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

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Suppressor Capacity of Copper Nanoparticles Biosynthesized Using Crocus Sativus L. Leaf Aqueous Extract on Methadone-Induced www.nature.com/scientificreports 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] SCIENTIFIC REPOrTs | (2020) 10:11631 | https://doi.org/10.1038/s41598-020-68142-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ In the last two decades, attention to nanomaterials, especially metal nanomaterials, has grown signifcantly due to their interesting and unique ­properties1–5. 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 vari- ous ­sciences11–15. Te demand for conventional healthcare treatment is rising every day. Iranian traditional medi- cine 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, hepato- protective, 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 neu- roprotective 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 neuroprotec- tive 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 con- centrations 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
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