu .p < 0.05), which is contradictory to the reports of Wang et al. [Except for acute and late radiation damage to the heart and ipsilateral lung, the delivery of low-dose irradiation to healthy tissue, especially to the contralateral breast and lung, has been estimated to double the risk of subsequent malignancy, and this risk is enhanced with increasing dose . Based og et al.  and can g et al. , the CN g et al. , the homThese results show that VMAT is the optimal technique for all PMRT patients, especially considering the complexity of the target and patient geometry. In our study, to find the balance between the dose to the target area and normal tissue in VMAT plans, it is possible to reduce the low-dose radiation to normal tissue while the target coverage and conformality reach the level of IMRT plans. We adopted the following in VMAT planning. (1) EUD parameters were used to control the dose of OARs during optimization, the EUD has more advantages in controlling the mean dose of OARs . (2) VirAnother clinical advantage of the use of VMAT is that it generally takes fewer MUs and improves the efficiency of plan delivery, compared with IMRT plans. Our results showed that the total MUs for VMAT plans were decreased by an average of 31.5%, and the treatment time was decreased by an average of 71.4%, compared with that of IMRT, consistent with other studies , 18.Finally, we acknowledge that the impact of respiratory movement on the CW was not considered in this study, which is the major drawback for this work. Deep inspiration breath-hold (DIBH) during breast treatment radiation had been demonstrated to not only eliminate the effects of breathing, but also to reduce considerably the dose to the heart –44. For The VMAT and IMRT techniques were evaluated for PMRT with the left-sided breast cancer patients in this study. Our dosimetric analyses demonstrate that VMAT plans confer advantages in terms of the PTV of CW dose coverage and homogeneity, reduce the mean dose and volumes both in the low- and medium-dose regions of the lung and heart and decrease MU and treatment time, as compared with IMRT plans. Based on estimated risks for OARs, VMAT was the appropriate PMRT technique for PMRT patients who are prone to developing radiogenic side effects. Overall, the VMAT plan is superior to the IMRT plan.


Many dairy products are discarded and useless after end of shelf-life, which causes economic and environmental challenges. The objective of this study was to study the compositional characteristics of some dairy products before and after shelf-life, and develop a process to utilize those dairy products after end of shelf-life in non dairy applications (cosmetic cream and soap). Several dairy products, such as sterilized milk, yogurt, soft cheese, hard cheese, cream, and butter were collected from markets in Egypt before shelf-life and after three months of shelf-life. Electrophoresis analysis was conducted to estimate the changes in the protein fractions of protein products  before and after expiration. Also, gas chromatography (GS) was performed to compare the fatty acids of fat products (cream and butter) before and after end of shelf-life. Sterilized milk, yogurt, soft, and hard cheese were turned into powder  to be used as a raw material in manufacturing of cosmetic creams. The fat was separated from cream, butter, and hard cheese  to be utilized in making soap. The formulated cosmetic creams were examined in vitro. Functional properties of cream were determined, such as appearance, spreadability, irritancy, and pH. Additionally, the soap quality was tested after manufacture. We found that dairy products, such as milk, yogurt, and cheese after shelf-life can be utilized as raw materials for the production of cosmetic creams, as well as production of soap from butter and cream. The produced products were similar to those in commercial markets. This study is an endeavor to conquer the dairy industry challenges, which are considered a huge loss from the economic and environmental aspects.  •Several dairy products were collected from markets before and after shelf-life.•The protein fractions were estimated in those dairy products before and after expiration.•Sterilized milk, yogurt, soft, and hard cheese after end of shelf-life were turned into powder.•This powder can be used as a raw material in manufacturing of cosmetic creams.•The fat of expired dairy products was used in making soap. Inaccurate detection methods for milk spoilage force milk producers to use excessively conservative expiration dates to avoid the legal and economic consequences for consumers from drinking spoiled milk . The staIn Egypt, any product that exceeded shelf-life (as shown on the label) is considered no longer suitable for human consumption. Consumers determine dairy products' shelf-life by checking the "sell by" and or "best if used by" dates on the labels. They do not prefer to buy products that about to be expired . In otheCodex refers to shelf-life as “date of minimum stability” which means the same as (“best before”). The date signifies the end of the period under any stated storage conditions, in which the product remains fully marketable and maintains its characteristics. However, beyond the date, the food may still be perfectly satisfactory . As a reThe total world milk production was about 915 million tons in 2018. Developed countries give one-third of world milk production , where mFat can be extracted from expired dairy products (EDPF) especially fatty products, such as cream and butter  to manuf22.1Sterilized milk, yogurt, soft cheese, hard cheese, cream, and butter samples were obtained from the main stores of Dairy Factories in Egypt. Those products were analyzed before shelf-life. New batches of those products were stored at 4°C until the end of shelf-life, then they were analyzed again after end of shelf-life.2.2The total protein using Kjeldahl method , and fat2.3Electrophoretic separation of proteins was performed at a pH of 8.6 using disc electrophoresis . Milk, y2.4The lipid extraction was done from cream, butter, and hard cheese products using chloroform methanol  at 2:1 v/v . The non-1, hydrogen flow rate was set at 33 ml min-1, and airflow rate was set at a rate of 330 ml min-1. The chart speed was 0.4 cm min-1. Peak identifications were established by comparing the retention times obtained with standard methyl esters. The areas under the chromatographic peak were measured with electronic integrator.The methyl esters of fatty acids were prepared from aliquots of total lipids using 5 ml of 3% sulfuric acid  in absolute methanol and 2 ml benzene . The con2.5Sterilized milk was centrifuged using centrifugal separators , and then skim milk was collected, placed into Petri dishes (7.0 cm diameter) and dried completely in an air forced oven  for 36 h at 60°C. Then, dried milk was grinded by a Blender mill and stored in the refrigerator (at 4–7°C) for further analyses.Yogurt was prepared by using gravity separation methods and diluted with 80°C distilled water in the ratio of 1:3, respectively. The diluted yogurt was then mixed well in a blender and kept at room temperature for about 8 h without stirring. As a result, three different layers  were obtained A Ma and. Fat was2.5. 1Moisture using a forced draft oven  in 2.5 ml water by heating (aqueous phase). Then 1.25 g of beeswax , 1.25 g soft paraffin , and 5 ml liquid paraffin  were added to a porcelain dish (oily phase). The W/O emulsion was prepared by the addition of aqueous phase to the oily phase drop by drop on a constant stirring over a thermostatically controlled boiling water bath with a shaker . Finally, the dish was removed from the water bath and the dairy product powder was added. Methylparaben  and rose water  were then added and stirred well until semi homogenous cream was obtained and the cream cooled to room temperature .2.7. 2The cream was evaluated for appearance, spreadability, irritancy test, and pH. The appearance of the cream was judged by its color, texture, roughness, and odor . The spr2 area on the left hand surface. A half gram was applied to the specified area. Irritancy, erythema, and edema were monitored for 24 h  for 10 min, followed by adding 4.0 g sodium hydroxide  in 10 ml water with continuous stirring. Saponification takes place gradually in 15 min (200 mg of NaOH used for each 1 kg of EDPF). The thick solution was pulled into a model holder and left overnight to ensure complete saponification. The pH of resulted soap was 11 whereas the typical pH of soap is 9.0. The soap was subjected to washing by dissolving in a minimum amount of water followed by separation through salting using sodium chloride salt three times to decrease the pH.2.9Compositional data were analyzed using R software  to determine the differences among means of products before and after shelf-life. An ANOVA was performed using the GLM procedure available in R software. When a significant difference (P < 0.05) was detected, differences among means were compared using the LSD test.33.1-1) and hard cheese (3.5 log cfu g-1), as well as, in butter (4.5 log cfu g-1). Those values have decreased to 3.2, 3.1, and 4.2 log cfu g-1 in soft cheese, hard cheese, and butter, respectively.The chemical composition and TBC of dairy products before and after shelf-life are shown in The composition of EDPP produced from expired products  is presented in 3.2The iodine value, saponification number, and free fatty acids in cream, butter, and hard cheese before and after expiration are presented in -1. These results are not similar to those reported by another researcher who found that the iodine values of buffalo and cow ghee samples are 22.6 and 50.6 mg100 g-1, respectively  state that the iodine values for the extracted fat should be in the range of 26.4–43.1 g 100 gectively . It is c-1. The saponification numbers of all fat samples .The fatty acids profile in cream and butter before and after shelf-life is shown in -1, in C8:0 and C10:0 by 0.5 mg g-1, and in both C14:0, C18:0 about 1.4, 1.2 mg g-1, respectively. Also, a very noticeable increase in C12:0 (Lauric acid) about 4.2 mg g-1 was detected. There was also a shortage of both C16:0 and C18:2 by 1.2 mg g-1 and 2.3 mg g-1, respectively. A very noticeable lack of C18:1 (Oleic acid) was noticed at a rate of 5.0 mg g-1 (In the butter sample, there was an increase after end of shelf-life in C6:0, C18:2T by 0.1 mg g0 mg g-1 . Our res3.5Formulated cream was evaluated for the parameters like visual appearance, spreadability, irritancy test, and pH. The appearance of cream was white color having a smooth greasy texture with no grainy particles . Irritan4According to the food legislation, all these products became not suitable for human consumption from the safety and quality point of view, but chemically they contain considerable amounts of undeteriorated or unrecompensed components, such as fat, protein, sugars, and minerals. No much differences were found in the composition of these products before and after shelf-life. Disposal of these products by burying them as wastes in the desert or the drainage channels represents a huge economic loss. As a result, it is useful to direct these substances into nonfood areas or nonfood industries. The study reveals that it is possible to formulate an effective cold cream containing expired dairy products and soap from expired dairy products fat.Nermeen N. Nasralla: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Nanis H. Gomah: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Morsy M. Aly: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation. Jelan A. Abdel-Aleem: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation. Ahmed R.A. Hammam: Writing – original draft, Writing – review & editing. Dina M. Osman: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation. Yaser M.A. El-Derwy: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The detected values for DTN, DON, and Urea-N in the lockdown period decreased by 9.6%, 30.4%, and 28.97%, respectively, compared to 2019. The reduction in human activities is the reason for the decrease. The urban travel intensity in Nanyang city reduced from 6 to 1 during the lockdown period; the 3 million population which should normally travel out from city were in isolation at home before May. The fertilization action to wheat and orange were also delayed.Atmospheric nitrogen deposition (AND) may lead to water acidification and eutrophication. In the five months after December 2019, China took strict isolation and COVID-19 prevention measures, thereby causing lockdowns for approximately 1.4 billion people. The Danjiangkou Reservoir refers to the water source in the middle route of South-to-North Water Diversion Project in China, where the AND has increased significantly; thus, the human activities during the COVID-19 period is a unique case to study the influence of AND to water quality. This work monitored the AND distribution around the Danjiangkou Reservoir, including agricultural, urban, traffic, yard, and forest areas. After lockdown, the DTN, DON, and Urea-N were 1.99 kg · hm HighlightThe DTN, DON and Urea-N in AND decreased during epidemic lockdown.The decrease can be explained by traffic stopping and fertilization being delayed.The nitrogen deposition flux in the Danjiangkou area is at a medium level in China.Solid waste and air pollution are important sources of pollution caused by human activities to the natural environment ,2,3. HumOn 30 January 2020, China implemented strict quarantine measures, and the entire country was under comprehensive and strict control, i.e., the country was in lockdown. At the end of April 2020, the country gradually entered the state of unsealing, wherein personnel began to move, and industrial and agricultural production resumed in an all-round way. For three months, approximately 1.4 billion Chinese people and their related activities stopped, which is also a unique example in human history [After the COVID-19 outbreak, global carbon emissions were reduced by 2.5 billion tons. The lockdown in various countries have led to the stopping of factories and vehicles, and the air quality has improved significantly ,7.2 column in eastern China decreased by 65% compared with the same period in 2019, mainly caused by the change of fossil fuel combustion after the epidemic response period [Since the 20th century, in addition to the carbon emission, human activities have also discharged nitrogen-containing compounds into the atmosphere. Asia , Western Europe, and North America have become the three largest nitrogen deposition concentration areas . The sige period .−1.DON is an important component of nitrogen deposition, approximately 15–30% of total deposition flux. The research on DON can be traced back to the early 20th century. The Lausanne experimental station in the UK has researched the total DON in rainwater for several years. The results show that the maximum concentration of DON in rainwater is 2.50 μmol/L, average 1.4 μmol/L, and its contribution to total nitrogen is roughly 25% . In the To further understand the impact of DON on the ecosystem, the composition of DON needs to be analyzed. Urea is an organic nitrogen compound often detected in AND. Urea is easily absorbed and utilized by organisms and is one of the main factors causing water eutrophication . In phosThe Danjiangkou reservoir is located between Hubei and Henan province. It is the water source of the middle route of the South-to-North Water Diversion Project in China. The South-to-North Water Diversion Project is a major water conservancy project to alleviate the drought and water shortage in northern China and to ensure the safety of the water supply . The watStudies on nitrogen deposition in the Danjiangkou reservoir are few . NitrogeAs shown in literature , the govSo, the periods of fighting COVID-19 can be defined as three stages. The early epidemic period is before January 2020, in which the cities were about to lockdown . The midIn this work, the nitrogen deposition around the reservoir was detected from January 2019 to May 2020. The DTN, DON and Urea-N in this period will be compared with the early epidemic period, middle epidemic period and after the epidemic lockdown. Furthermore, the nitrogen deposition data will be compared with the same period in 2019 to investigate the impact of human activity on the nitrogen deposition.4+—N, and NO3—N.Six sampling stations are set around Xichuan reservoir area, as shown in Taocha (S1) station is located around the canal head with large surrounding population and the developed traffic status. The location is defined as urban area, where the main pollution sources are daily waste and road traffic pollution.Songgang (S2) station is located at the wharf. The location is defined as traffic area, where the main pollution source is traffic and waste pollution.Tumen (S3) station is close to wheat and corn farmland. The location is defined as agricultural area, where pesticides and fertilizers are sprayed. There are pollution sources of livestock manure and waste.Daguanyuan (S4) station is a scenic spot. There is a large flow of people and vehicles on holidays. Its main pollution sources are traffic and waste pollution. The location can be defined as traffic area.Dangzikou (S5) station is around the orange yard. Wulongquan (S6) station is in forest area, as shown in The automatic samplers were placed on the different functional areas, as shown in Dry deposition samples are collected once a week and four times a month. Wet deposition samples are collected once in one rainfall. If there are multiple rainfalls a day, they shall be combined into one sample. The collected samples are placed in a brown polyethylene bottle, stored in a refrigerator at −20 °C, and the analysis is completed within one week. The dimension of the collector is length 1.2 m × width 0.8 m × height 1.5 m, consisting of a dust tank (d = 150 mm) and a rainfall funnel (d = 300 mm).The dry and wet deposition fluxes of atmospheric nitrogen around the reservoir area are obtained by Formulas (1) and (2) .dF is the monthly dry deposition flux, kg · hm−2 · month−1, dK is the conversion coefficient, 10−2, C is the nitrogen concentration in the collected solution, mg/L, V is the volume of collected liquid, L, dS is the area of dust cylinder, 0.0177 m2, and dK/dS = 0.5659 hm−2.Dry deposition fluxwF is the monthly wet deposition flux, kg · hm−2 · month−1, wK is the conversion coefficient, 10−2, iC is the nitrogen concentration in rainfall collected in one month, mg/L, iV is the volume of each rainfall, L, wS is the area of rainfall funnel, 0.0707 m2, and wK/wS = 0.1415 hm−2.Wet deposition flux3—N was measured by phenol disulfonic acid spectrophotometry [−1; NH4+-N was measured by Nessler reagent spectrophotometry [−1. DTN was measured by potassium persulfate digestion ultraviolet spectrophotometry [−1. Organic nitrogen = Total nitrogen—Tnorganic nitrogen (NO3—N + NH4+—N). Urea-N is determined by diacetyl MONOOXIME thiosemicarbazone spectrophotometry [NOotometry  , and the monthly average DTN during the middle epidemic lockdown period was 1.99 kg · hm−2 · month−1 (variation coefficient 0.14–0.45), which is an increase of 8.7%. After the epidemic lockdown, the amount of DTN in May was 2.56 kg · hm−2 · month−1 (coefficient of variation 0.24), an increase of 28.41%. Compared with the same period in 2019, the average monthly DTN during the middle epidemic lockdown period decreased from 2.21 kg · hm−2 · month−1 to 1. 99 kg · hm−2 · month−1, a decrease of 9.6%.In 2020, the DTN in the early lockdown period was 1.83 kg · hmAs shown in By Pearson correlation coefficient, the correlation between DTN and rainfall is 88.66% in 2019 and 89.89% in 2020. The average rainfall from February to April 2019 is 20 mm, compared with 34 mm in 2020, which is one of the reasons for the rise of DTN during the epidemic in 2020.−2 · month−1 (variation coefficient 0.329), and the monthly average DON during the middle epidemic lockdown period was 0.80 kg · hm−2 · month−1 (variation coefficient 0.19–0.56), a decrease of 4.8%, DON was 1.06 kg · hm−2 · month−1 (variation coefficient 0.25) in May. After the epidemic lockdown, DON increased by 31.86%. Compared with the same period in 2019, the average monthly DON during the middle epidemic period decreased from 1.15 kg · hm−2 · month−1 to 0.8 kg · hm−2 · month−1, a decrease of 30.4%.In 2020, the DON in the early lockdown period was 0.84 kg · hmThe data shows that the total amount of DON decreased during the middle epidemic period, lower than that in the same period in 2019. After the lockdown, the amount of DON increased significantly, but it was also far lower than that in the same period in 2019.As shown in −2 · month−1. During the middle epidemic lockdown period, the monthly average Urea-N was 0.15 kg · hm−2 · month−1, a decrease of 9.85%. After the lockdown was finished, the amount of Urea-N was 0.17 kg · hm−2 · month−1, up 9.3%. Compared with the same period in 2019, the average monthly Urea-N during the middle epidemic period decreased from 0.22 kg · hm−2 · month−1 to 0.15 kg · hm−2 · month−1, a decrease of 28.97%.Before the epidemic lockdown, the monthly Urea-N was 0.17 kg · hmAs shown in In the early epidemic lockdown period, the Urea-N/DON of the current month was 0.20. During the middle lockdown period, the monthly average Urea-N/DON was 0.20. After the epidemic lockdown from the end of April, the Urea-N/DON was 0.16. Compared with the same period in 2019, the average monthly Urea-N/DON during the middle lockdown period decreased from 0.21 to 0.20, a decrease of 4.99%. It shows that during the middle lockdown period, the change of Urea-N/DON is smaller than that in the early/later lockdown periods and the same period in 2019.Urea-N in the atmosphere comes from multiple sources such as agricultural activities, biomass combustion, excretion of final nitrogen products of animal metabolism, and decomposition of protein substances ,31,32.3/s.The Danjiangkou reservoir is located between Henan and Hubei province, which is the upstream of the Hanjiang River, a tributary of the Yangtze River. The reservoir location area is the north subtropical monsoon climate, which has the characteristics of sufficient precipitation, abundant heat and four distinct seasons. The annual sunshine number is 1950 h and the sunshine rate is 44%. Each square centimeter of land receives 104.8 kcal of radiant energy throughout the year. The average temperature is 15.6–16.0 °C, with an annual range of 24.7 °C. The highest temperature is in July, with an average temperature of 27.8 °C; The lowest temperature is January, and the average temperature is 3.1 °C. The frost-free period in the Danjiangkou reservoir area is 254 days. The average annual precipitation is 750–900 mm, and the interannual precipitation varies greatly. The precipitation in summer is 30–49% of the annual precipitation; Only 4–6% in winter; The precipitation in spring and autumn is similar, the dam elevation is 176.6 m, and the out flow is 500 mThe sources of DON, including industrial and agricultural activities, aquaculture and animal husbandry production, combustion of coal and biomass, waste treatment, direct volatilization of soil humus and plant pollen, animals and plants, and products of photochemical reaction between active NOx and hydrocarbons in the atmosphere, are complex and diverse.−1 · yr−1) and through fall (0.5–1.2 kg · hm−1 · yr−1) deposition of inorganic N in the three national parks. The estimated total N deposition levels reported for the forest stands studied in the three parks (1.3–2.1 kg · hm−1 · yr−1). Fenn et al.  shown th3 emissions for the continental United States (U.S.) On-road NH3 emissions were determined from on-road CO2 emissions data and empirical NH3: CO2 vehicle emissions ratios. Emissions of NH3 from on-road sources in urbanized regions are typically 0.1–1.3 t · km−2 · yr−1, while NH3 emissions in agricultural regions generally range from 0.4–5.5 t· km−2 · yr−1, with a few hotspots as high as 5.5–11.2 t · km−2 · yr−1, as shown in Donna et al.  provide −1) from Land use class of Forest, semi natural vegetation/grassland, Croplands and Other land [Total deposition of NHx-N, NOy-N and total N on the world’s terrestrial ecosystems for the year 2010. The Total N was up to 70.3  [The total N deposition was estimated at 80–100 kg N · ha07, 2010 ), and 29 et al.) .Total N deposition of 43 sites was ranked by land use as urban > rural > background sites  .−1 · yr−1 at five sites in Yangtze River Basin, China.Xiao et al.  reported−2 · a−1, The monitoring results in this work are obviously higher than the above research data.The data of European Monito-ring and Evaluation Programme  shows that the nitrogen wet deposition flux in Europe from 2003 to 2005 is 1.04–18.4 kg · hmIt can be seen the nitrogen deposition flux in this region is at a medium level in China, but much higher than that in remote areas with less human interference, indicating that the nitrogen deposition is greatly affected by human activities.The areas with high concentration in the epidemic situation have decreased significantly, and the areas with high deposition have been transferred to the orchard and tourism areas. The forest area belongs to deciduous broad-leaved forest, and its active nitrogen emission mainly comes from soil organic matter, animal and plant decay, plant allelochemicals, and pollen particles.The land around Xichuan reservoir area is mainly farmland and forest. The farmland is 45% (including about 10% orange yard), mostly distributed in hilly areas, with 34% of forest area, mostly distributed in mountainous areas, followed by grassland and bushes area, accounting for about 11%, and urban area and traffic area accounting for about 10%. The East and North banks of the reservoir are mainly agricultural area, and the West Bank is mainly forest area, as shown in −2/month, followed by S1 station, 2.24 kg · hm−2/month, and the minimum value was at S5 station, 0.866 kg · hm−2/month. The variation coefficient of between the six stations was 0.398.In 2020, before the epidemic lockdown, the maximum value of DTN was at S4 station, 3.12 kg · hm−2/month, followed by S4 station, 2.26 kg · hm−2/month, and the minimum value was at S6 station, 1.32 kg · hm−2/month. The maximum value of DTN decreased and transferred from traffic area to urban area. The variation coefficient between the six stations decreased, indicating that the variation of DTN in each region decreased.In the middle epidemic lockdown period, the average maximum value of DTN was at S1 station, 2.67 kg · hm−2/month, followed by S4 station, 2.33 kg · hm−2/month, and the minimum value was at S6 station, 1.43 kg · hm−2/month, with a variation coefficient of 0.12. The DTN increased after lockdown finish and the highest place is still in urban area.After the epidemic lockdown period, the maximum DTN was at S1 station, 2.72 kg · hm−2/month to 2.03 kg · hm−2/month, and S2 decreased from 2.70 kg · hm−2/month to 1.74 kg · hm−2/month, a decreased of 1 kg · hm−2/month. S5 is yard area and S2 is traffic area.As shown in −2/month, followed by S3 station, 0.98 kg · hm−2/month, and the minimum value was at S1 station, 0.49 kg · hm−2/month. The variation coefficient between the six stations was 0.329.In the early lockdown period in 2020, the maximum value of DON was at S4 station, 1.37 kg · hm−2/month, followed by S3 station, 0.95 kg · hm−2/month, and the minimum value was at S6 station, 0.49 kg · hm−2/month. The variation coefficient of six locations was 0.257. The maximum value of DON decreased and shifted from traffic area to yard area. The locations variation coefficient decreased, indicating that the change of DTN in each region decreased.In the middle lockdown period, in 2020, the average maximum value of DON was at S5 station, 1.09 kg · hm−2/month, followed by S5 station, 1.14 kg · hm−2/month, the minimum value was at S6 station, 0.69 kg · hm−2/month, and the variation coefficient was 0.258. The DON value increased after the lockdown finished, and the highest values occurred in urban areas.After the epidemic lockdown, the maximum value was at S1 staiton, 1.55 kg · hm−2/month to 0.66 kg · hm−2/month, and S5 data decreased from 1.74 kg · hm−2/month to 1.05 kg · hm−2/month, a decrease of 0.7 kg · hm−2/month, respectively.As shown in S2 refers to traffic area; the lockdown activity results in nearly zero traffic.S5 location is yard area; DON deposition mainly comes from agricultural activities and fossil fuel combustion. Around S5, the station is surrounded by a large orange yard. Germination fertilizer is applied in February, pre-flowering fertilizer is applied in March, and stable fruit fertilizer is applied in May. Plants and soil humus directly volatilize a large quantity of DON into the atmosphere, resulting in a large quantity of DON deposition.Due to the epidemic lockdown, the orange fertilization, land tillage, wheat fertilization and travel in the city cannot be carried out, so nitrogen deposition is reduced.−2/month, followed by S3, 0.20 kg · hm−2/month, and the minimum value was at S4 station, 0.07 kg · hm−2/month. The variation coefficient between the six stations was 0.26.In early lockdown period, the maximum value of Urea-N was at S3 station, 0.215 kg · hm−2/month, followed by S3, 0.22 kg · hm−2/month, and the minimum value was S4 yard, 0.07 kg · hm−2/month. The variation coefficient of six locations was 0.41. The maximum value of Urea-N decreased and shifted from agricultural area to urban area. The variation coefficient increased, indicating that the Urea-N in each region was quite different.In the middle lockdown period, the average maximum value of Urea-N was S1, 0.25 kg · hm−2/month, followed by S6, 0.23 kg · hm−2/month, and the minimum value was S6, 0.69 kg · hm−2/month, with a variation coefficient of 0.34. the Urea-N increased after lockdown and the highest place is still in urban area.After the epidemic, the maximum value was at S1, 0.23 kg · hm−2/month to 0.080 kg · hm−2/month.It can be seen from 2 and PM2 during the US epidemic lockdown period and found that the concentration decreased in cities but did not change significantly in countryside areas.S4 Daguanyuan is a traffic area, indicating that traffic travel has been significantly affected by lockdown. Berman et al.  studied Urban travel intensity refers to the ratio of the population traveling in the city to the population living in the city. S4 is Daguanyuan, the main scenic spot in the city, and S2 is Songgang, the main wharf. It is the main destination and transit place for travel in the city. The impact of the epidemic lockdown has caused a sharp reduction in the intensity of urban wind travel, and the traffic area in the two places has a great impact. As shown in Spring Festival transportation is a major migration of population with Chinese characteristics. China’s annual traffic reaches 2–3 billion people in 40 days. Nanyang is a city where population outflows, which means a large population work in southern China and back home during Spring Festival. The legal holiday of Spring Festival in China is 7 days. The returning population began to leave their hometown 6 days after the festival day. After the epidemic lockdown in January 2020, a large number of people stayed in their hometown. According to the statistics of Nanyang City Government, the permanent resident population of Nanyang in 2019 was 10.03 million, including 3.1 million people returning home during the Spring Festival, as shown in As shown in After the lockdown in 2020, the traffic flow within three months is almost zero. Therefore, nitrogen emissions have been significantly reduced.(1)In December 2019, the COVID-19 outbreak broke out worldwide. On 30 January 2020, China had entered a state of comprehensive and strict control, and population movement was in lockdown. The periods of lockdown in China can be identified as three stages, the early epidemic period (January 2020), the middle epidemic period (February–April 2020), and the later epidemic period (May 2020). The impact of human activities on AND was investigated in January to May in 2019 and 2020.(2)The Danjiangkou reservoir is the source of the middle route of South-to-North Water Diversion Project in China. The water quality of the Danjiangkou reservoir is directly related to the water safety of more than 20 large cities, such as Beijing and Tianjin. The AND has become a risk to the reservoir water quality.(3)The lockdown activity resulted in a decrease of DTN, DON and Urea-N from the atmosphere in February to April 2020, decreasing by 9.6%, 30.4%, and 28.97%, respectively, compared to 2019. During the middle lockdown period, the DON decreased from 0.84 kg · hm−2·month−1 to 0.80 kg · hm−2 · month−1, and the Urea-N decreased from 0.17 kg·hm−2 · month−1 to 0.15 kg · hm−2 · month−1.(4)The nitrogen deposition flux in this region is at a medium level in China, but it is much higher than that in remote areas with less human interference, indicating that the nitrogen deposition is greatly affected by human activities.(5)The decrease can be explained by traffic stopping and fertilization being delayed, with the peak values of detected data between positions occurring in urban area and farmland. Necessary fertilization activities shall be carried out according to the growth cycle of crops, especially the addition of nitrogen-containing fertilizer, which has a significant impact on atmospheric Urea-N deposition.


Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC50 of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC50 of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than −7.0 kcal·mol−1; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen–Chuanxiong herbal pair have been successfully characterized through a spectrum–effect relationship analysis and an LC–MS analysis.In this study; a spectrum–effect relationship analysis combined with a high-performance liquid chromatography–mass spectrometry (LC–MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma–Chuanxiong Rhizoma (Danshen–Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC–MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid;  Thrombin (THR) and factor Xa (FXa), which are members of the Serine Protease family, play critical roles in the coagulation cascade . In throSalvia miltiorrhiza–Carthamus tinctorius herbal pair through a spectrum–effect relationship analysis −, at m/z 179.06 for [M − H − C7H10O5]− and at m/z 135.03 for [M–H − C8H10O7]−. Therefore, peak 4 is suspected to be chlorogenic acid −. Peak 5 is tentatively identified as caffeic acid −. According to the reference −. Therefore, peak 17 is suspected to be Z-ligustilide −. Therefore, peak 61 is suspected to be tanshindiol B −. Therefore, peak 62 is tentatively identified as methyl dihydronortanshinonate −. Therefore, peak 67 is suspected to be tanshinone I − and at m/z 248.99 for [M + H − H2O − CO]−. Therefore, peak 71 is suspected to be tanshinone IIA [Peak 4 gives a [M − H]nic acid . Peak 5 eic acid . Peak 9 eference , peak 9 ustilide . Peak 61indiol B . Peak 62hinonate . Peak 6750 value of 64.94 µM. Chlorogenic acid with an IC50 value of 185.08 µM exhibits a better inhibitory activity than Z-ligustilide with an IC50 value of 1460.65 µM on THR. As shown in 50 value of rivaroxaban, as the positive control drug [50 values of 112.59 µM and 138.19 µM, respectively. In addition, caffeic acid and ferulic acid do not show a significant inhibitory effect with IC50 values of 3789.96 µM and 2488.90 µM, indicating that they may not act directly on the active center of FXa.In order to confirm the feasibility of the spectrum–effect relationship method for screening of active compounds, in vitro enzymatic activity assays were performed. Argatroban, a well-known THR inhibitor , was selrol drug , is 47.2Z-ligustilide are less than −5.0 kcal·mol−1, indicating that they are potential THR inhibitors. Chlorogenic acid is combined with THR through Van der Waals forces and ionic bonds. Z-ligustilide can enter THR catalytic activity pockets with a strong affinity by forming H-bonds with residues of SER214 and GLY226. The 2D interaction diagrams of chlorogenic acid and Z-ligustilide with THR can be observed in −1, can interact with FXa by forming H-bonds with residues of SER214, GLY219 and GLN192. In addition, caffeic acid and ferulic acid, which have binding energies of −5.82 kcal·mol−1 and −6.35 kcal·mol−1, are combined with FXa through Van der Waals and other forces. It is worth mentioning that the molecular docking results of the four active components are consistent with their activity in in vitro tests. Tanshinone I and tanshinone IIA with stronger affinity exhibit better inhibition effects. The 2D interaction diagrams are shown in Molecular docking can be used to reveal the possible modes and sites of compounds interacting with THR and FXa. As shown in Z-ligustilide, caffeic acid, ferulic acid, tanshinone I and tanshinone IIA  were purchased from Purechem-standard Co., Ltd. . HPLC-grade acetonitrile was purchased from Shanghai Titan Scientific Co., Ltd. . Water used for all the experiments was purified by a water purification system . All the other chemicals and solvents, such as ethanol, potassium dihydrogen phosphate and disodium phosphate, were obtained from Chron Chemicals Co., Ltd. .Thrombin was purchased from Sigma-Aldrich . Substrate S-2238  was bought from Adhoc International Technology Co., Ltd. . Factor Xa and substrate S-2765  were obtained from Adhoc International Technology Co., Ltd. . Argatroban was bought from Huazhong Haiwei Gene Technology Co., Ltd. . Rivaroxaban was from Aladdin Biochemical Technology Co., Ltd. . The reference compounds of chlorogenic acid, Salvia miltiorrhiza Bge. (number SM2020050101) and Ligusticum chuanxiong Hort. (number CF2020050101) were deposited at the Pharmaceutical Engineering Laboratory in School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, China.Crude drugs  of Danshen and Chuanxiong were purchased from Kangmei Pharmaceutical Co., Ltd. in May 2020. Their origins are from Shandong and Sichuan, respectively. The voucher specimens of w/w, dried extract/crude herb). All solutions were stored in the dark at 4 °C. Before the HPLC analysis, the extract was dissolved in methanol (5.0 mg/mL) and filtered through a 0.22 μm membrane filter .Crude drugs of DS and CX were pulverized, then stored in a dry box after filtrating through a 50 mesh sieve (about 0.29 mm). Nine different ratios of DC herbal pairs  were prepared for reflux extraction. First, 2.5 g of DS and CX mixed powder was extracted through ultrasonic extraction at room temperature for 15 min with 20 mL of 70% ethanol in a 250 mL round-bottom flask. Then, it was further extracted in a water bath at 80 °C for 30 min. The above process was repeated three times. After the extract solutions were filtered through filter paper and combined, they were evaporated in a rotary evaporator  at 55 °C under reducing pressure to remove the ethanol solvent. Finally, the extracts were further dried by lyophilization with a freezing–drying system  to obtain the DC extracts at a yield of about 20% , which is equipped with a diode array detector (DAD), controlled with Agilent ChemStation software. Chromatographic separation was operated on an Agilent ZOBAX SB-Aq column  preceded by a guard column  at a column temperature of 35 °C. The mobile phase, which consists of 0.1% aqueous acetic acid solution (A) and acetonitrile (B), was at a flow rate of 1.0 mL/min. The gradient program was programmed as follows: 0–2 min, 5% B; 2–10 min, 5%–15% B; 10–24 min, 15%–22% B; 24–35 min, 22%–29% B; 35–42 min, 29%–37% B; 42–60 min, 37%–60% B; 60–62 min, 60%–5% B; and 62–70 min, 5% B. The injection volume for all samples was 10.0 μL.1 data and MS2 data were recorded in the range of m/z 100 to 1000. An LC–MS software package (Agilent) was employed for data collection and processing.An Agilent 6545 equipped with electrospray ionization was used for LC/Q-TOF-MS analyses . The LC conditions were the same as described previously. Nitrogen was used as the atomizing dry gas at a flow rate of 8.0 L/min. The Q-TOF-MS analysis was used in both negative and positive ion modes. The key parameters were set as follows: ion source temperature, 320 °C; sprayer pressure, 35 psi; spray voltage, 1000 V; and TOF acquisition speed, 0.5 s. For compound identification, MS2HPO4 and 0.24 g of KH2PO4 in 800 mL of water for immediate use (pH was adjusted to 8.0 by 1.0 M NaOH). A stock solution of THR (125 U/mL) was prepared by dissolving 4.16 mg of THR in 1.0 mL of PBS. A stock solution of FXa (0.5 IU/mL) was prepared by dissolving 5.0 mg of FXa in 1.0 mL of PBS. Stock solutions of S-2238 (2 mg/mL) and S-2765 (2.5 mg/mL) were prepared in PBS. The DC extract samples (10 mg/mL) were prepared in a PBS buffer and diluted to 5.0 and 2.5 mg/mL as necessary.A PBS buffer was prepared by dissolving 1.44 g of NaTHR and FXa inhibitory activity assays were performed with an iMark™ Microplate Absorbance Reader . A DC extract solution of 50 μL and a 50 μL THR solution were mixed and incubated for 10 min at 37 °C. Subsequently, a 100 μL S-2238 (2 mg/mL) solution as the chromogenic substrate was added to start the reaction in the microplate reader. The absorbance was monitored at 405 nm every 30 s for 10 min, and each group was measured three times. As a control group, the operation process was similar to the experimental group but without a DC extract. Except for the enzyme solution and chromogenic substrate, the procedure for assessing the FXa inhibitory activity was exactly the same as for THR. The inhibition rate (%) of THR and FXa was calculated using Formula (1).A spectrum–effect relationship analysis was performed by transferring the DC fingerprint peak area and inhibition rate of THR and FXa into SPSS software  for a canonical correlation analysis (CCA). The chromatograms of nine ratios of DC samples were calculated and generated by “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine” composed by Chinese Pharmacopoeia Committee (Version 2004A). A CCA was used to establish the spectrum–effect relationships between the areas of 71 peaks in the fingerprint and inhibition rate of THR and FXa, respectively, to screen potential active components.x, y, z) = , and the grid centers of THR and FXa were set to  =  and  = , respectively. The Lamarckian genetic algorithm (LGA) was employed and the number of GA runs was equal to 50 for finding the most favorable ligand binding orientations. Finally, Discovery Studio 4.5  was used to observe the interaction of active compounds with THR and FXa.AutoDock 4.2  was employed for molecular docking studies to predict the site and mode of action of active compounds from THR and FXa, respectively. The brief steps for molecular docking are as follows. First, the crystal structures of THR (No. 1DWC) and FXa (No. 2W26) from the PDB library were downloaded, which were output in PDBQT format after the following operations: the water molecules and the ligands of argatrobana and rivaroxaban were deleted while polar hydrogen was added and the point charge was calculated. Second, the 3D chemical structures of active compounds were drawn by ChemDraw 3D (Ver. 15.0) to minimize energy to prepare ligands. Semi-flexible docking was used to simulate the docking process. The grid size was set to (All data are presented as the mean ± standard deviations (SD) of at least three different experiments. The statistical analysis was performed by SPSS.Z-ligustilide) and four potential THR inhibitory active compounds  were predicted. Furthermore, chlorogenic acid, tanshinone I and tanshinone IIA were verified to possess good THR and FXa inhibitory activity with IC50 values of 185.08 µM, 112.59 µM and 138.19 µM, respectively. In addition, the molecular docking results, which are consistent with their in vitro activity verification, show that tanshinone I and tanshinone IIA can interact with FXa by forming H-bonds with residues of SER214, GLY219 and GLN192. In this study, the potential THR and FXa inhibitory components in the DC herbal pair were screened by a spectrum–effect relationship analysis. Combined with a canonical correlation analysis, in vitro THR and FXa inhibitory activity test and LC–MS analysis, two potential THR inhibitory active compounds (chlorogenic acid and