Suppressor Capacity of Copper Nanoparticles Biosynthesized Using Crocus Sativus L. Leaf Aqueous Extract on Methadone-Induced

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OPEN Suppressor capacity of copper nanoparticles biosynthesized using Crocus sativus L. leaf aqueous extract on methadone‑induced cell death in adrenal TICLE phaeochromocytoma (PC12) cell line AR Peng Zhang1, Jian Cui2*, Shirin Mansooridara3, Atoosa Shahriyari Kalantari4, Akram Zangeneh5,6, Mohammad Mahdi Zangeneh5,6, Nastaran Sadeghian7, Parham Taslimi8, Ramazan Bayat9 & Fatih Şen9

In this research, we prepared and formulated a neuroprotective supplement (copper nanoparticles in aqueous medium utilizing Crocus sativus L. Leaf aqueous extract) for determining its potential against methadone-induced cell death in PC12. The results of chemical characterization tests i.e., FE-SEM, FT-IR, XRD, EDX, TEM, and UV–Vis spectroscopy revealed that the study showed that copper nanoparticles were synthesized in the perfect way possible. In the TEM and FE-SEM images, the copper nanoparticles were in the mean size of 27.5 nm with the spherical shape. In the biological part of the present research, the Rat infammatory cytokine assay kit was used to measure the concentrations of infammatory cytokines. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test was used to show DNA fragmentation and apoptosis. Caspase-3 activity was assessed by the caspase activity colorimetric assay kit and mitochondrial membrane potential was studied by Rhodamine123 fuorescence dye. Also, the cell viability of PC12 was measured by trypan blue assay. Copper nanoparticles-treated cell cutlers signifcantly (p ≤ 0.01) decreased the infammatory cytokines concentrations, caspase-3 activity, and DNA fragmentation and they raised the cell viability and mitochondrial membrane potential in the high concentration of methadone- treated PC12 cells. The best result of neuroprotective properties was seen in the high dose of copper nanoparticles i.e., 4 µg. According to the above results, copper nanoparticles containing C. sativus leaf aqueous extract can be used in peripheral nervous system treatment as a neuroprotective promoter ETRACTEDand central nervous system after approving in the clinical trial studies in humans.

R 1 Department of Neurosurgery, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Medical College of Henan University, Zhengzhou 450003, Henan, China. 2Department of Neurosurgery, Xi’an No. 1 Hospital, No. 30 South Street Powder Lane, Beilin District, Xi’an 710002, Shaanxi, China. 3Medical Sciences Research Center, Faculty of Medicine, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran. 4Department of Neurology, Faculty of Medicine, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. 5Department of Clinical Sciences, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran. 6Biotechnology and Medicinal Plants Research Center, Ilam University of Medical Sciences, Ilam, Iran. 7Department of Chemistry, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey. 8Department of Biotechnology, Faculty of Science, Bartin University, 74100 Bartin, Turkey. 9Sen Research Group, Department of Biochemistry, University of Dumlupınar, 43000 Kütahya, Turkey. *email: [email protected]

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In the last two decades, attention to nanomaterials, especially metal nanomaterials, has grown signifcantly due to their interesting and unique properties 1–5. Te properties of nanoparticles in nano sizes have created unique features that have made their application in various sciences attractive6–10. Meanwhile, metal oxide nanomaterials, especially copper oxide nanoparticles, have shown more unique properties, which has increased their use in various sciences11–15. Te demand for conventional healthcare treatment is rising every day. Iranian traditional medicine is one of the traditional and accepted methods of conventional medicine all over the world16–22. Numerous medicinal supplements and drugs are developed per annum and manufactured from traditional medicinal plants. One of these plants is Crocus sativus L. C. sativus is from Plantae kingdom, Magnoliophyta division, Liliopsida class, Asparagales order, Iridaceae family, and Crocus genus 23. Te most antioxidant compounds of C. sativus are crocin, picrocrocin, and safranal. C. sativus is used in the medicine for its antioxidant, anti-infammatory, antinociceptive, antibacterial, antifungal, antiviral, anti-parasitic, gastro-protective, entro-protective, hepatoprotective, nephron-protective, spleno-protective, anticancer, and especially anxiolytic and relaxant properties. As a neuroprotective supplement, C. sativus is involved in the treatment of nervous disorders in conventional medicine23. An in vitro study displayed the inhibitory impact of C. sativus on amyloid beta-peptide fbrillogenesis 24 and its defensive function against the toxicity of H 2O2-triggered human neuroblastoma cells . In addition, C. sativus was administered for one week to normal and aged mice (60 mg/kg body weight), and markedly increased memory and learning 25. In addition, in vitro studies in amnesic and ischemic rat models have verifed the neuroprotective efects of C. sativus and its components 26. C. sativus has been declared to inhibit peroxidized lipid formation in cultured adrenal pheochromocytoma (PC12) cells, in moderation restored superoxide dismutase (SOD) activity and preserved morphology of neurons. Although C. sativus antioxidant impact was comparable to that of α-tocopherol, at certain concentrations it was much more prominent 25. Methadone is an artifcial, long-acting opioid with qualitatively similar morphine-like pharmacological actions and is active through oral and parenteral administration. Methadone is a small molecule that contains hydrophobic and hydrophilic units. Methadone can be considered aTICLE small amphipathic agent for mitochondria which acts as an ion carrier 26,27. N-methyl-d-aspartate (NMDR) and μ opioid receptors are considered to have a greater impact on the treatment of chronic pain and neuropathic pain caused by the activity of methadone 28–32. In a study on the SH-SY5Y cell line, this undiferentiated human neuroblastoma cell line led to severe cell deaths using methadone at high concentrations33. Increasing the production of free radicals and increasing the amount of calcium afect increasing cell deaths. Methadone does not directly produce reactive oxygen species, however, an increment in intracellular calcium can ARcause toxicity26. Te decomposition activity of methadone may result from the ability of small ions to transfer to the phospholipid membrane. In addition, methadone, like other opioids, has been shown to leak into the membranes of lipophlipids made up of phospholipids and form pores27,34. Methadone-exposed cells lie behind the mitochondrial outer membrane and BAX transfer ’s high permeability and also release cytochrome C from mitochondria33. A study conducted on rat liver mitochondria that have been treated using methadone, methadone induced mitochondrial disruption and also showed that it has the ability ions transferred, therefore, it causes cellular death due to remarkably reducing cellular ATP levels35,36. One group of materials that can remove the neurotoxicity activities of the psychedelic drugs such as methadone is the metallic nanoparticles synthesized by plants37. Recently, scientists have used the neuroprotective potentials of medicinal plants for synthesizing the metallic nanoparticles containing natural compounds. So far, the neuroprotective efects of Salvia Ofcinalis, Hypericum perforatum, Lavandula angustifolia, Opuntia fcus-indica, Curculigo orchioides, Ficus religiosa, Angelica sinensis, Cassia fstula, Dichrostachys cinerea, Panax ginseng, Aerva lanata, Juglans regai, Crocus sativus, Pongamia pinnata, Polygala paniculata, Cipura paludosa, Carum carvi L., Cymbopogon winteri-anus, Mentha spicata L., Cassia siamea, Galanthus nivalis L., Tymus vulgaris L., and Curcuma longa have been proved38. Attention to nanotechnology and synthesis of diferent types of nanomaterials increase in recent years 2,4–6,39. Tis increasing is relative to unique properties and wide range application of nanomaterails 7–9,13,40. In between, attention to metal oxide nanoparticles show more interest between researchers due to large surface area and catalytic activity10–12,41,42. Copper nanoparticles (CuNPs) have a special role between all metallic nanoparticles39,40. CuNPs have been recently described as having signifcant neuroprotective potential and being able to treat the central and peripheral nervous systems37. One study investigated the positive activity of CuNPs on cerebral microvascular endothelial cells of rats. Previous research, copper nanoparticles 1.56–50 µg/ml exposing at concentrations increases proliferation of rat cerebral microvascular endothelial cells. Copper nanoparticles (40 and 60 nm) the release of prostaglandin E2 was increased noteworthy manner (3 times, 8 h). Tere were no changes in the extracellular levels of both IL-1b and TNF-a afer exposure to copper nanoparticles Rat brain as an indi- cation of increased permeability of the microvascular endothelial cells, P-an aspect ratio greater than about ETRACTEDtwofold for copper nanoparticles37. In addition, neurotoxic agents in the peripheral nervous system and central nervous system may be removed by copper nanoparticles37. About the neuroprotective properties of CuNPs green-synthesized by plants, the neuroprotective efects of Achillea biebersteinii leaf aqueous extract synthesized R CuNPs against methamphetamine-induced cell death in PC12 were investigated by Wang et al. (2020). Tey indicated when copper salt is combined with plant extract, herbal nanoparticle with signifcant antioxidant activities is synthesized and probably neuroprotective properties of copper nanoparticles green-synthesized by plants are related to these antioxidant efects43. Based on the neuroprotective properties of C. sativus and CuNPs, we have attempted to determine the CuNPs efects comprising C. sativus leaf aqueous extract on cellular and molecular variables like viability and cell death index in methadone-induced PC12 cell death. In these cells, the activity of caspase-3, infammatory cytokine concentration, and mitochondrial membrane potential were also examined to efectively fgure out the functioning mechanism of the CuNPs.

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Experimental Material. Antimycotic antibiotic solution, decamplmaneh fetal bovine serum, Dulbecco’s Modifed Eagle Medium (DMED), carbazole reagent, 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH), 4-(Dimethyl- amino)benzaldehyde, phosphate bufer solution (PBS), borax-sulphuric acid mixture, Ehrlich solution, hydro- lysate, and dimethyl sulfoxide (DMSO), all of this was obtained from Sigma-Aldrich, USA.

Synthesis of copper nanoparticles. Te C. sativus used in this study was obtained in Kermanshah, Iran, at the elevation of 1,400 m hereinabove sea level, in spring 2019, afer it was identifed. Extraction was obtained by dissolving 250 g of C. sativus leaf powder in 2,500 mL of distilled water and holding it in an orbital shaker for 48 h. It then concentrated the extract in a rotary evaporator. In addition, galenic extracts were lyophilized for 37 24 h at − 48 °C and stored for later use . With a 20 mL of Cu(NO3)2·3H2O reaction mixture in the concentration of 0.05 M and 200 mL of aqueous extract solution of C. sativus leaf (20 μg/mL) in the proportion 1:10 in a coni- cal fask, the green synthesis of the copper nanoparticles was launched. Te solution mixture was held at room temperature under a magnetic stirring for 12 h. Te dark green colored solution of Cu was produced at last time of reaction. Te mixture obtained at the end of the reaction was centrifuged at 10 × 103 rpm for 15 min. Te sedi- ment was washed with water at three times, then centrifuged44,45. Te that organic chemistry techniques, i.e., for the study of CuNPs FE-SEM, FT-IR, TEM, and UV–Vis spectroscopy was used. TEM and FE-SEM microscopic methods analyzed the morphological properties of CuNPs in terms of shape and size. In addition, CuNPs were primarily tested utilizing UV–Vis spectroscopy at a scanning range of 450–750 nm of the FT-IR spectrophotometer wavelength to classify biomolecules involved in CuNP reduction. Te XRD pattern of CuNPs was obtained in the 2θ range of 30°–80° by a GNR EXPLORER device at 40 kV, 30 mA, and Cu-Kα radiation (1.5406 Å). TICLE Neuroprotective analyses of copper nanoparticles. Cell culture. PC12 (ATCC CRL-1721TM) cells used in this experiment were cultured according to protocol rules in the Gibco RPMI1640 cell culture environment. 100 IU/mL penicillin (Sigma), 100 µg/mL streptomycin and 10% fetal bovine serum supported in cell 2 cultures and standard case in T-25 cm tissue culture bottles (5% incubated at 37 °C in CO2). It led to changes in the morphology of methadone cells, so apoptotic cells led to the cell being treated with diluted methadone with 100 mM water. For Cu(NO3)2, C. sativus leaf aqueous extract, CuNPs solutions in the RPMI1640 water cell cultural environment were frst dissolved in DMSO andAR added to the cultural environment with a fnal volume of 0.1%. Te cells were then washed by PBS (37 °C) 12 h afer the plating and divided into eight classes, including I) Methadone: Cell culture contains 100 μM methadone; II) Control: Cell culture medium without methadone, Cu(NO3)2, C. sativus leaf aqueous extract, and CuNPs; III) T1: Cell culture contains 100 μM methadone and 2 μg of Cu(NO3)2; IV) T2: Cell culture contains 100 μM methadone and 4 μg of Cu(NO3)2; V) T3: Cell culture contains 100 μM methadone and 2 μg of C. sativus leaf aqueous extract; VI) T4: Cell culture contains 100 μM methadone and 4 μg of C. sativus leaf aqueous extract; VII) T5: Cell culture contains 100 μM methadone and 2 μg of CuNPs; VIII) T6: Cell culture contains 100 μM methadone and 4 μg of CuNPs.

Caspase‑3 activity. For plating PC12 cells, the well cell culture plate containing the PRMI1640 medium was used. Afer 12 h, the plate was washed by PBS. Ten, the diferent treatments of I-VII were added to the cells. Trypsin was used for separating the cells from the fask. For removing the supernatant, all samples were centrifuged for 10 min, then the centrifuging was done by adding lysate bufer and fnal they were transferred to the well cell culture plate. Aferwards, 5 µl of N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline Caspase-3 Substrate DEVD- pNA, the wells cell culture plate was added and was allowed within 2 h of incubation at 37 °C. Subsequently, the release of pNA on the result of caspase-3 activity was measured and recorded43.

Secretion of infammatory cytokines. Pro-infammatory cytokines TNFa, IL-6 and IL-1β concentrations were measured using the Rat V-Plex Kit.

Cell death index. For determining of PC12 cell death index in the diferent treatments of I-VII, TUNEL stain- ing was used. Eight random wells with a fuorescence microscope were chosen to count TUNEL positive cells. Te ratio of cell death index to apoptotic cells to total cells is equal 43.

Cell viability. Trypan blue was used for assessing cell viability. Vital Dye, whose membranes penetrate broken ETRACTEDdamaged or Dead Cells, is seen by penetrating dead cells in the Blue lobes in the Neubauer Lamela. For 12 h, 96 wells with a 5 × 104 cell/mL density were coated on the cultural plate, then, they were cultured with various I-VII treatments and incubated at 37 °C at 5% CO 2 for 48 h. Since the cells were trypsinized, Neubauer Lamela R suspended 200 μL of cell suspension 2–3 min since combining with 0.4% 40 μL of Trypan blue. Te cell viability of all samples was determined by following the formula below46: Cell viability: Non-colored cells number/Total cells number

Mitochondrial membrane potential (MMP). For half an hour diferent treatment were exposed to 10 mg/mL rhodamine-123. Te cell was then washed using PBS. 900 μL Triton X-100 was subsequently added to each well and held for 2 h at 4 °C. Te solutions were taken into microtubes to be centrifuged at 16,000 rpm for 20 min. Fluorescent absorbing in cells was performed using a fuorescent microplate reader (488 nm excitation and 520 nm emissions)47.

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TICLE Figure 1. FE-SEM image of copper nanoparticles.

Evaluation of the antioxidant activity of copper nanoparticles with DPPH. Troughout the start of the study, 100 mL of methanol (50%) was appliedAR to DPPH’s 39.4 g. Also reported were some amounts of Cu(NO3)2, C. sativus leaf aqueous sample, and CuNPs, i.e., 0–1,000 μg/mL. Te following DPPH has been applied to Cu(NO3)2, C. sativus leaf aqueous sample, and CuNPs at diferent concentration. At a temperature of 37 °C all samples were transferred to an incubator. For this analysis, the negative and positive controls were methanol (50%) and butylated hydroxytoluene (BHT), respectively. Te antioxidant properties of Cu(NO3)2, C. sativus leaf aqueous extract, and CuNPs were defned according to the following formula 44,48,49: DPPH free radical scavenging (%) = (Control − Test/Control) × 100

Statistical analysis. Te data obtained at the end of the study were fed to the SPSS-22 program and the Tukey post-hoc test (p ≤ 0.01) afer the one-sided ANOVA test. Results and discussion In this study, copper nanoparticles were prepared and synthesized in a watered environment using C. sativus leaf extract as decreasing and stabilizing agents. Also, we investigated their potential against methadone-induced cell death in PC12.

Chemical characterization of copper nanoparticles. Te surface size and morphology of these nanoparticles were determined using FE-SEM in Fig. 1. Te pictures showed that the biosynthesized copper nanoparticles are homogeneous, well dispersed and homogeneous. A collection tendency is seen for synthetic nanoparticles. Metallic nanoparticles, such as copper nanoparticles, silver nanoparticles, gold nanoparticles, and titanium nanoparticles synthesized using environmentally friendly methods have been reported in previous studies 49–51. In our analysis of the literature, various sizes have been recorded for biosynthesized copper nanoparticles utilizing herbal extracts. For instance, 10–100 nm for Punica granatum peels extract52.; 21.50–34.23 nm for the aqueous oak fruit hull extract53; 20–60 nm for extracting cofee powder54; 5–20 nm for extract Z. spina-christi55; 20–50 nm for the synthetic copper nanoparticles using Olea europaea leaf extract 56; 20–40 nm for leaf extract ETRACTED57 58 from Solanum lycopersicum ; and 15–20 nm for extract of Punica granatum . In addition, the average nanoparticles scale (27.5 nm) determined by means of TEM images and ImageJ sofware package (Fig. 2). In addition, the histogram graph showed in TEM image that biosynthesized copper R nanoparticles are in particle size distribution in the range of 14 to 38 nm. In previous experiments in the ranges of 10–50 nm the scale of copper nanoparticles developed by aqueous extract of medicinal plants with the shape of spherical was measured 45,59. Te fndings reported in such studies refect the existing research outcomes. UV–Vis. spectrums of biosynthesized copper nanoparticles utilizing the aqueous extremity of the C. sativus leaf are shown in Fig. 3. UV–Vis. spectroscopy was used for the surface plasmon resonance of copper nanoparticles. Te appearance of a band with a wavelength of 545 nm indicates that the formation of copper nanoparticles has taken place. Previous studies have reported that copper oxide is on a hill in the wavelength range of 540–580 nm for biosynthesis 51,58. Peaks between 450 and 700 cm−1 in the FT-IR spectrum (Fig. 4), 450 to 700 cm−1 in the region belong to metal–oxygen vibrations. Te development of copper oxide nanoparticles in this sample is confrmed by the

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TICLE Figure 2. TEM image of copper nanoparticles. AR

Figure 3. Te UV–Vis spectrum of biosynthesized copper nanoparticles.

inclusion of a hill at 488 cm−1 Cu(II)–O belongs to the bending vibration60,61 and the bending vibration of −138. Cu(I)–O at 618 cm Simultaneous biosynthesis of CuONPs and Cu2ONPs using herbal extracts has already been reported previously. Copper nitrate was used using the FT-IR method to examine the existence of second- ary metabolites responsible for closure and growing its predecessor to CuONPs. In the C. sativus leaf extract was observed the existence of various IR bands involved in the existence of diversifed functional groupings. For example, peaks in the range of 1,402 to 1601 cm−1 provisions with C=C and C=O yawning peaks at 3,409 and 2,924 cm−1 relate to O–H and aliphatic C–H stretching and can be attributed to the peak –C–O–C yawn ETRACTED−1 at 1,088 cm . Tese peaks can be used to confrm compounds with the help of compounds, such as previously reported phenolic, favonoid and carboxylic experiments obtained at the end 62. R XRD is used for the phase identifcation and characterization of copper nanoparticles’ crystal structure (Fig. 5). In the case of Cu comprising the sample, it is possible to index the XRD peaks at 43.2°, 50.3° and 74.1° to (111), (200) and (220) Bragg ’s face-centered cubic (fcc) structure of metallic Cu, similar to the Joint Committee on Powder Difraction Specifcations (JCPDS No. 04–0,784). Also previously the peaks are reported at diferent degrees 37,45. Te mean crystal size of CuNPs was calculated using the Scherrer equation for the difraction of the X-ray: k D = βcosθ

CuNPs average crystal size has been found as 28.92 nm.

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Figure 4. FT-IR spectra of copper nanoparticles which are biosynthesized.

ETRACTED R

Figure 5. XRD pattern of biosynthesized copper nanoparticles.

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Figure 6. EDS pattern of biosynthesized copper nanoparticles.AR

Te prepared nanoparticles’ energy-dispersive X-ray (EDX) profle shows strong elemental copper signals and confrms the presence of copper in a nano form, as shown in Fig. 6. It indicates that the CuNPs prepared comprise only metallic copper, without any other impurities. Te EDX pattern obviously demonstrations that the CuNPs are crystalline. Te detected signals like CuLα below 1 keV; CuKα around 8 keV; and CuKβ below 9 keV were confrmed the presence of copper in synthesized nanoparticles. Such signals match an earlier analysis of synthesized CuNPs utilizing many medicinal plants as well37,45. Te other signals like Okα and CKα correspond to the organic molecules existing in C sativus extract that connected to CuNPs.

Antioxidant activities of copper nanoparticles synthesized using C. sativus leaf aqueous extract. Previously, synthesized copper nanoparticles also demonstrated higher antioxidant activity to form free radicals within the living system. Since CuNPs possess redox properties, they take an important role in neutralizing free radicals in living systems57. In our research, the antioxidant efects of synthesized copper nanoparticles utilizing C. sativus leaf aqueous extract were tested by DPPH assay dig up concentration-addicted results i.e. an rise in copper nanoparticles concentration induces an increase in antioxidant function. Te best results were found in the tested concentrations at the highest concentration of 1,000 μg/mL (Fig. 7). Te results of the comparative study of individual antioxidant research have identifed major diferences in the usage of radical scavenging efects. Between all materials tried (Cu(NO3)2, C. sativus leaf aqueous extract, and copper nanoparticles), Copper nanoparticles have demonstrated more outstanding results on DPPH inhibitions. Conversely, standard (butylated hydroxytoluene) represented lowly antioxidant efects in proportion to the copper nanoparticles. Te IC 50 of C. sativus leaf aqueous extract, butylated hydroxytoluene, and copper nanoparticles were 342, 252, and 188 µg/mL, respectively. Recently, researchers have made studies to evaluate the antioxidant activity properties of plants and bio- ETRACTEDmediated synthesized copper nanoparticles. Te cause of green or biosynthesized copper nanoparticles with antioxidant activity can be caused by the presence of metabolite compounds such as phenol compounds, favonoids, sugar and other carbohydrates 63–66. In addition, many researchers have noted that phenolic and favonoids that R are bound to copper nanoparticles show antioxidant activity. Te C. sativus leaf has previously been documented to be high in antioxidant compounds such as picrocrocin, crocin, and safranal. Various studies have been conducted in the feld of nanotechnology using diversifed medicinal plants, but there are yet no reports on copper nanoparticles synthesized using C. sativus leaf aqueous extract.

Neuroprotective activities of copper nanoparticles. As a result of long-term use of methadone used to treat chronic and acute pain and opioid dependence, it causes neurotoxic efects, the etiology of which is not known67,68. Organs involved in excretion and metabolism of methadone can show side efects of this drug69,70, however, direct efects on the central nervous system due to their negative efects on the central nervous system71,72. Li et al. (2016) argued that methadone harms the entirety of the white matter of the methadone consumer for a long time in a dose-dependent order73. As a result of radiological studies, methadone afects the

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Figure 7. Te antioxidant properties of Cu(NO3)2, Crocus sativus L. leaf aqueous extract, CuNPs, and BHT against DPPH.

structure of the brain in the deep white matter of the cerebellum, basal ganglia and cerebral hemispheres at high concentration and long-term consumption74,75. For methadone two signal pathways were proposed: one interacting with opioid receptors, and the other functioning as an antagonist in NMDR76. Tis NMDR path seems to have more pronounced efects on methadone toxicity in cortical cell cultures. In the feld of immunology, one study has shown that people with opioid- dependent methadone maintenance therapy afect immune system activities thus, prolonged infammation in the nervous system may also occur. A previous studies found that prolonged methadone maintenance treatment raises the rates of IL-1β cytokine, and large dose methadone cytokinine improves rates of IL-6 and TNF-a77. High concentrations of methadone toxicity have a reducing efect on the potential for cell proliferation between nerve cells, leading to cell death77. Friesen et al. (2008) methadone revealed that apoptosis and cell proliferation inhibition in leukemia cells causes cell death through inhibition. Many processes took part in this path. For instance, in apoptosis, caspase-9 and 3 are activated by methadone. In addition, apoptosis due to the Bcl-xl and X chromosome is arranged down and poly (ADP-ribose) polymerase decolletage78. Te results of our study confrmed the fndings. Tey showed that high concentrations of methadone had a signifcant lowering efect on cell viability (p ≤ 0.01) and infammatory cytokine concentrations and caspase-3 activity. Treatment of ETRACTEDthese cells improved the capacity for cell viability and cell proliferation for both doses of copper nanoparticles owing to decreased cytotoxicity of the cells (Figs. 8, 9, 10, 11). Ligand-receptor system is used by Methadone in the activation of mitochondrial apoptosis. It can also directly R rouse up apoptosis. Members of the Bcl2 family regulate mitochondrial apoptosis 79,80. Tis form of apoptosis causes many replace in mitochondria. For instance, free oxygen radicals are churn out that cause pore formation in the mitochondrial membrane. As noted below, cytochrome C activates Caspase 3 using caspase 2 or 9, releas- ing factors that induce apoptosis from the mitochondrial membrane to the cytoplasm 79,81. Apoptosis complexes are formed of Apaf-1, Caspase 9 and Cytochrome C are efectuated for activation of Caspase-3 and suction of apoptosis80. Increased absorption of Rhodamine123 indicates enhancement mitochondrial membrane potency with enhancement activity of proton pumps. Active proton pumps control membrane function in the mitochondria and inhibit the activation of apoptosis80. Te reduction in the potential of mitochondrial membrane causes c release and cascade activates of the caspase81. Apoptosis study conducted with TUNEL testing has shown in our study that methadone causes fragmentation of DNA and causes apoptosis in nerve-like PC12 cells. Our fndings

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Figure 8. Te caspase 3 absorption in diferent treatments afer 48 h. M: Methadone, C: Control, T1: 100 μM methadone and 2 μg of Cu(NO3)2, T2: 100 μM methadone and 4 μg of Cu(NO3)2, T3: 100 μM methadone and 2 μg of Crocus sativus L. leaf aqueous extract, T4: 100 μM methadone and 4 μg of Crocus sativus L. leaf aqueous extract, T5: 100 μM methadone and 2 μg of CuNPs, T6: 100 μM methadone and 4 μg of CuNPs. *It indicates the substantial gap (p ≤ 0.01) for methadone treatment trial studies.

also show that copper nanoparticles signifcantly (p ≤ 0.01) increase the mitochondrial membrane potential and reduce the rate of DNA degradation in methadone-treated PC12 cells (Fig. 12). Treatment with C. sativus extract (5 and 25 mg/ml) may reduce the neurotoxic impact of biochemical param- eters in PC12 cells, in accordance with our research. Te results showed that glucose (13.5 and 27 mg/ml) decreased the viability of PC12 cells whereas cell death decreased with pretreatment of C. sativus82. Another study revealed that for 45 days administration of C. sativus extract (200 mg/kg) and honey syrup (500 mg/kg) decreased neurotoxicity in mice to aluminum chloride-induced83. Certain experiments have shown that C. sativus has a certain benefcial impact on multiple oxidative injury factors in ischemic rat hippocampal tissue and in hippocampal tissue following administration of quinolinic acid (QA)84. C. sativus also decreased extracellular ETRACTEDglutamate and aspartate concentrations (excitatory amino acids) in anesthetized rat hippocampus following administration of kainic acid 85. With respect to the impact of C. sativus on neuronal damage and apoptosis, it was proposed that the neu- R roprotective impact of C. sativus on brain injury in animal experiments are attributed to its ability to suppress apoptosis at the early phases of the injury and its ability to induce angiogenesis at the sub-acute level as motivated by greater endothelial vascular growth factor receptor-2 levels (VEGFR) and serum response factor (SRF)86. Recent research showed that C. sativus (50 mg/kg) blocked apoptosis of retinal ganglion cells (RGCs) following retinal ischemia/reperfusion injury through the signaling pathway for phosphatidylinositol 3-kinase/AKT (PI3K/AKT). Additionally, the Bcl-2/BAX ratio improved with C. sativus87. C. sativus (10 μM) can suppress proapoptotic mRNA expression induced by tumor necrosis factor-alpha (TNF-α) that releases cytochrome c from mitochondria, and it has been proposed that crocin prevents neuronal cell death triggered by both internal 88 and external apoptotic stimulus . In addition, C. sativus can inhibit RGC-5 cell death caused by H 2O2 and suppress caspase-3 and caspase-9 development 89. Te lipid peroxidation may increase in serum/glucose-deprived

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Figure 9. Te cytokine concentration in diferent treatments afer 48 h. M: Methadone, C: Control, T1: 100 μM methadone and 2 μg of Cu(NO3)2, T2: 100 μM methadone and 4 μg of Cu(NO3)2, T3: 100 μM methadone and 2 μg of Crocus sativus L. leaf aqueous extract, T4: 100 μM methadone and 4 μg of Crocus sativus L. leaf aqueous extract, T5: 100 μM methadone and 2 μg of CuNPs, T6: 100 μM methadone and 4 μg of CuNPs. *It indicates the substantial gap (p ≤ 0.01) for methadone treatment trial studies.

cells, which may be blocked by C. sativus. Tis plant may have been able to suppress caspase-8 activation and its antioxidant properties are more pronounced at the same concentration than α-tocopherol90. Furthermore, C sativus blocked caspase-8 activation triggered by lack of serum /glucose91. In serum-deprived and hypoxic PC12 cells, C. sativus actively prevented membrane lipid peroxidation, caspase-3 activation, and cell death, which was more diferentiated than tricrocin. It has been reported that C. sativus has several related glucose esters92. C. sativus suppressed syncytin-1 and nitric oxide (NO)-induced cytotoxicity of astrocytes which oligodendrocytes and decreased neuropathology with slightly less neurological impairment in experimental autoimmune encephalomyelitis (EAE). Syncytin-1 has led to mortality and neuroinfammation of the oligodendrocytes93. Syncytin-1 is strongly expressed in lesions with multiple sclerosis in astrocytes, microglia, and the glial cells 94. Te stimulation of endoplasmic reticulum (ER) is directly related to the infammatory pathways. It has been shown that the transcript rates of the ER tension genes XBP-1/s are enhanced by EAE95. C. sativus administration decreased the repression of ER stress and infammatory gene expression in the spinal cord on day 7 afer EAE induction, as well as the release of stress genes from ER XBP-1/s96. In accordance with our work, extracts from C sativus aqueous (80–320 mg/kg) and ethanolic (400–800 mg/ kg) have minimized symptoms of opioid withdrawal induced by naloxone in mice 97. Crocin (200 and 600 mg/ ETRACTEDkg) can even decrease the withdrawal signal without reducing locomotor activity 98. It seems that copper nanoparticles, due to its antioxidant potential, dramatically (p ≤ 0.01) enhancement cell survival and mitochondrial membrane potential, and decreased infammatory cytokines concentrations, cas- R pase-3 activity, and DNA fragmentation in the high concentration of methadone-treated PC12 cells. Antioxidants

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TICLE

Figure 10. Te apoptosis index of diferent treatmentsAR afer 48 h. M: Methadone, C: Control, T1: 100 μM methadone and 2 μg of Cu(NO3)2, T2: 100 μM methadone and 4 μg of Cu(NO3)2, T3: 100 μM methadone and 2 μg of Crocus sativus L. leaf aqueous extract, T4: 100 μM methadone and 4 μg of Crocus sativus L. leaf aqueous extract, T5: 100 μM methadone and 2 μg of CuNPs, T6: 100 μM methadone and 4 μg of CuNPs. *It indicates the substantial gap (p ≤ 0.01) for methadone treatment trial studies.

cell cytotoxicity reducing efect, which is the reason for blocking the production of reactive oxygen species and oxidative stress in cells23. Conclusions Te recent research determined the chemical characterization, neuroprotective, cytotoxicity, and antioxidant properties of copper nanoparticles using Crocus sativus L. leaf aqueous extract. Tis was done using TEM, FT-IR, FE-SEM and UV–Vis tests to make chemical characterization of nanoparticles. Te resulting data showed that CuNPs were synthesized in the best probable state. In the FT-IR analysis, the inclusion in copper nanoparticles of many antioxidant compounds with related bonds induced excellent copper-reduction state. In the images of TEM and FE-SEM the copper nanoparticles had a spherical shape at an average size of 27.5 nm. Also, UV–Vis revealed the copper nanoparticles formation by a clear peak in the wavelength of 569 nm. Assessment of the antioxidant properties of copper nanoparticles was done with the common free radical scavenging test i.e., DPPH in the existence of butylated hydroxytoluene as the positive control. Te copper nanoparticles inhibited half the concentration of 188 μg/ml of DPPH molecules. Copper nanoparticles increased cell survival and Rh123 absorption in methadone-treated PC12 cells. Tese nanoparticles reduced the caspase 3 absorption, IL-1β, IL-6, and TNF-α infammatory cytokines concentration, and apoptosis index. Tese events show that copper nanoparticles are suppressed in a dose-dependent manner ETRACTEDby methadone-induced cell death in PC12 cells. In clinical studies, afer confrmation of the above-mentioned R results, copper nanoparticles can be applied as neuroprotective support in the treatment of human neurotoxicity.

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TICLE

Figure 11. Afer 48 h the cell viability for multiple therapies. M: Methadone, C: Control, T1: 100 μM methadone and 2 μg of Cu(NO3)2, T2: 100 μM methadone and 4 μg of Cu(NO3)2, T3: 100 μM methadone and 2 μg of Crocus sativus L. leaf aqueous extract, T4: 100 μM methadone and 4 μg of Crocus sativus L. leaf aqueous extract, T5: 100 μM methadone and 2 μg of CuNPs, T6: 100 μM methadone and 4 μg of CuNPs. *It indicates the substantial gap (p ≤ 0.01) for methadone treatment trialAR studies.

ETRACTED R Figure 12. Te mitochondrial membrane potential of diferent treatments afer 48 h. M: Methadone, C: Control, T1: 100 μM methadone and 2 μg of Cu(NO3)2, T2: 100 μM methadone and 4 μg of Cu(NO3)2, T3: 100 μM methadone and 2 μg of Crocus sativus L. leaf aqueous extract, T4: 100 μM methadone and 4 μg of Crocus sativus L. leaf aqueous extract, T5: 100 μM methadone and 2 μg of CuNPs, T6: 100 μM methadone and 4 μg of CuNPs. *It indicates the signifcant diference (p ≤ 0.01) for methadone treatment trial studies.

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Received: 2 April 2020; Accepted: 18 June 2020

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