Biosynthesis of Copper Oxide Nanoparticles by Marine Streptomyces MHM38 and their Preventive Ecacy Against Paracetamol-Inducing Hepatic Damage of Albino Rats

Biosynthesis methods, employing microorganisms, have emerged as an eco-friendly, clean and viable alternative to chemical and physical methods. The present study reports the biosynthesis of copper oxide nanoparticles (CuONPs) using cell-free culture supernatant of marine Streptomyces sp. MHM38. For the optimized production of copper oxide nanoparticles, the inuence of some parameters such as concentration of copper sulphate, reaction time, ltrate to substrate ratio and pH were studied. 5mM CuSO 4 was optimal for NP production. Well-dened CuONP formation occurred after 60 min incubation when equal volume of ltrate (cell-free supernatant) to substrate (CuSO 4 solution) was added. NPs remained stable in aqueous solution with increasing time at pH 8. CuONPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD) and nally the nature of the nanoparticles was identied by elemental analysis (EDX). Uv-vis spectroscopy of CuONPs exhibited peak at 550 nm which corresponds to the Surface Plasmon Resonance of CuONPs. Most of the particles are spherical in shape and size ranges from 1.06 – 6.5 nm analyzed using Transmission Electron Microscope (TEM). Antimicrobial activity of CuONPs was performed by well diffusion method against Enterococcus faecalis ATCC 29212, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8939), fungi (Rhizoctonia solani, Fusarium solani, Aspergillus niger) and yeast (Candida albicans ATCC 10237) .The highest antimicrobial activities recorded were against Candida albicans ATCC 10237, were as Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 8939 showed the lesser activity. The preventive ecacy of CuONPs was evaluated against the oxidative stress induced by paracetamol (PAC) in albino rats. The biochemical ndings of CuONPs groups appeared a signicant (p (cid:0) 0.05) diminish in the levels of ALT, AST, ALP, LDH, total and direct bilirubin, Urea, and Creatinine as compared to paracetamol group. Non-enzymatic and enzymatic antioxidants of CuONPs groups were signicantly elevated (p (cid:0) 0.05) in SOD and GSH levels and signicantly low NO and MAD levels compared to the paracetamol group. Also, the histopathological examination of the CuONPs groups assured that the impact of improving CuONPs against paracetamol-induced liver damage.


Introduction
Because of their special electrical , optical, catalytic and magnetic characteristics, metal nanoparticles are synthesized and used (Zhang et  the synthesis of inorganic nanoparticles. Nevertheless, the value of biological synthesis is realized globally as dangerous, expensive and nonenvironment-friendly chemical methods (Shantkriti and Rani, 2014). It is thus important to develop fast, cost-effective, ecological and easy-to-scale synthetic approaches. The development of green roads to manufacture nanoparticles of metal using biological systems is therefore important. It has been well established that microbes develop mechanisms to survive in toxic metals by turning toxic metal ions into their corresponding nontoxic forms as metal sul de / oxides, in harsh conditions (Tenkouano, 2017). The speci cs of nano-transformation mechanisms are not well known. For nanoparticles synthesis, a wide range of biological tools can be used such as bacteria, yeasts, fungi, algae and plants. A large number of the world's population uses nanoparticles to treat some diseases. Recent studies have indicated that CuNPs have broad biological properties (Murthy, 2007).
Paracetamol is a medication with antipyretic and analgesic impacts that is broadly utilized by the wider community. Paracetamol is taken freely without supervision, however high doses of paracetamol cause liver damage. Paracetamol is expected to be a major factor in the cause of severe liver damage (Nerdy and Ritarwan, 2019). This present study has been designed to use marine actinobacterial strains namely, Streptomyces sp. that was screened for biosynthesis, optimization and characterization of CuNPs produced as well as to evaluate their inhibitory action against some pathogenic microorganisms and conducted as an attempt to see if animal tissue accepts CuONPs and whether CuONPs have antioxidant and hepatoprotective activity.

Material And Methods
Microorganisms and cultural conditions Streptomyces sp. MHM38 was isolated by Dr. Moaz M. Hamed (Marine Microbiology Lab., Marine Environmental Division, National Institute of Oceanography and Fisheries, Egypt) from the marine sediment sample in Suez gulf, and deposited in the Genbank as Streptomyces sp. MHM38 with accession number KU764745. This marine actinobacterial isolate was maintained on slopes containing starch nitrate agar medium (SNM) of the following composition (g/l): Starch, 20; K 2 HPO 4 , 1; KNO 3 , 2; MgSO 4 , 0.5; agar, 18. Components were dissolved in 0.5-liter distilled water and 0.5-liter seawater (El-Sersy et al., 2010). The 50 and 20 μg mL-1 tetracycline and nystatin as antibacterial and anti-fungal agents to prevent bacterial and fungal infection were applied following autoclaving and solidi cation. Strain was incubated for a period of 7 days at 30-32 0 C. The isolate was stored as spore suspension in 20% (v/v) glycerol at -20 0 C for subsequent investigation.
Inoculum preparation 250 ml Erlenmeyer ask containing 50ml of medium consisting of (g/l): Starch, 20; K 2 HPO 4 , 1; KNO 3 , 2; MgSO 4 , 0.5. Components were dissolved in 0.5liter distilled water and 0.5liter seawater. This ask was inoculated with old stock culture grown on starch nitrate agar medium. The ask was incubated for 5 days in a rotator incubator shaker at 30-32 0 C, 200 rpm, and was used as inoculums for subsequent experiments.

Extracellular synthesis of CuONPs
In order to screen Streptomyces sp. MHM38 for the synthesis of CuONPs, the isolate was freshly inoculated in an Erlenmeyer ask containing 50ml of the production medium consisting of (g/l): Starch, 20; K 2 HPO 4 , 1; KNO 3 , 2; MgSO 4 , 0.5. Components were dissolved in 0.5liter distilled water and 0.5liter seawater. The inoculated ask was incubated for 5 days in a rotator incubator shaker at 30-32 0 C and 200 rpm. The culture was centrifuged at 10,000 rpm at the end of the incubation, and supernatants were used to detect copper nanoparticles. A volume of 15 ml of 1mM CuSO 4 was added to 15 ml of each supernatant in 100 ml Erlenmeyer asks. Flasks were incubated at 30-32 0 C and observed for color change. Two controls were used, rst control (sterile media mixed with 1mM Copper sulphate) to establish that media components cannot reduce the copper ions to copper nanoparticles and a negative control (copper sulphate solution) to con rm that no color change is observed by time. For any visual color change the asks were observed daily. Usage of UV-visible spectroscopy in the range 200-800 nm was measured for asks with color adjustment (Shantkriti and Rani, 2014).

Optimization of different factors on the production of CuONPs by Streptomyces sp.MHM38
Effect of copper concentration on NPs production In 15 mL of 1mM to 10 mM CuSO 4 , 15 mL of cell-free supernatant were added. The blend was incubated as above and UV-vis-spectroscopy was used for the collection and study of CuONPs.

Effect of reaction time on NPs production
Due to time, nanoparticles synthesis and stability are signi cant. 15 mL of cell-free supernatant was added to 15 mL of 5mM CuSO4 solution. The mixture has been incubated for various periods as above; for CuONPs, a UV vis analysis has been performed and analyzed.
Effect of substrate to ltrate ratio on NPs production In order to study the effect of different substrate to ltrate ratio on the CuONPs formation, three asks were prepared one contained 15 mL of cell-free supernatant was added to 15 mL of 5mM CuSO 4 solution; other one 15 mL of cell-free supernatant was added to 7.5 mL of 5mM CuSO 4 solution and the last one 15 mL of cell-free supernatant was added to 30 mL of 5mM CuSO 4 solution. The mixture was incubated static for 60 min and CuONPs formed were analyzed by UV-vis spectroscopy.

Effect of pH on NPs production
Cell-free supernatant was exposed to 5 mm CuSO 4 at pH 6, 7 and 8 and incubated for 60 min in order to investigate the impact of pH on CuNP 's production. UV-vis spectroscopy has analyzed the formed CuONPs.

UV-visible spectral analysis
Color changes were observed for biosynthesized copper nanoparticles using the free cell supernatant. CuNPs was characterized by UV-visible spectroscopy in range of 200-800 nm, at regular intervals.

Transmission Electron Microscope analysis
CuONPs solution was diluted and sonicated. A drop was placed on carbon coated grid and water evaporated. TEM measurements were performed on a JEM-100-CX, worked at a voltage acceleration of 80 KV dry, then examined with a transmission electron microscope at the Faculty of Science, Alexandria University.
Energy Dispersive X-ray (EDX) spectroscopy analysis The technique described by (Jyoti et al., 2016) is used mainly to determine the elementary structure of the sample and to con rm that the suspension of the nano part contained only copper (Roy et al., 2013). This analysis was done by using powder of lyophilized copper nanoparticles. A sample was analyzed using Oxford instrument attached to scanning electron microscope at the Electron Microscope Unit, Faculty of Science, Alexandria University.

X-ray Diffraction (XRD) analysis
CuONPs sample was dried for XRD pattern analysis recorded in the transmission mode on Shimadzu XRD7000 instrument (at the Central Laboratory, City of Scienti c Research and Technological Applications, Egypt) operating at 40KV current 30mA with Cu Ka radiation (λ=1.5404 A) (Fayaz et al., 2011). A monochromatic X-ray beam with wave length lambda was used to analyze the crystalline nature of the sample (Karthik et al., 2014) Biotechnological application of copper nanoparticles Antimicrobial activity of CuNPs by agar diffusion method Biosynthesized Cu-NPs were examined for antimicrobial activity against Gram-positive bacterial pathogens (Enterococcus faecalis ATCC 29212), Gramnegative bacteria (Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8939), fungi (Rhizoctonia solani, Fusarium solani, Aspergillus niger) and yeast (Candida albicans ATCC 10237) using the well-diffusion method on Mueller-Hinton Agar (MHA) was used for bacteria, Sabouraud Dextrose Agar (SDA) was used for C. albicans and potato D-glucose agar (PDA) used for fungi. A 100 μl bacterial suspension was used in the preparation of bacterial lawns for each bacterial test organism. The staff of the National Institute of Oceanography and Fisheries (NIOF), Alexandria branch received all the bacterial and yeasts cultures, The Assiut Mycological Center (AUMC) University, Assiut, Egypt, supplied fungal cultures as well. An 8mm diameter agar well was made by a sterilized cork borer in stainless steel. The wells were loaded with 100 μl of 200 mg/mL concentrations of CuO nanoparticles solution and 100 μl of culture broth from Streptomyces sp. MHM38 cell free supernatant and a DMSO was used as a solvent. The plates were incubated for 24 hours at 37 o C for bacteria and 120 hours at 28 o C for fungi then examined for inhibition zones. The diameter of such inhibition areas was measured and the mean value was recorded in millimeters for each organism (El-Naggar and Abdelwahed, 2014).

CuO NPs against oxidative stress Animal and Experimental Design
Eighty healthy, 8-week-male albino rats (Sprague -Dawley strain) weighing 180-200g was obtained and housed in the biology lab in Agricultural Chemistry Department, Faculty of Agriculture, Minia University at a controlled temperature of (25 ± 2°C) with 12 hr. dark/light photoperiod for an adaptation period of two weeks. The study protocol was approved by the Agricultural Chemistry Department Ethics Committee, Minia University Faculty of Agriculture; rats were randomly divided into eight groups (10 rats in each group); and subjects for 21 days to one of the following treatments: After 24 hours, rats were sacri ced under light ether narcosis followed by decapitation obtain biomaterials (blood and liver) for research. The blood sample was collected from the portal vein in the tubes that do not contain anticoagulants. The blood samples obtained were centrifuged at 1200 g for 15 minutes to separate the serum. The serum obtained was used to estimate biochemical analyses following: alanine aminotransferase (ALT), aspartate aminotransferase (AST), Lactate dehydrogenase (LDH) , alkaline phosphatase (ALP), total and direct bilirubin, urea, and creatinine which were measured Homogenization of the liver was done in homogenization buffer PBS (pH 7.4) and then homogenates were centrifuged at 10,000 g for 30 min (+4 °C) to obtain supernatants. The supernatants of liver tissues of rats were used to analyze for glutathione (GSH) levels according to (Davies et al., 1984), nitric oxide (NO) according to (Montgomery and Dymock, 1961), malondialdehyde (MDA) according to (Ohkawa et al., 1979), and superoxide dismutase (SOD) according to (Kakkar et al., 1984).
Liver tissues were also xed in neutral buffered formalin10 %, dehydrated, cleared and para n ionized for para n blocks and 5 micron sections were obtained, mounted on a glass slide and stained with Hematoxylin and Eosin (H&E), and Prussian blue reaction according to (Bancroft and Gamble, 2008) Statistical analysis Data are presented as mean ± standard error of the means (SEM). One-way analysis of variance (ANOVA) followed by Tukey post-hoc test using the Statistical Package for the Social Sciences (SPSS software V. 18.0) were performed for statistical analysis. P values < 0.05 were considered as the signi cant level.

Results And Discussion
Evaluation of biosynthesis of Copper Oxide nanoparticles by Streptomyces sp.MHM38 Streptomyces sp. MHM38 had the potential to reduce the copper ions to copper nanoparticles. The biosynthesis of copper nanoparticles was indicated by changing the faint blue reaction blend to green after 1 % (v / v) of a 1 mM aqueous CuSO 4 was added to the cell-free Streptomyces sp. MHM38 supernatant. No color change, by comparison, was observed in aqueous CuSO 4 incubated under the same conditions without free cell supernatant (Fig.1).
The formation of colors depends on the surface vibration of Plasmon on the surface (Shantkriti and Rani, 2014). Our results agree with (Shantkriti and Rani, 2014) who mentioned that when cell-free supernatant of Pseudomonas uorescens was added to CuSO 4 solution and incubated for 48 h, the reaction mixtures color changed from blue to dark green.

UV-Visible spectral analysis
The presence of nanoparticles with the UV-visible spectrophotometry within the 200-800 nm range has been con rmed. The copper nanoparticles formed by the Streptomyces sp. MHM38 showed an absorption peak of 550 nm ( g. 2) of the different Surface Plasmon Resonance spectra (SPR), indicating the existence of CUNPs. Depending on individual particle properties like size , shape and capping agents, the exact location of the SPR Band can vary (Mott et al., 2007). (Brause et al., 2002) reported that surface-plasmon resonation is dominant in the optical absorption of metal nanoparticles and particle size is linked to the absorption pick. Copper nanoparticles in aqueous solution have the Surface Plasmon Resonance increase to longer wavelengths, with particle size increasing. The position and form of copper nanocluster absorption of plasmon is strongly dependent on the particle size, stabilizing molecules or surface adsorbed particles, and bioelectricity of the media. (Krishnaraj et al., 2010). Our results agree with those of (Hamid, 2015) who mentioned that the copper Surface Plasmon Resonance (SPR) band of Salmonellatyphimurium occurred at 565 nm. (Shantkriti and Rani, 2014) who mentioned that the copper Surface Plasmon Resonance (SPR) band of Pseudomonas uorescens exhibits a distinct absorption peak in the region of 550-650 nm.

Optimization of CuONPs by Box-Behnkin design
Effect of copper concentration on NPs production From Fig. 3, it is clear that the rate of formation for CuONPs increased with the increase of substrate concentration reaches its maximum at 5 mM, as it was shown here the best observation for CuONPs is at concentration 5mM of

Effect of pH on CuONPs production
Altering the pH is said to help control nanoparticles' shape and size (Gurunathan et al., 2009). The peaks of acidic pH of 6 were not typical of CuONPs.
The alkaline pH has provided a high absorption peak at 550 nm. While (Shantkriti and Rani, 2014), characteristic peak of CuONPs formed by Pseudomonas uorescens at neutral pH. After optimization we can estimate the most optimum conditions for CuONPs was substrate concentration 5mM, equal volume of ltrate and substrate were added, pH adjusted at 8 and incubated static for 60 min. As showed in Fig.7, the color of solution changed to green color when we applied the preview conditions, which indicates high production of CuONPs.

Characterization of CuONPs Transmission Electron Microscope (TEM)
Many studies have classi ed copper nanoparticles in their shape and size based on TEM structures (Singh et al., 2015). The present work reveals the spherical form of nanoparticles in the TEM images of copper nanoparticles (Fig. 8). TEM analysis of CuONPs produced by Streptomyces sp. MHM38 are relatively uniform in shape. In general, spherical particles appear with an average dimensional size of 1.09 -6.54 nm. These are smaller than CuNPs Energy dispersive X-ray (EDX) spectroscopy analysis EDX and elementary mapping determined the purity and elemental composition of the nanoparticles. In the current research EDX spectroscopy analysis was performed for CuONPs produced by Streptomyces sp. MHM38 (Fig.10), which con rmed the presence of elemental copper by the signals. In the EDX spectrum, the nanoparticles displayed a peak at 8 keV, which is due to the absorption of copper oxide nano crystallites corresponding to surface plasmon resonance (Maqbool et al., 2018). The optical absorption band peak for nanoparticles produced by Streptomyces sp. MHM38 was in the range of 1 to 9 keV is typical for the absorption of copper oxide nano crystallites. The main component observed was copper oxide; other elements were observed, such as calcium, chloride, phosphate and carbon. The carbon distribution was triggered by the use of a TEM network. However; there were other EDX peaks for calcium, phosphorus and chloride and that also represents an essential ingredient in bacterial structural proteins that have functional groups suggesting that they were mixed precipitates from the centrifuged supernatant/metal solution.

Antimicrobial activity of biosynthesized CuONPs
Nanoparticles have elevated surface-volume ratio, tiny size and elevated dispersion characteristics that enable them to interact with microbial surfaces. The Cu-NPLs ' large surface area enhances their interaction with the microbes in order to perform wide antimicrobial operations (Shende et al., 2015). However, the few reports on Cu-NPLs antimicrobial research have shown that Cu-NPLs are effective against multiple pathogenic microorganisms (Hassanien et al., 2018). The anti-microbial activity of CuONPs was carried out on pathogenic bacterial strains Gram-positive bacteria (Enterococcus faecalis ATCC 29212), Gram-negative bacteria (Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8939), fungi (Rhizoctonia solani, Fusarium solani, Aspergillus niger) and yeast (Candida albicans ATCC 10237) using the method of well diffusion, and the inhibition area values are shown in (Table 1 & gure 11). In each plate and DEMSO as control, cell-free supernatant broth with no CuSO 4 addition is maintained. The highest antimicrobial activity was observed against Candida albicans ATCC 10237 and Pseudomonas aeruginosa ATCC 9027, whereas a lower activity was found against Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 8939. Such ndings are consistent with those of previous studies which examined Candidaalbicans and Pseudomonasaeruginosa antimicrobial activity of Cu-NPs. (Hassanien et al., 2018). The appearance of the inhibition area showed that in these locations there was no growth of bacteria. This shows how biosynthesized Cu-NPs interact with a smaller part / high surface area, of which Cu-NPs have been adsorbed onto the surface of the microorganism cell wall. As a result of this, cell walls that destroy human pathogens were demolished and disrupted by the resistance property of biosynthesized Cu-NPs. The antibacterial activity of copper metal is licensed by the United States as a microbial. EPA Agency (Environmental Protection Agency) (Hassan et al., 2019). The inhibitory action of CuO-NPs can be due to their small size and high volume-to-volume surface area, allowing it to interact with microbial cell membrane (Usman et al., 2013). In addition, their inhibitory action is related to the production of hydroxyl radicals that ruin the helical structure of DNA by binding it and harm vital proteins by binding amino sulfhydryl and carboxyl amino acid groups and then inactivating important enzymes (Yoon et al., 2007). Espirito Santo et al. indicated that ‚ CuO-NPs inhibitory action associated with inactivated surface protein responsible for transporting material across cytoplasmic membranes and destroying selective permeability (Santo et al., 2008). Marine actinomycetes have recently revealed biosynthesis of CuNPs and their applications against pathogenic microbes (Rasool and Hemalatha, 2017).
The effect of CuO-NPs produced by Streptomyces sp. MHM38 against paracetamol-induced liver and kidney damage The tests for ALT, AST, ALP, and bilirubin are important in diagnosing the condition of the liver. When liver cells (hepatitis) deteriorate, they are released into the bloodstream and level above the normal range. Urea and creatinine are both metabolic wastes excreted by the kidneys through urine, and only a small amount remains in the blood. If there is a disorder of kidney function, then there is an expansion in these two parameters. LDH is a conspicuous marker and a diagnostic tool for tissue injury. As shown in the Table.2, there were no signi cant differences in ALT, AST, ALP, LDH, total and direct bilirubin, Urea, and Creatinine between the groups treated with Cu-NPs compared with the control group. Although these parameters increased with increasing dose of CuO-NPs. Conversely, signi cant increases were demonstrated in the ALT, AST, ALP, and LDH, total and direct bilirubin, Urea, and Creatinine levels in paracetamol treated rats compared to the controlled value. Meanwhile, pretreatment with CuO-NPs recorded suppression with these values as shown in Table 2. These results were in agreement with (Ravindran et al., 2013) who explained that paracetamol increased levels of ALT, AST, ALP, and bilirubin, Urea, and Creatinine. Paracetamol turns into water-soluble products by drug metabolism enzymes and excreted in the urine in a therapeutic dose (Hinson et al., 2010). Excess paracetamol is oxidized to N-acetyl-pbenzoquinone imine (NAPQI) which is a toxic and is done by the hepatic cytochrome p450 system (CYP450) (Yen et al., 2007). Detoxi cation is normally expelled from NAPQI by GSH.GSH depletion occurs when using high doses of paracetamol and consequently the toxic NAPQI accumulation that binds to cellular proteins via cysteinyl sulfhydryl groups and forms NAPQI-protein adducts Paracetamol caused an increase in the level of LDH. Paracetamol works to accumulate Ca 2+ intracellular, which activates anaerobic glycolysis and phosphofructokinase, forming lactate and thus increasing LDH (Landowne and Ritchie, 1971). Returning these enzymes to normal levels demonstrates the protective effect of CuO-NPs against liver and kidney damage and their ability to regenerate liver and kidney cells. These results agree with (Zhang et al., 2019).Green synthesized nanoparticles exhibit a bene cial effect against liver and kidney degradation because the bacteria used in the synthesis of nanoparticles have medicinal properties. These results are consistent with (Ghaffar et al., 2014). The protective activity of CuO-NPs may be attributed to its role in preventing cellular leakage and losing the functional integrity of the cellular membrane in hepatocytes and kidney.
The effect of CuO-NPs produced by Streptomyces sp. MHM38 against paracetamol-induced oxidative stress In this study, the oral administration of biosynthesized CuO-NPs alone did not induce any obvious changes in the most biochemical parameters compared with the control group. The administration of paracetamol produced a signi cant increase in NO and MAD content accompanied by a marked inhibition of GSH and SOD activities compared with the control group. The administration of biosynthesized CuO-NPs led to a signi cant decrease in NO and MAD content and an increase of GSH and SOD levels compared to the paracetamol group (Table 3).
Non-enzymatic antioxidants (GSH) and enzymatic antioxidants (SOD) in natural conditions regulate the removal and production of free radicals and thus maintain the level of ROS. Thus, this antioxidant protects the body from oxidative stress. (Ravindran et al., 2013) explain that consuming high doses of paracetamol reduces the activity of these enzymatic antioxidants and makes cells more vulnerable to injury caused by free radicals. (Madkour and Abdel-Daim, 2013) showed that high doses of paracetamol cause oxidative stress and damage the liver, causing increased levels of MDA and NO and a decrease in the activities of SOD compared with the control group. Cytotoxicity occurs due to oxidative stress when the level of free radicals is increased to the point that cells are unable to remove them and prevent their formation. An increased level of MDA and NO and decreased level of SOD and GSH is an indication of tissue damage and failure of an antioxidant system.
It is assumed that the high antioxidant state by CuO-NPs (1, 2 and 5 mg/kg.b.wt) provides protection against lipid peroxide by scavenging free radicals. From these results, we conclude that the CuO-NPs positively modify the state of the antioxidants and restore them to an almost normal rate (Table 3) Treatment with CuO-NPs reduced the pathological changes caused by paracetamol, which enhanced its ability to protect the liver from paracetamol toxicity (Figures 11(F, G and H). These results are consistent with (Keshari et al., 2018). (Bhuvaneswari et al., 2014) appeared that nanoparticle ameliorates liver tissue for CCl4-treated rats. The drug's ability to reduce injuries or maintain the physiological liver function after induction of poisoning is an indication of its hepatoprotective impact (Yadav and Dixit, 2003).

Conclusions
The results appeared that copper nanoparticles biosynthesized from mrine Streptomyces sp. MHM38 has no toxic effect on the liver of the studied rats; it also mitigated the adverse effects of paracetamol, however. This shows that it can be used for several bene cial purposes, including prophylactic impacts on the liver.

Declarations
Ethics approval and consent to participate Approval Consent for publication We are agree

Availability of data and materials
The datasets generated during the current study are available from the corresponding author on reasonable request.

Competing interests
There are no con icts of interest   Results are expressed as mean ± SE (n=10) where mean signi cant at p < 0.05. a compared with normal group; b compared with paracetamol groups.    Effect of reaction time on the production of CuONPs by Streptomyces sp. MHM38.

Figure 5
Effect of ratio between ltrate (cell-free supernatant) to substrate (CuSO4 solution) concentration on the production of CuONPs by Streptomyces sp. MHM38.

Figure 6
Effect of pH on the production of CuONPs by Streptomyces sp. MHM38.   Shows X-Ray diffraction pattern with regards to the biosynthesized CuONPs by Streptomyces sp. MHM38.   Section of livers rats.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Abs.docx