Synthesis, characterization, and evaluation of cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing effects of copper nanoparticles using the aqueous extract of Strawberry fruit and l -Ascorbic acid

Journal Pre-proofs Synthesis, characterization, and evaluation of cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing effects of copper nanoparticles using the aqueous extract of Strawberry fruit and L-Ascorbic acid Saba Hemmati, Ahmad Ahmeda, Yaser Salehabadi, Akram Zangeneh, Mohammad Mahdi Zangeneh PII: DOI: Reference:

S0277-5387(20)30082-6 https://doi.org/10.1016/j.poly.2020.114425 POLY 114425

To appear in:

Polyhedron

Received Date: Revised Date: Accepted Date:

22 August 2019 18 January 2020 29 January 2020

Please cite this article as: S. Hemmati, A. Ahmeda, Y. Salehabadi, A. Zangeneh, M.M. Zangeneh, Synthesis, characterization, and evaluation of cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing effects of copper nanoparticles using the aqueous extract of Strawberry fruit and L-Ascorbic acid, Polyhedron (2020), doi: https://doi.org/10.1016/j.poly.2020.114425

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Elsevier Ltd. All rights reserved.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Synthesis, characterization, and evaluation of cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing effects of copper nanoparticles using the aqueous extract of Strawberry fruit and L-Ascorbic acid Saba Hemmatia, Ahmad Ahmedab, Yaser Salehabadic, Akram Zangeneh*,d,e, Mohammad Mahdi Zangeneh*,d,e a Department

of Chemistry, Payame Noor University, Tehran, Iran. of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar. MSC in Toxicology, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. d Department of Clinical Sciences, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran. e Biotechnology and Medicinal Plants Research Center, Ilam University of Medical Sciences, Ilam, Iran. b Department

*Corresponding authors: [email protected]; [email protected]

ABSTRACT Fragaria ananassa, also known as “Strawberry” is a common species in Iran and widely used for its antiinflammatory, anti-ulcer, astringent, anti-allergic, antibacterial, antifungal, and antidiarrheal activities and also in the treatment of skin wounds. The purpose of the study was chemical characterization and assessment of cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing properties of copper nanoparticles (CuNPs) using the aqueous extract of Strawberry fruit and L-Ascorbic acid as reducing and stabilizing agents. These nanoparticles were characterized by FT-IR, UV-visible spectroscopy, EDS, FE-SEM, and TEM analysis. TEM images exhibited a uniform spherical morphology and diameters of 10-30nm for the biosynthesized nanoparticles. DPPH free radical scavenging test revealed similar antioxidant properties for Strawberry, CuNPs, and butylated hydroxytoluene. The Strawberry and synthesized CuNPs had great cell viability dose-dependently against HUVEC cell line. In the microbiological part of this study, CuNPs showed higher antibacterial and antifungal properties than all standard antibiotics (p≤0.01). Also, CuNPs prevented the growth of all bacteria at 2-8mg/mL concentrations and destroyed them at 2-16mg/mL concentrations (p≤0.01). In the case of antifungal property of CuNPs, they inhibited the growth of all fungi at 2-4mg/mL concentrations and destroyed them at 2-8mg/mL concentrations (p≤0.01). In vivo design, the use of CuNPs ointment in the treatment groups substantially remarkably raised (p≤0.01) the wound contracture, hydroxyl proline, hexosamine, hexuronic acid, fibrocyte, and fibrocytes/fibroblast rate and reduced (p≤0.01) the wound area, total cells, neutrophil, macrophage, and lymphocyte compared to Strawberry, CuSO4, tetracycline, Eucerin basal, and untreated control groups. In conclusion, the results of chemical characterization confirm that the Strawberry fruit can be consumed to produce copper nanoparticles with a remarkable amount of remedial effects without any cytotoxicity against HUVECs. Keywords: CuNPs; Strawberry; antioxidant; cytotoxicity; antimicrobial; cutaneous wound healing.

1. Introduction Nanobiotechnology is a capable technology that deals with nanomaterials in several scientific domains such as medicine, materials science, chemistry, physics, nanotechnology, and biotechnology [1]. Metallic nanoparticles as the most important products of nanobiotechnology field, have gained significant attention in the area of biomedical technology [2]. Because of its high surface area, metallic nanoparticles are being widely used in various fields including the medical and engineering sciences [2]. Wounds can be defined as acute or chronic ruptures of any soft parts of the body, with or without damage to its functions underlying factors, and caused by external and/or internal factors. They can be classified, according to their etiology, complexity and time of existence [3]. The healing of a wound is a dynamic process to restore the structure of the injured tissue [4]. There are several diseases interfere negatively in the tissue repair process, such as diabetes mellitus, systemic sclerosis, anemia, and malnutrition, among others. Additionally, many conditions make this process difficult for resolution, preventing or slowing down the complete restoration of the tissues. By somehow hindering the tissue repair, these diseases contribute to the potential for increased morbidity and mortality [3,4]. According to the above reasons,

the supplements and drugs finding for the treatment of wounds is the priority of every country. In this regard, plant nanomaterial’s have an important role. A list of ethnomedicinal plants that synthesized by nanoparticles for enhancing the antioxidant and wound healing properties includes Astragali Radix, Tragacanth gum, Calendula officinalis, Spartium junceum L., Tecomella undulata, Centella asiatica, Cassia roxburghii, Arnebia nobilis, Biophytum sensitivum, Cellulose gum, Coleus forskohlii, Drosera binate, Ficus religiosa, Naringi crenulata, Citrus reticulate, Moringa oleifera, Potato starch, Guar gum, Nyctanthes arbor-tristis L., Bryonia laciniosa, Cassia auriculata, Lansium domesticum, Phytophthora infestans, Azadirachta indica, Piper nigrum, Catharanthus roseus, Orchidantha chinensis, Momordica charantia, Aloe vera, Cal. Officinalis, Dendrocalamus hamiltonii, Bambusa bambos, Danggui Buxue, Pluchea indica, Silybum marianum L., and Fraxinus angustifolia [5,6]. In Iranian traditional medicine, herbal medicine is one of the most common treatment methods. Moreover, in Iran, many new drugs are extracted from natural resources, most of which are rooted in ancient medicine [7-10]. Recently, due to problems associated with the use of modern medicine, there is a great tendency to replace it with traditional medicine and natural resources [11-12]. It is assumed that many medicinal plants are useful for the treatment of microbial diseases and wounds [13-15]. One of these plants that have been highly important in ancient medicine is Strawberry (Fragaria ananassa). It has been revealed that Strawberry provide notable health benefits because of their high levels of fibers, minerals, vitamins, and polyphenols [16]. The main polyphenolic compounds in Strawberry are flavan-3-ols, flavonols, hydrolyzable tannins (ellagitannins and gallo-), and anthocyanins [16]. According to the above compounds, it can be having considerable therapeutic effects against several diseases. Given the pharmaceutical properties of this plant and the significance of nanoparticles produced by plants, the present research attempted to find low-risk and more desirable methods for the production of bioactive copper nanoparticles through appropriate modeling. This research investigated the property of Strawberry aqueous extract in the production of copper nanoparticles and antioxidant, cytotoxicity, wound healing, and antimicrobial effects of the produced nanoparticles (CuNPs).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

2 Experimental

27

2.1. Material

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

All materials were obtained from Sigma Aldrich chemicals.

47

2.4. Determining the properties of the CuNPs

48

2.4.1. UV-vis analysis

2.2. Preparation of Strawberry fruit extract In this study, Strawberry as collected from an altitude of 1400m above the sea level in Kermanshah city, Iran in spring 2019 (Fig. 1). The plant fruits were kept and dried in dark/light conditions and powdered for extraction. Then, 10g of the powder of fruits of the plant was separated and poured into a decanter. Next, distilled water (100 mL) was added gradually until all the powder was soaked in the decanter and distilled water covered the sample. Seventy-two hours after the extraction, the solvent was isolated from the extract by a rotary machine using a vacuum pump [17,18]. Fig. 1 2.3. Green synthesis of CuNPs the solutions of L-Ascorbic acid (5 mL, 0.001M) and Strawberry extract (10 mL) were added to the CuSO4 (50 mL, 0.04M) solution under rapid stirring. Then the solutions of NaOH (0.01M) was added to the mixed copper salt solution under stirring for adjust the pH 8. The initial blue color of the reaction mixture eventually turned to dark brown color. Stirring was continued for another 1h to complete the reaction. The precipitate was washed twice with ethanol and water after filtration and then dried to obtain CuNPs in 90% yield.

1 2 3 4 5

Reduction of copper ions was observed in the reaction solution by measuring the UV absorption range of the solution using an optical spectrometer (X-ma 2000, UV-vis, Humancorp) at the wavelength of 300-800nm. After adding the extract to CuSO4 solution at different volumetric concentrations, the solution was changed to dark blue and then browned. Therefore, the absorption of the reaction solution was observed at a pH range of 5-10 and volumetric rations of 0.05-0.6 at different periods for the formation of copper nanoparticles [17,18].

6 7 8 9 10 11

2.4.2. FE-SEM and EDS analysis In FE-SEM, the electron is radiated on the surface of the sample and reflected and collected by a detector. These responses are then changed into optical photons to create a visible image. This microscope produces many images from the surface structure of particles. In this study, EM3200 microscope (KYKY Co) was used. EDX analysis was conducted with the same instrument to confirm the elemental composition of the sample [17,18].

12 13

2.4.3. TEM analysis

14 15 16 17 18

Transmission Electron Microscopy (TEM) (LEO912-AB, LEO) was sued to determine the shape and size of the nanoparticles. After dissolving the nanoparticles in deionized water, one drop of the copper nanoparticles were placed on the copper-coated carbon grades. The samples placed on the TEM carbon grades were allowed to dry. Then, the shape and size of the particles were analyzed and distribution of the size of particles was done by a particle sizer [17,18].

19 20

2.4.4. FTIR analysis

21 22 23

FTIR analysis was used to determine the plant factors influencing the reduction of copper ions. The dried powder of the nanoparticles produced as well as the powder of the extract was plated along with KBr and analyzed by FT-IR (Spectrum 65, Perkin Elmer) [17,18].

24 25

2.4.5. XRD analysis

26 27 28 29 30

The X-ray diffraction method was used to study the metal nature of the nanoparticles produced. After bioreduction, the copper nanoparticles solution was centrifuged at 8000rpm for 5min. The resulting plate was then dissolved in 10mL sterile deionized water and centrifuged three times, and the obtained plate was dried in a vacuum oven for 24h. The structure and composition of the nanoparticles obtained were analyzed by XRD (Philips Xpert Pro, Netherlands) [17,18].

31 32

2.5. Measurement of antioxidant activity of CuNPs by DPPH

33 34 35 36 37 38

To determine the trapping potential of DPPH, different concentrations of the CuSO4, Strawberry, and CuNPs were mixed with 2mL 0.004% DPPH solution. The control solution contained 2mL DPPH and 2mL ethanol. The solutions were kept in darkness at room temperature for 30min. Then, the absorption rate of the samples was measured at 517nm by the following formula compared to the control sample [19]: DPPH free radical scavenging (%) = (Control – Test/Control) × 100

39

2.6. Evaluation of cytotoxicity assay of CuNPs by MTT

40 41 42

Human umbilical vein endothelial cells (HUVECs) was used to investigate the efficacy of copper nanoparticles in the culture medium. To this end, the cell line was placed in T25 flasks along with complete culture medium. After cell density reached 80%, the sample was exposed to 1% of EDTA-trypsin solution. After 3 min incubation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

at 37°C along with 5% CO2 in the cell culture incubator and observing the cells detached from the plate floor, the sample was centrifuged for 5min at 5000rpm and the cell deposition was trypsinized by adding the culture medium. Then, the cell suspensions were counted by Neobar slide after trypan blue staining, and cell toxicity test was done by MTT assay. For this reason, 10000 HUVEC cells along with 200µL complete culture medium were added to each 98-plate culture plate. To achieve cells with single layer density, the plate was incubated again at 37°C along with 5% CO2. After 80% of cell growth was achieved, the culture medium was removed and the surface of the cells was irrigated with FBS, and 100µL double concentration culture medium was added afterward. Then, 100µL CuSO4, Strawberry, and CuNPs solution soluble in PBS was added to the well 1 (1000µg/mL). After mixing CuSO4, Strawberry, and CuNPs in the culture medium, 100µL of the first well was removed and added to the second well. Next, 100µL of the second well was removed and added to well 3. This process was continued up to well 11 so that half of the CuSO4, Strawberry, and CuNPs were added to each well. Well 12 only contained the cell and single concentration complete culture medium and remained as control. The plate was incubated at 37°C for 24h at the presence of 5% CO2, after which cell toxicity was determined by tetrazolium staining. After that, 10µL of tetrazolium stain (5mg/mL) was added to the wells, including the control, and the plate was incubated at 37°C for 2h at the presence of 5% CO2. Then, the stain was removed from the wells and 100µL of DMSO was added to the wells. The plate was wrapped in an aluminum foil and shaken for 20min in a shaker. Finally, cell viability was recorded by an ELISA reader at a wavelength of 570nm according to the following formula [20]: Percentage of cell viability (%) = (Sample Absorbance/Control absorbance) × 100

21

2.7. Evaluation of antibacterial and antifungal activities of CuNPs

22 23 24 25 26 27 28 29 30 31 32 33 34 35

In this study, eight bacteria, namely, Staphylococcus aureus, Staphylococcus saprophyticus, Bacillus subtilis, Streptococcus pneumonia, Escherichia coli O157:H7, Salmonella typhimurium, Proteus mirabilis, and Pseudomonas aeruginosa and five fungal species, namely Candida guilliermondii, Candida parapsilosis, Candida albicans, Candida krusei, and Candida glabrata were used. The prepared microbial suspension with 0.5McFarland turbidity standard was cultured onto Mueller Hinton Agar and Sabouraud Dextrose Agar in completely sterile conditions. Then, 60 µl of different dilutions of CuSO4, Strawberry, and CuNPs were added to the wells and disks. In this study, distilled water was negative control and nine standard antibiotics were positive controls. The growth inhibition zone (GIZ) was recorded after 24 hours of incubation at 37 °C. Minimum Inhibitory Concentration (MIC), Minimum Bacterial Concentration (MBC), and Minimal Fungicidal Concentration (MFC) of CuSO4, Strawberry, and CuNPs were determined according to the Clinical and laboratory standards institute (CLSI) protocol [21].

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

The study protocol was performed according to the ethical considerations of the International Committee on Laboratory Animals. A total of 60 healthy male rats with a weight range of 215±5 g (15 weeks age) were used in this study. The rats were prepared from the animal house of Iran Pasteur Institute and kept in special cages. No experiment was done on the rats for one week to avoid stress and to let the rats become compatible with the environment. All animals were kept in similar environmental and nutritional conditions (temperature, humidity, light, diet, and frequency of meals) and 12/12 dark/light cycle. The rats were fed special pellet for laboratory animals with free access to water. To perform all surgical procedures described as followed, the animals were anesthetized intraperitoneally (i.p.) with 10% ketamine hydrochloride, associated with 2% xylazine hydrochloride at doses of 100 mg/kg and 10 mg/kg, respectively. After induction of anesthesia, a wound (2×2cm) was made by a scalpel, which involved the removal of all cutaneous layers (Fig. 2). After surgery, the animals received an intraperitoneal single dose of Dipirone, 50 mg/kg bodyweight, diluted in saline for postoperative analgesia. At the end of the surgical procedure, the animals received subcutaneously (s.c.) 1mL saline for fluid replacement and were maintained in a heated environment until complete recovery of anesthesia. Then, the rats were randomly divided into six groups: untreated control, treatment with Eucerin basal ointment, treatment with 3% tetracycline ointment, treatment with

2.8. In vivo design

1 2 3 4 5 6 7 8 9

0.2% Strawberry ointment, treatment with 0.2% CuSO4 ointment, and treatment with 0.2% CuNPs ointment. The ointment was applied to the wound bed for 10 consequent days. On day 10, after complete anesthesia by 10% ketamine hydrochloride, associated with 2% xylazine hydrochloride at doses of 100 mg/kg and 10 mg/kg, respectively, a sample was taken from the wound in each group. In the histopathological study, the number of total cells, blood vessel, fibrocyte, fibroblast, neutrophil, macrophage, and lymphocyte and ratio of fibrocyte to fibroblast were measured by an optic microscope. For the determining of biochemical parameters (hexuronic acid, hexosamine, and hydroxyl proline concentrations), the ELISA method was done on the second half of the samples [22]. Fig. 2

10 11

2.9. Statistical Analysis

12 13 14

The obtained results were fed into SPSS-22 software and analyzed by one-way ANOVA, followed by Duncan post-hoc test (P≤0.01). In all tables, non-identical letters reveal a notable shift between the experimental groups (p≤0.01).

15 16

3. Results and Discussion

17 18 19 20 21 22 23 24

3.1. Chemical characterization of CuNPs

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

The UV-Vis. spectra of biosynthesized CuNPs using the aqueous extract of Strawberry is shown in Fig. 3. The surface plasmon resonance of CuNPs was confirmed by UV-Vis. spectroscopy. The appearance of a band at the wavelength of 581 nm approve the formation of CuNPs. The previous studies have reported a peak with a wavelength range of 570-590 nm for the biosynthesized of copper oxide [18]. We guess that the presence of biomolecules (such as polysaccharides and protein) of the extract can act as stabilizing agents to inhibit the Cu cluster from aggregation through the ion-dipole intermolecular forces Fig. 3 The size and morphology of the CuNPs synthesized sample were investigated using FE-SEM that were shown in Fig 4. The shape of particles is almost spherical and their average size is not possible to estimate, because biomolecule layers of extract stabilizing agent coat it. Fig. 4 The composition of Strawberry extract stabilized nanoparticles prepared from CuSO4 is further probed by EnergyDispersive X-ray (EDX) analysis and is shown in Fig. 5 which indicates the presence of Cu phase and C, N and O, which attributed these elements as the evidence for showing the stabilization of nanoparticles by capping agents. Fig. 5 In TEM image, the particles formed were spherical (Fig. 6). The nanospherical formed were shown to have at high surface area. Formed nanoparticles were in the range of 10-30nm in size with 20nm average size. The particles were monodispersing with thin layers of extract in their surface. Fig. 6 To identify the biomolecules responsible for the stabilizing of CuNPs, FT-IR was performed on biosynthesized CuNPs (Fig. 7). The 572cm-1 absorption band is relevant to the Cu-O functional group resonance. This confirms that nano-sized Cu particles are present in the nanocomposite. An intense and thick band emerged in the 3200-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

3500cm-1 region, matching the hydroxyl functional groups stretching mode. Sp2-Carbon groups are generally the reason for the band around 1450cm-1, while carbonyl functional groups include the 1625cm-1 band. Fig. 7

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

The in vitro DPPH radical inhibitory assay is based on an antioxidant’s hydrogen donating ability to reduce DPPH radical in methanol to form the non-radical DPPH-H [3]. In the present study, Strawberry extract and CuNPs similar to BHT demonstrated a remarkable concentration-dependent DPPH radical scavenging activity. The interaction between the Strawberry extract and CuNPs and DPPH might have occurred through the transfer of electrons and hydrogen ions to 2,2-diphenyl-1-picrylhydrazyl radical to form a stable 2,2-diphenyl-1picrylhydrazine molecule (DPPH) [18]. The DPPH radical usually has a strong absorbance at the wavelength of 517 nm. However, upon acceptance of an electron or hydrogen atom from an antioxidant compound, it becomes a stable diamagnetic molecule with decreased absorbance at 517 nm. The resulting color change from purple to pale yellow determines the anti-radical power of an antioxidant [4]. A stable diamagnetic free radical, DPPH, has been widely applied as a sensitive and rapid tool for estimation of free radical scavenging activities of both lipophilic and hydrophilic antioxidants [3]. The IC50 values of BHT, Strawberry, and CuNPs were 592, 718, and 442, respectively (Fig. 9). The antioxidant activity exhibited by Strawberry can be attributed to the presence of various phytochemicals that are thought to function interactively and synergistically to neutralize ROS and RNS.[60] In the previous study determined that Strawberry was rich of antioxidant compounds such as flavan-3ols, flavonols, hydrolyzable tannins (ellagitannins and gallo-), and anthocyanins [16]. These bioactive compounds have been shown to maintain the redox homeostasis through multiple-step processes of antioxidant reactions which involves initiation, propagation, branching, and termination of free radicals [16].

34 35 36 37 38 39 40 41 42 43 44

Fig. 9 3.3. Cytotoxicity activities of CuNPs The cytotoxicity activities of CuSO4, Strawberry, and CuNPs were assessed against HUVEC cells (Fig. 10). HUVECs are the human normal cells that are separated from umbilical vein endothelial cells. These cells are applied for determining the cytotoxicity potentials of metallic nanoparticles such as copper nanoparticles [3]. In our study, the absorbance rate was measured at 570nm, which revealed extraordinary viability on HUVEC even up to 1000μg/mL for CuSO4, Strawberry, and CuNPs. The absence of any significant toxicity of CuSO4, Strawberry, and CuNPs has numerous safe applications in medicinal and pharmaceutical fields. In the previous research has been reported when metallic salts such as Cu(NO3)2 are combined with plant extracts, their cytotoxicity capacity significantly decreases against HUVECs [18].

45 46 47 48 49

About cytotoxicity and anticancer properties of copper nanoparticles, increased copper levels result in the loss of membrane integrity, which causes essential nutrients, including glutamate and potassium to leak from cells and cause apoptosis. Shafagh et al. (2015) revealed apoptosis and cytotoxicity of copper oxide nanoparticles on a chronic myeloid leukemia K562 cell line [23]. CuNPs indicated selectivity towards the K562 cell line and are potentially a suitable anti-cancer supplement since it does not remove normal cells [24]. Copper nanoparticles

XRD is used for the phase identification and characterization of the crystal structure of the copper nanoparticles (Fig. 8). In the case of Cu containing sample, The XRD peaks at 43.1°, 50.2° and 74.1° can be indexed to the (1 1 1), (2 0 0) and (2 2 0) Bragg’s reflections of face center cubic (fcc) structure of metallic Cu respectively similar to Joint Committee on Powder Diffraction Standards (JCPDS No.04-0784). Fig. 8 3.2. Antioxidant activities of CuNPs One option for increasing the antioxidant capacity of plants is combining them with metallic salts that are called herbal nanoparticles. In the previous studies have been indicated when plants are combined with gold, silver, titanium, copper, zinc, and iron, their antioxidant potentials increase significantly [1]. In this regards, we decided to investigate the antioxidant properties of CuSO4, Strawberry, and CuNPs in comparison to BHT as a common positive control.

1 2

also induced apoptosis in a human skin melanoma cell line [25]. Similar results have been revealed in a glial cancer cell line by Kukia et al. (2018) [25].

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Fig. 10 3.4. Antifungal and antibacterial effect of CuNPs As is seen in Tables 1-6, there is no remarkable change in GIZ of all fungi and bacteria between various concentrations of CuNPs and standard antibiotics. There was an increase in the GIZ in many of the samples when CuNPs increased. The widest GIZ in agar well and disk diffusion tests were seen at 64mg/mL concentration. In agar well diffusion, no inhibitory effect of CuNPs was seen at 1mg/mL concentration in cases of P. mirabilis, S. typhimurium, E. coli, S. aureus, S. pneumonia, and C. albicans (p≤0.01). CuNPs prevented S. saprophyticus/C. parapsilosis/C. krusei/C. guilliermondii, E. coli/P. aeruginosa/S. aureus/S. pneumonia/B. subtilis/C. albicans/C. glabrata, and P. mirabilis/S. typhimurium growth at 2, 4, and 8mg/mL concentrations, respectively and destroyed S. saprophyticus/C. guilliermondii, P. aeruginosa/B. subtilis/C. glabrata/C. parapsilosis/C. krusei, S. typhimurium/E. coli/S. aureus/S. pneumonia/C. albicans, P. mirabilis at 2, 4, 8, and 16mg/mL concentrations, respectively. Thus, the findings showed excellent antifungal and antibacterial properties of CuNPs against all of the tested fungi and bacteria. Moreover, CuNPs had the highest antibacterial effect on S. saprophyticus (p≤0.01). About antimicrobial effects of metal nanoparticles, they can generate hydrogen peroxides (H2O2) and these peroxides react with membrane proteins and lipid bilayers of the bacterial cell [26]. The antimicrobial action of copper nanoparticles may involve both the production of reactive oxygen species (ROS) and the accumulation of metal nanoparticles itself in the cytoplasm. It is well known that ROS results in cell membrane damage and dysfunction, finally leading to cell death [27,28]. Metal nanoparticles ranging from 1-10 nm easily enters the mitochondria through various transporters that leads to mitochondrial oxidative stress and apoptosis [29]. Liu et al. (2009) found that metal nanoparticles might alter and damage bacterial cell membranes, allowing for the leakage of intracellular components, leading to cell death [30]. In the antibacterial analysis of CuNPs, the effect of copper nanoparticles is higher on gram-positive bacteria than gram-negative bacteria. This difference may be because 90% of the wall of gram-negative bacteria is made of lipid. Moreover, a thin layer of peptidoglycan exists on the internal membrane, on which lipoprotein structures exist, followed by the external membrane only was seen in gram-negative bacteria with lipopolysaccharide structures. Such structures do not exist in gram-positive bacteria [31]. Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 3.5. Cutaneous wound healing potential of CuNPs As shown in Table 7, the CuNPs ointment significantly reduced the wound area compared to other studied groups. It has been shown that antioxidant compounds ameliorate the shearing wounds. These compounds eliminate the free radicals from the wound area and prevent the production of pus and delayed wound healing [320]. Since the CuNPs ointment had remarkable antioxidant effects in the recent study, improvement of wound surface by this ointment seems to be normal.

43

Table 7

44 45 46 47 48 49 50

The results of microscopic and biochemical studies showed that CuNPs ointment significantly decreased the number of total cells, neutrophils, and lymphocytes and significantly increased the number of blood vessels, fibrocyte, fibroblasts, fibroblast/fibrocyte ratio, and concentration of hydroxyproline, hexosamine, and hexuronic acid compared to other studied groups (Tables 7-9; Fig. 11,12). Studies have shown that the amount of angiogenesis at the wound site increases with the incidence of the wound, which causes more migration of fibroblasts to this site [33,34]. Then, the fibroblasts induce the production of collagen (proline, hydroxyl proline, and glycine) and extracellular matrix compounds like hexosamine and hexuronic acid [35,36]. After a while, the

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

fibroblast is changed into fibrocyte. The ability of fibrocyte in wound healing is by far higher than that of fibroblast [37]. Further, inflammatory cells, i.e., macrophages, neutrophils, and lymphocytes migrate to the wound site after angiogenesis [38]. Due to the accumulation of free radicals especially reactive oxygen species (ROS) in the wound site, the excessive presence of these cells causes extreme inflammation and pus [32,39]. Antioxidant compounds such as CuNPs ointment prevent this accumulation by eliminating free radicals and increase the rate of wound healing. In detail, ROS have also been implicated as important mediators of cell signaling and inflammation in wound repair [40]. Platelet-derived growth factor, a chemotactic recruiter of cells key to wound repair, has been shown to generate hydrogen peroxide and superoxide in human hepatoma cells [40] and human aortic smooth muscle cells [41], respectively; the latter then induces other inflammatory mediators such monocyte chemoattractant protein-1 (MCP-1) [39]. In MCP-1-deficient mice, a decreased monocyte population at the wound results in a decrease in hydrogen peroxide production and subsequent decrease in wound angiogenesis [42]. ROS-stimulated release of VEGF was also found in human keratinocytes [42]. Hydrogen peroxide is implicated in the induction of neutrophil chemotaxis [43]. Additionally, hydrogen peroxide has been shown to indirectly induce matrix metallopeptidase 1 via activator protein-1 [44]. Matrix metallopeptidase 1 degrades extracellular matrix proteins, thereby allowing wound cells to migrate. All these studies underscore the pro-wound effects of ROS when levels are controlled. Although ROS production is physiologic, excessive production can be harmful. ROS are a normal byproduct of cellular metabolism. It has been estimated that 2–5% of the daily basal oxygen use in humans is converted to ROS [43,44]. Inflammatory cells produce ROS as a defense against invading pathogens. The respiratory burst produced by neutrophils and other leukocytes such as macrophages and monocytes creates large quantities of ROS mainly through the enzyme complex NADPH oxidase [45]. These ROS include the superoxide anion and the dismutated, nonradical product hydrogen peroxide. ROS play a role in cellular signaling through orchestration of cytokines, growth factors and hormones vital for wound repair [46]. The nonradical metabolites such as hydrogen peroxide have the potential to be harmful at excessive levels [46]. Radicals have the potential to be damaging through lipid peroxidation, protein modification and DNA modification. Thus to control possible damage from physiologic generation of ROS, physiologic defenses have evolved. Physiologic antioxidant defenses include the ROS-detoxifying enzymes superoxide dismutase (SOD), catalase, glutathione peroxidases and peroxiredoxins [47]. Endogenous and exogenous low-molecular-weight antioxidants such as glutathione, vitamin E, vitamin C and phenolic are nonenzymatic defenders against ROS [46]. These lowmolecular-weight antioxidants ‘sacrifice’ themselves to be oxidized and become radicals themselves, though less reactive and therefore less damaging than the radicals they scavenge [46]. In a continuing chain reaction, antioxidants work together to regenerate these ‘sacrificed’ antioxidants to be available again in their reduced forms for cell defense [47]. Abnormally low levels of these antioxidants have been associated with impaired wound healing [45]. As interest in the use of antioxidants continues to grow, several animal and human studies have already shown the efficacy of these compounds in promoting healing [45-47].

35

Table 8

36

Table 9

37

Fig. 11

38

Fig. 12

39 40

4. Conclusion

41 42 43 44 45 46 47

Biosynthesized CuNPs are of great interest due to their eco-friendliness, economic prospects, feasibility, and short synthesis time. In this study, we reported an eco-friendly and cost-effective biological process to synthesize CuNPs using L-Ascorbic acid and Strawberry fruit aqueous extract as reducing and stabilizing agents. The biosynthesized CuNPs showed excellent distribution with a size of 10-30nm. XRD indicated that synthesized CuNPs were high in purity, and which was powder or crystalline in nature. This method could be used for the large-scale industrial synthesis of CuNPs as antioxidant, antifungal, antibacterial, and cutaneous wound healing agents, using Strawberry fruit aqueous extract. These nanoparticles may have a wide range of medical

1 2

applications in the pharmaceutical industry for the development of new formulations against microbial strains, which are developing resistance to currently available antibiotics.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

References [1] (a) L. Becquemont, Pharmacogenomics J. 2009, 10, 961–969; (b) H. Veisi, S. Razeghi, P. Mohammadi, S. Hemmati, Mat. Sci. Eng. C, 2019, 97, 624-631; (c) H. Veisi, S.B. Moradi, A. Saljooqi, P. Safarimehr, Mat. Sci. Eng. C, 2019, 100, 445-452; (d) H. Veisi, M. Ghorbani, S. Hemmati, Mat. Sci. Eng. C, 2019, 98, 584-593; (e) H. Veisi, N. Hajimoradian Nasrabadi, P. Mohammadi, Appl. Organometal. Chem. 2016, 30, 890; (f) H. Veisi, P. Safarimehr, S. Hemmati, Mat. Sci. Eng. C, 2019, 96, 310-316; (g) H. Veisi, S. Azizi, P. Mohamadi, J Cleaner Prod. 2018, 170, 1536-1543; (h) H. Veisi, L. Mohammadi, S. Hemmati, T. Tamoradi, P. Mohammadi, ACS Omega, 2019, 4, 13991. [2] R. W. Raut, N. S. Kolekar, J. R. Lakkakula, V. D. Mendhulkar, S. B. Kashid, Nano‐Micro Lett. 2010, 2, 106. [8] (a) R. S. Varma, Curr Opin Chem Eng. 2012, 1, 123; (b) (a) H. Veisi, S. Hemmati, P. Safarimehr, J. Catal. 2018, 365, 204; (c) H. Veisi, S.A. Mirshokraie, H. Ahmadian, Int. J. Biol. Macromol. 2018, 108, 419; (d) G. Shaham, H. Veisi, M. Hekmati, Appl. Organometal.Chem. 2017, 31, e3737; (e) M. Shahriary, H. Veisi, M. Hekmati, S. Hemmati, Mat. Sci. Eng. C. 2018, 90, 57; (f) H. Veisi, S. Hemmati, H. Shirvani, H. Veisi, Appl Organometal Chem. 2016, 30, 387; (j) K. Zomorodian, H. Veisi, S.M. Mousavi, M.S. Ataabadi, S. Yazdanpanah, J. Bagheri, A. Parvizi Mehr, S. Hemmati, H. Veisi, Int J Nanomedicine. 2018, 13, 3965. [3] A. Khorasgni, A. H. Karimi, M. R. Nazem, Pak. Vet. J. 30 (2010) 72-74. [4] F. Malekzadeh, Appl. Microbiol. 16 (1986) 663-664. [5] K. Dastmalchi, Damien, H. Dormana, P. Oinonena, Y. Darwisd, I. Laaksoa, R. Hiltunen, LWT- Food. Sci. Techno. 41 (2008) 391-400. [6] M. Hajialyani, D. Tewari, E. Sobarzo-Sánchez, S. M. Nabavi, M. H. Farzaei, M. Abdollahi, Int. J. Nanomed. 13 (2018) 5023–5043. [7] M. M. Zangeneh, A. Zangeneh, R. Tahvilian, R. Moradi, Arch. Bio. Sci. 70 (2018) 655-664. [8] S. Goorani, N. Shariatifar, N. Seydi, A. Zangeneh, R. Moradi, B. Tari, F. Nazari, M. M. Zangeneh, Orient. Pharm. Exp. Med. 2019. DOI: 10.1007/s13596-019-00361-5. [9] S. Goorani, M. M. Zangeneh, M. K. Koohi, N. Seydi, A. Zangeneh, N. Souri, M. S. Hosseini, Comp. Clin. Pathol. 2018. DOI: 10.1007/s00580-018-2866-3. [10] L. Hagh-Nazari, N. Goodarzi, M. M. Zangeneh, A. Zangeneh, R. Tahvilian, R. Moradi, Comp. Clin. Pathol. 26 (2017) 455–463. [11] A.R. Jalalvand, M. Zhaleh, S. Goorani, M.M. Zangeneh, N. Seydi, A. Zangeneh, R. Moradi, J. Photochem. Photobiol. B. 192 (2019) 103-112. [12] R. Moradi, M. Hajialiani, S. Salmani, M. Almasi, A. Zangeneh, M. M. Zangeneh, Comp. Clin. Pathol. 2018. DOI: 10.1007/s00580-018-2834-y. [13] T. Sayyedrostami, P. Pournaghi, S. Ebrahimi Vosta-Kalaeea, M. M. Zangeneh, J. Essent. Oil. Bear. Pl. 21 (2018) 164-174. [14] M. M. Zangeneh, Appl. Organometal. Chem. 2019, DOI:10.1002/aoc.4963. [15] M. M. Zangeneh, S. Bovandi, S. Gharehyakheh, A. Zangeneh, P. Irani, Appl. Organometal. Chem. 2019, DOI:10.1002/aoc.4961. [16] S. H. Nile, S. W. Park, Nutrition. 30 (2015) 134–144. [17] S. Hemmati, A. Rashtiani, M. M. Zangeneh, P. Mohammadi, A. Zangeneh, H. Veisi, Polyhedron. 158 (2019) 8–14. [18] (a) R. Tahvilian, M.M. Zangeneh, H. Falahi, K. Sadrjavadi, A. R. Jalalvand, A. Zangeneh, Appl. Organometal. Chem. 33 (2019) e5234. DOI: 10.1002/aoc.5234. (b) M.M. Zangeneha, H. Ghaneialvar, M. Akbaribazm, M. Ghanimatdan, N. Abbasi, S. Goorani, E. Pirabbasig, A. Zangeneh, J. Photochem. Photobiol. B. 197 (2019) 111556. (c) S. Hemmati, M.M. Zangeneh, A. Zangeneh. Polyhedron. 177 (2020) 114327. (d) M. Hamelian, M. M. Zangeneh, A. Amisama, K. Varmira, H. Veisi, Appl. Organometal. Chem. 32 (2018) e4458. [19] S. J. Hosseinimehr, A. Mahmoudzadeh, A. Ahmadi, S. A. Ashrafi, N. Shafaghati, N. Hedayati, Cancer. Biother. Radiopharm. 26 (2011) 325–329.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

[20] V. Arulmozhi, K. Pandian, S. Mirunalini, Colloids. Surf. B. Biointerfaces. 110 (2013) 313–320. [21] Clinical and laboratory standards institute (CLSI), M7-A7. 26 (2006). [22] A. Ghashghaii, M. Hashemnia, Z. Nikousefat, M. M. Zangeneh, A. Zangeneh, Pharm. Sci. 23 (2017) 256– 263. [23] M. Shafagh, F. Rahmani, N. Delirezh, Iran. J. Basic. Med. Sci. 18 (2015) 993-1000. [24] R. Chakraborty, T. Basu, Nanotechnol. 28 (2017) 105101. [25] N. Kukia, A. Abbasi, S. Froushani, Dhaka. Univ. J. Pharm. Sci. 17 (20180 105-111. [26] L. Zhang, Y. Jiang, Y. Dingh, K. Daskalakis, J.L. Povey, A.J. O’Neil, J. Nanopart. Res. 12 (2010) 16251636. [27] R.K. Dutta, B.P. Nenavathu, M.K. Gangishetty, A.V.R. Reddy, Colloids. Surf. B. Biointerfaces. 94 (2012) 143-150. [28] M.J. Akhtar, M. Ahamed, S. Kumar, M.M. Khan, J. Ahmad, S.A. Alrokayan, Int. J. Nanomedicine. 7 (2012) 845-857. [29] T. Xia, M. Kovochich, A.E. Nel, Front. Biosci. 12 (2007) 1238-1246. [30] Y. Liu, L. He, A. Mustapha, H. Hu, Z.Q. Li, M. Lin, J. Appl. Microbiol. 107 (2009) 1193-1201. [31] A. Mai-Prochnow, M. Clauson, J. Hong, A. B. Murphy, Sci. Rep. 6 (2016) 38610. [32] M. Hajialyani, D. Tewari, E. Sobarzo-Sánchez, S. M. Nabavi, M. H. Farzaei, M. Abdollahi, Int. J. Nanomed. 13 (2018) 5023–5043. [33] G. D. Phillips, R. A. Whitehe, D. R. Kinghton, Am. J. Anat. 192 (1991) 257–262. [34] G. S. Lazarus, D. M. Cooper, D. R. Knighton, D. J. Margolis, R. E Pecoraro, G. Rodeheaver, M. C. Robson, Arch. Dermatol. 130 (1994) 489–493. [35] H. Azhdari-Zarmehri, S. Nazemi, E. Ghasemi, Z. Musavi, Z. Tahmasebi, F. Farsad, A. Farzam, JBUMS. 16 (2014) 42-48. [36] G. F. Caetano, M. Fronza, M. N. Leite, A. Gomes, M. A. C. Frade, Pharm. Biol. 54 (2016) 2555–2559. [37] D. Dwivedi, M. Dwivedi, S. Malviya, V. Singh, J. Tradit. Complement. Med. 7 (2017) 79–85. [38] B. S. Nayak, G. Isitor, E. M. Davis, G. K. Pillai, Phytother. Res. 21 (2007) 827–831. [39] T. J. Koh, L. A. DiPietro, Expert. Rev. Mol. Med. 13 (2011) e23. [40] Y.S. Bae, J.Y. Sung, O.S. Kim, Y.J. Kim, K.C. Hur, A. Kazlauskas, S.G. Rhee, J. Biol. Chem. 275 (2000) 10527–10531. [41] T. Marumo, V.B. Schini-Kerth, B. Fisslthaler, R. Busse, Circulation. 96 (1997) 2361–2367. [42] C.K. Sen, S. Khanna, B.M. Babior, T.K. Hunt, E.C. Ellison, S. Roy, J. Biol. Chem, 277 (2000) 33284–33290. [43] I.V. Klyubin, K.M. Kirpichnikova, I.A. Gamaley, Eur. J. Cell. Biol. 70 (1996) 347–351. [44] J. Wenk, P. Brenneisen, M. Wlaschek, A. Poswig, K. Briviba, T.D. Oberley, K. Scharffetter-Kochanek, J. Biol. Chem. 274 (1999) 25869–25876. [45] Y.U. Inoue, J. Asami, T. Inoue, Mol. Cell. Neurosci. 39 (2008) 95–104. [46] M. Schäfer, S. Werner. Pharmacol. Res. 58 (2008) 165–171. [47] P. Bossolasco, L. Cova, C. Calzarossa, S.G. Rimoldi, C. Borsotti, G.L. Deliliers, V. Silani, D. Soligo, E. Polli, Exp. Neurol. 193 (2005) 312–325.

1 2 3 4

Fig. 1. Strawberry image.

5 6

Fig. 2. Excision model in rats (S show wound area).

7 8 9 10 11

Fig. 3. UV-vis spectrum of biosynthesized CuNPs.

12

Fig. 5. EDX data of biosynthesized CuNPs.

Fig. 4. FE-SEM image of biosynthesized CuNPs.

13 14 15 16 17 18 19 20 21 22

Fig. 6. TEM image of biosynthesized CuNPs.

23 24 25

Fig. 10. Percentage viability measured on HUVEC cells after treatment with CuSO4, Strawberry, and CuNPs@Strawberry.

26 27

Fig. 11. Longitudinal section of wounds of the control (A), basal ointment (B), tetracycline ointment (C), CuSO4 ointment (D), Strawberry ointment (E), and CuNPs@Strawberry ointment (F) on 10 days post-injury.

28

Scale bar: 150 μm.

29

Magnification ×200.

30 31 32

Fig. 12. Longitudinal section of wounds of the control (A), basal ointment (B), tetracycline ointment (C), CuSO4 ointment (D), Strawberry ointment (E), and CuNPs@Strawberry ointment (F) on 10 days post-injury.

33

Scale bar: 600 μm.

34

Magnification ×800.

35 36 37 38 39 40 41 42 43 44

Fig. 7. FTIR spectrum of biosynthesized CuNPs. Fig. 8. XRD spectra of biosynthesized CuNPs. Fig. 9. Antioxidant potential of CuSO4, Strawberry, BHT, and CuNPs@Strawberry. BHT: Butylated hydroxyl toluene.

1 2 3 4

Table 1 The GIZ of bacteria in several dilutions of CuNPs@Strawberry, Strawberry, and CuSO4.

Dilution (mg/ml)

GIZ in disk diffusion (mm)

P. mirabilis Difloxacin (30) Chloramphenicol (30) Streptomycin (10) Gentamicin (10) Oxytetracycline (30) Ampicillin (10) Amikacin (25) CuNPs@Straw (64) CuNPs@Straw (32) CuNPs@Straw (16) CuNPs@Straw (8) CuNPs@Straw (4) CuNPs@Straw (2) CuNPs@Straw (1) Strawberry (64) Strawberry (32) Strawberry (16) Strawberry (8) Strawberry (4) Strawberry (2) Strawberry (1) CuSO4 (64) CuSO4 (32) CuSO4 (16) CuSO4 (8) CuSO4 (4) CuSO4 (2) CuSO4 (1) Distilled water

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

23.8±0.4b 23.8±0.4b 16.6±1.1bc 21.2±0.8bc 18.8±0.4bc 18.2±0.4bc 23±1.2b 32.4±1.3ab 26.4±1.3b 24.8±0.4b 17.4±0.8bc 13.2±1.3c 10.8±1c 8.4±0.5c 21.8±0.4bc 17.6±1.1bc 13±0.7c 11.2±1.3c 9.8±1c 0±0d 0±0d 13.2±0.8c 12.4±1.3c 9.2±0.8c 8.4±0.8c 0±0d 0±0d 0±0d 0±0d

Gram-negative bacteria S. E. coli typhimurium 25±0.7b 27.2±0.8b 25.4±0.5b 28.6±1.1b bc 16.8±0.4 13.4±0.5c 16±1.2bc 16.8±0.4bc 20.2±1.3bc 18.6±1.1bc bc 18.6±1.1 14.6±0.8c bc 22.4±0.5 22.8±1bc 35.2±1.3ab 37.8±0.4a 32.8±0.4ab 32.8±0.4ab b 25.8±0.4 27.2±0.8b 22.2±0.8bc 23.2±0.8b 14±1c 17.4±1.3bc c 12.8±0.4 13.2±0.8c c 8.6±1.1 10.2±0.8c 24.4±0.8b 26.8±0.4b 20±0.7bc 22.8±1bc c 14.8±1 16.6±0.8bc 12.8±0.4c 13.2±0.8c 10.4±0.8c 10.4±0.8c c 8.4±0.5 9.2±0.4c 0±0d 0±0d 15.2±0.8bc 15.2±0.8bc 12.2±1.3c 11.8±0.4c c 11.2±1.3 10.4±1.3c 9±1c 9.8±0.4c 8.6±1.14c 8.8±0.4c d 0±0 0±0d 0±0d 0±0d 0±0d 0±0d

P. aeruginosa 33.2±0.4ab 29.8±1b 19.2±0.8bc 25±1b 24.8±1b 23.4±0.5b 31.6±1.1ab 41.8±0.4a 37.2±0.8a 31±1.2ab 25.2±1.3b 22.8±0.4bc 15.6±0.8bc 12±1c 32±0.7ab 25.8±1b 22±0.7bc 20.2±0.8bc 15.6±0.8bc 12.2±0.4c 9.8±1c 17.2±0.4bc 13.2±0.4c 11.4±0.8c 9.6±0.8c 9±1.2c 0±0d 0±0d 0±0d

S. aureus 24.4±0.5b 19±1.2bc 12.4±0.5c 15.2±1.3bc 22±1.2bc 19.4±1.3bc 21.4±0.5bc 37.8±0.4a 31.2±0.8ab 27.8±1b 23.2±1.3b 15.4±0.8bc 12.4±0.5c 9.2±0.8c 27.8±0.4b 23.4±1.3b 20.6±1.1bc 16.6±1.1bc 12±1.2c 11.2±1.3c 0±0d 14.2±0.8c 11.2±1.3c 11.8±1c 9±1.2c 8.2±1.3c 0±0d 0±0d 0±0d

Gram-positive bacteria S. B. subtilis pneumoniae 22.8±0.4bc 27.8±1b 22.4±0.5bc 26.2±1.3b c 14.8±0.4 24.8±1b 22.2±0.8bc 13.2±0.8c 22.8±0.4bc 23.8±1b c 12.6±1.1 19.4±1.3bc b 25±1.2 28.2±0.4b 38.2±0.8a 42.8±1a 33.6±1.1ab 38.2±0.4a ab 30.8±1 31±1.2ab 24.8±0.4b 26.2±1.3b 18±1bc 19.2±1.3bc c 13.4±1.3 15.2±0.4bc c 10.2±1.3 11.8±0.4c 29.4±0.5b 34.2±1.3ab 24.6±1.1b 30.2±0.4ab bc 20.2±0.8 25.2±1.3b 18.8±0.4bc 21±1.2bc 12.4±1.3c 15.8±1bc c 11.8±0.4 12.2±0.8c 8.2±0.4c 10.4±1.3c 18±1.2bc 23.6±0.8b 14.6±1.1c 19.6±0.8bc c 12±0.7 13.8±1c 10.2±0.8c 12.4±1.3c 9.8±0.4c 10±1c d 0±0 8.2±0.4c 0±0d 0±0d 0±0d 0±0d

S. saprophyticus 34.6±1.1ab 19±1bc 12.2±1.3c 12.8±1c 21.8±0.4bc 17.6±0.8bc 17.8±0.4bc 45.4±0.8a 40.8±1a 32.4±1.3ab 25.8±0.4b 20.2±0.8bc 16.8±0.4bc 13.6±0.8c 37.6±0.8a 31.6±1.1ab 25.4±0.5b 21.4±0.8bc 17.8±0.4bc 13.6±0.8c 12±1.2c 23.8±0.4b 15.2±0.8bc 13.4±0.5c 11±0.7c 9.8±1c 8.4±0.8c 0±0d 0±0d

1 2 3 4

Table 2 The GIZ of bacteria in several dilutions of CuNPs@Strawberry, Strawberry, and CuSO4.

Dilution (mg/ml)

GIZ in well diffusion (mm)

P. mirabilis CuNPs@Straw (64) CuNPs@Straw (32) CuNPs@Straw (16) CuNPs@Straw (8) CuNPs@Straw (4) CuNPs@Straw (2) CuNPs@Straw (1) Strawberry (64) Strawberry (32) Strawberry (16) Strawberry (8) Strawberry (4) Strawberry (2) Strawberry (1) CuSO4 (64) CuSO4 (32) CuSO4 (16) CuSO4 (8) CuSO4 (4) CuSO4 (2) CuSO4 (1) Distilled water

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

25.8±0.4ab 21.2±0.8b 17.8±0.4bc 13.2±1.3c 9.2±1.3c 0±0d 0±0d 17.2±0.8bc 14.6±1.1c 11.2±1.3c 9±1.2c 0±0d 0±0d 0±0d 10.6±1.1c 9.8±0.4c 0±0d 0±0d 0±0d 0±0d 0±0d 0±0d

Gram-negative bacteria S. E. coli typhimurium 27.4±0.5ab 28.4±0.5ab 25.6±0.8ab 24.6±1.1b b 21.2±0.8 21.8±0.4b 16.8±1bc 17.4±0.5bc 12.4±0.5c 12.4±1.3c c 8.4±0.5 9.2±1.3c d 0±0 0±0d 19.2±0.8bc 22.6±1.1b 16.2±0.4bc 18±1.2bc c 11.6±0.8 14.8±0.4c 10.2±0.8c 11.4±1.3c 8.4±0.5c 9.8±0.4c d 0±0 0±0d d 0±0 0±0d 12.2±0.8c 12.2±0.4c 11.8±1c 10.4±0.5c c 9.2±0.83 8.2±1.3c 0±0d 0±0d 0±0d 0±0d d 0±0 0±0d 0±0d 0±0d 0±0d 0±0d

P. aeruginosa 34.6±0.8a 30.8±1a 23±1.2b 18.2±1.3bc 13.8±0.4c 11.8±1c 8.4±1.3c 27.8±0.4ab 19.4±1.3bc 16.6±1.1bc 13.6±1.1c 10±0.7c 8.6±1.1c 0±0d 16.4±0.8bc 12±1c 10.4±0.89c 8.2±1.3c 0±0d 0±0d 0±0d 0±0d

S. aureus 28.4±0.5ab 25.8±0.4ab 21.6±0.8b 15.8±0.4bc 12.6±0.8c 9.6±1.1c 0±0d 20.6±0.8b 16.4±0.8bc 15.2±0.8bc 12.2±0.8c 10.2±0.8c 0±0d 0±0d 11±1c 10.8±0.4c 10±0c 0±0d 0±0d 0±0d 0±0d 0±0d

Gram-positive bacteria S. B. subtilis pneumoniae 32±1a 37.2±1.3a 24.8±0.4b 26.2±0.8ab b 20.2±0.8 22.6±1.1b 16.4±0.5bc 19.4±1.3bc 11.8±1c 14.6±0.8c c 9.8±1 12.4±0.5c d 0±0 9.2±0.4c 25.2±0.8ab 27.6±1.1ab 21.2±0.8b 23.4±0.8b bc 17.4±0.5 20.6±1.1b 14.4±0.5c 14.2±0.4c 11.4±0.5c 10.6±1.1c c 9.4±1.3 9.4±1.3c d 0±0 0±0d 12.6±0.8c 15±1bc 11.2±1c 11.2±1.3c c 9.2±0.4 10.6±0.8c 8±0.7c 9.2±0.8c 0±0d 0±0d d 0±0 0±0d 0±0d 0±0d 0±0d 0±0d

S. saprophyticus 37.6±0.8a 29.8±0.4ab 25.8±0.4ab 21±1.2b 15.2±0.8bc 12.4±0.5c 10.4±0.8c 28±1.2ab 25±1.2ab 22.6±0.8b 17±0.7bc 12±1c 10±1c 8±1.2c 18.2±0.4bc 14.2±1.3c 12.4±0.5c 10±0.7c 8.2±0.83c 0±0d 0±0d 0±0d

1 2 3 4

Table 3 MIC and MBC of CuNPs@Strawberry, Strawberry, and CuSO4 against bacteria. Microorganism MICCuNPs@Straw (mg/ml) MBCCuNPs@Straw (mg/ml) MICStrawberry (mg/ml) MBCStrawberry (mg/ml) MICCuSO4 (mg/ml) MBCCuSO4 (mg/ml)

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

P. mirabilis

S. typhimurium

Gram-negative bacteria E. coli

P. aeruginosa

S. aureus

Gram-positive bacteria S. B. subtilis S. pneumoniae saprophyticus

8±0c

8±0c

4±0b

4±0b

4±0b

4±0b

4±0b

2±0a

16±0D

8±0C

8±0C

4±0B

8±0C

8±0C

4±0B

2±0A

16±0d

8±0c

8±0c

4±0b

8±0c

8±0c

4±0b

4±0b

16±0D

16±0D

16±0D

8±0C

16±0D

8±0C

8±0C

4±0B

32±0e

16±0d

16±0d

8±0c

16±0d

16±0d

8±0c

8±0c

32±0E

32±0E

32±0E

16±0D

32±0E

32±0E

16±0D

16±0D

1 2 3 4

Table 4 The GIZ of fungi in several dilutions of CuNPs@Strawberry, Strawberry, and CuSO4.

Dilution (mg/ml)

Fluconazole (60) Itraconazole (60) Miconazole (60) Amphotericin B (60) Nystatin (60) CuNPs@Straw (64) CuNPs@Straw (32) CuNPs@Straw (16) CuNPs@Straw (8) CuNPs@Straw (4) CuNPs@Straw (2) CuNPs@Straw (1) Strawberry (64) Strawberry (32) Strawberry (16) Strawberry (8) Strawberry (4) Strawberry (2) Strawberry (1) CuSO4 (64) CuSO4 (32) CuSO4 (16) CuSO4 (8) CuSO4 (4) CuSO4 (2) CuSO4 (1)

Distilled water

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

C. albicans

C. glabrata

41.8±0.4a 35±1.2ab 38.4±0.5a 38.8±0.4a 31.2±1.3ab 36.8±0.4ab 31.2±0.8ab 27.6±0.8b 22.8±1bc 15.8±0.4bc 10.2±1.3c 8.6±1.1c 23.2±0.4b 20.6±1.1bc 15±1bc 12.4±1.3c 10.8±0.4c 8±1.2c 0±0d 14.2±0.8c 12.6±1.1c 10.8±1c 8.2±1.3c 0±0d 0±0d 0±0d 0±0d

44.8±1a 36.8±0.4ab 43.4±0.5a 36.8±0.4ab 34±1.2ab 38±1a 34.4±0.5ab 32.8±0.4ab 30.8±1ab 23.2±0.8b 14.8±0.4c 10.2±0.8c 26.8±0.4b 22.4±1.3bc 19.8±1bc 15.2±1.3bc 13.8±0.4c 9.4±1.3c 8.4±0.5c 15.8±0.4bc 12.4±1.3c 10.2±0.8c 9.2±0.8c 8.6±0.89c 0±0d 0±0d 0±0d

GIZ in disk diffusion (mm) C. parapsilosis C. krusei

41.6±0.8a 40.8±0.4a 43.8±0.4a 36.2±0.8ab 37±1.2ab 41.8±1a 35.6±1.1ab 33±1ab 27.2±0.8b 23.2±1.3b 16.8±0.4bc 11±1.2c 28.6±0.8b 22.4±0.5bc 20.2±0.8bc 18.8±0.4bc 14.6±0.8c 11±1.2c 10.2±0.4c 16.2±0.8bc 12.2±0.4c 11.4±1.3c 9.8±0.4c 9.2±1.3c 0±0d 0±0d 0±0d

45.2±1.3a 40.8±1a 43.4±0.5a 37.8±0.4ab 38.2±1.3a 41.2±0.8a 32±1.2ab 29±1.2b 25.8±0.4b 21.6±0.8bc 14.4±0.5c 11.8±1c 28±1.2b 25.8±0.4b 21.6±1.1bc 17.2±0.8bc 14.4±0.8c 11.6±1.1c 9±1.2c 16±1bc 12.8±0.4c 11.2±0.8c 10.4±1.3c 9.2±0.8c 0±0d 0±0d 0±0d

C. guilliermondii

48.8±0.4a 45.8±1a 50±1a 41.8±0.4a 40.4±1.3a 45.8±0.4a 41.8±0.4a 35.4±0.8ab 32.2±1.3ab 23.2±0.8b 15.6±1.1bc 13.8±0.4c 32.2±1.3ab 30.2±0.8ab 24.4±0.8b 21.4±0.5bc 16.8±0.4bc 14.2±0.8c 11.8±1c 18.2±0.8bc 12.8±0.4c 11.6±0.8c 10.4±1.3c 10.8±0.4c 0±0d 0±0d 0±0d

1 2 3 4

Table 5 The GIZ of fungi in several dilutions of CuNPs@Strawberry, Strawberry, and CuSO4.

Dilution (mg/ml)

CuNPs@Straw (64) CuNPs@Straw (32) CuNPs@Straw (16) CuNPs@Straw (8) CuNPs@Straw (4) CuNPs@Straw (2) CuNPs@Straw (1) Strawberry (64) Strawberry (32) Strawberry (16) Strawberry (8) Strawberry (4) Strawberry (2) Strawberry (1) CuSO4 (64) CuSO4 (32) CuSO4 (16) CuSO4 (8) CuSO4 (4) CuSO4 (2) CuSO4 (1)

Distilled water

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

C. albicans

C. glabrata

27.4±0.8ab 23.8±0.4b 20.2±0.8b 15.8±0.4bc 12±1.2c 9.2±1.3c 0±0d 18.2±0.8bc 14.6±1.1c 11.2±0.8c 10.8±0.4c 8.8±1c 0±0d 0±0d 10.6±1.1c 9.4±1.3c 8.4±0.5c 0±0d 0±0d 0±0d 0±0d 0±0d

30.2±1.3a 25.8±0.4ab 22.8±1b 16.2±1.3bc 13±1c 12.6±1.1c 8.8±0.4c 19.6±0.8bc 14.2±0.8c 12±1.2c 10.2±1.3c 10.4±0.5c 0±0d 0±0d 12±0.7c 11.6±1.1c 9.8±1c 8.6±0.8c 0±0d 0±0d 0±0d 0±0d

GIZ in well diffusion (mm) C. parapsilosis C. krusei

34.2±1.3a 30.4±0.5a 22±1.2b 19.8±0.4bc 15±1.2bc 14.8±1c 10.8±0.4c 22±1.2b 18.6±0.8bc 14.4±0.5c 11.2±1.3c 10.8±0.4c 8.2±0.8c 0±0d 12.6±0.8c 10.8±1c 10.2±0.4c 8.2±0.8c 0±0d 0±0d 0±0d 0±0d

34.8±1a 32.4±1.3a 24.8±0.4b 17.6±0.8bc 14.2±0.8c 12.4±0.5c 10.4±0.5c 23.4±1.3b 15.8±1bc 13.6±1.1c 11.8±0.4c 9.2±0.8c 9.6±1.1c 0±0d 13.4±1.3c 11.2±0.8c 10.2±0.4c 9.4±0.5c 0±0d 0±0d 0±0d 0±0d

C. guilliermondii

36.4±0.8a 33.8±0.4a 31.2±0.8a 24.8±0.4b 18.4±1.3bc 13.8±1c 12.2±0.8c 26±1ab 23.2±0.4b 18.2±1.3bc 15±0.7bc 14.4±0.5c 10.2±0.8c 0±0d 16.6±1.1bc 12.8±1c 12±1.2c 9.8±0.4c 0±0d 0±0d 0±0d 0±0d

1 2 3 4

Table 6 MIC and MFC of CuNPs@Strawberry, Strawberry, and CuSO4 against fungi. Microorganism MICCuNPs@Straw (mg/ml) MFCCuNPs@Straw (mg/ml) MICStrawberry (mg/ml) MFCStrawberry (mg/ml) MICCuSO4 (mg/ml) MFCCuSO4 (mg/ml)

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

C. albicans

C. glabrata

C. parapsilosis

C. krusei

C. guilliermondii

4±0b

4±0b

2±0a

2±0a

2±0a

8±0C

4±0B

4±0B

4±0B

2±0A

8±0c

8±0c

8±0c

4±0b

4±0b

16±0D

8±0C

8±0C

8±0C

4±0B

16±0d

16±0d

8±0c

8±0c

8±0c

32±0E

16±0D

16±0D

16±0D

8±0C

1 2 3

Table 7 The level of macroscopic parameters in experimental groups. Parameters Wound area (cm2) Wound contractures (%)

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Control

Basal ointment

Groups (n=10) Tetracycline CuSO4 ointment ointment

Strawberry ointment

CuNPs@Straw ointment

2.5±0.2d 37.5±2.5d

2.5±0.1 37.5±1.5

1.6±0.1b 60±1.5b

1.5±0.2b 62.5±2.5b

0.9±0.2a 77.5±2.5a

2.1±0.1c 47.5±1.5c

1 2 3

Table 8 The level of microscopic parameters in experimental groups. Parameters Total cell (n) Vessel (n) Fibrocyte (n) Fibroblast (n) Fibrocyte to Fibroblast (ratio) Lymphocyte (n) Macrophage (n) Neutrophil (n)

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Control

Basal ointment

Groups (n=10) Tetracycline CuSO4 ointment ointment

Strawberry ointment

CuNPs@Straw ointment

1523.5±30c 5.4±0.3b 2.3±0.3d 13.2±0.6c 0.17±0.01d

1498.8±41.8c 5.8±0.3b 2.4±0.1d 12.1±0.8c 0.19±0.03d

1109.7±23.9a 9.5±0.3a 6.5±0.3b 18.7±0.9b 0.34±0.04b

1269.7±31.8b 8.9±0.6a 4.5±0.3c 17.8±0.8b 0.25±0.02c

1232.9±31.1b 9.3±0.4a 7.2±0.5b 19.1±0.9b 0.37±0.02b

1093.7±32.8a 9.6±0.5a 11.2±0.6a 25.4±1.3a 0.44±0.04a

26.5±2.1c 5.6±0.5b 28.7±2.6d

24.4±1.7c 4.9±0.3b 21.7±1.6c

13±1.4b 1.7±0.2a 12.4±0.9b

14.1±2.2b 2.1±0.3a 11.6±0.9b

13.6±1.6b 1.9±0.1a 11.7±1.4b

6.7±0.9a 1.6±0.4a 3.2±0.4a

1 2 3

Table 9 The level of biochemical parameters in experimental groups. Parameters Hydroxyproline (mg/g of tissue) Hexosamine (mg/100mg of tissue) Hexuronic acid (mg/100mg of tissue)

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Control

Basal ointment

Groups (n=10) Tetracycline CuSO4 ointment ointment

Strawberry ointment

CuNPs@Straw ointment

13.7±0.8d

15.2±1.7d

25.1±0.7b

19.9±0.7c

27.6±2.1b

39.8±3.2a

0.19±0.02d

0.19±0.01d

0.29±0.03b

0.24±0.0c

0.32±0.5b

0.43±0.03a

0.07±0.0d

0.08±0.01d

0.18±0.03b

0.12±0.1c

0.19±0.0b

0.26±0.3a

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Fig. 1.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fig. 2.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fig. 3.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Fig. 4.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Fig. 5.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Fig. 6.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Fig. 7.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Fig. 8.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Fig. 9.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Fig. 10.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fig. 11.

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fig. 12.

1 2 3 4 5 6 7

Synthesis, characterization, and evaluation of cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing effects of copper nanoparticles using the aqueous extract of Strawberry fruit Saba Hemmati a, Sheida Ahany Kamangar a, Akram Zangeneh*,b,c, Mohammad Mahdi Zangeneh*,b,c,

1 2 3



That the submission” Synthesis, characterization, and evaluation of cytotoxicity, antioxidant, antifungal,

4

antibacterial, and cutaneous wound healing effects of copper nanoparticles using the aqueous extract of

5

Strawberry fruit” is original, submitted solely to this journal, and not currently under consideration for publication

6

or already published elsewhere.

7



That no any sentence is copied from other sources. See our editorial policies on plagiarism and text recycling.

8



That the submitting author takes responsibility for the submission on behalf of all authors as the corresponding

9 10 11 12 13

author. 

That all authors have reviewed, approved, and consented to the submission, and they are accountable for all aspects of its accuracy and integrity in accordance with ICMJE criteria.