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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 3  |  Page : 341-348

Anti-Oxidant and anti-cancer activities of biogenic synthesized copper oxide nanoparticles


1 PG & Research Department of Chemistry, Holy Cross College (Autonomous), Tiruchirappalli, Tamilnadu, India
2 Department of Chemistry, Shrimati Indira Gandhi College, Affiliated to Bharathidasan University, Tiruchirappalli, Tamilnadu, India

Date of Submission29-May-2022
Date of Decision02-Jun-2022
Date of Acceptance16-Jul-2022
Date of Web Publication17-Sep-2022

Correspondence Address:
Aranganathan Agila
Holy Cross College (Autonomous), Trichy, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_136_22

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  Abstract 


Background: The nature acts like a large “bio-laboratory” comprising plants, algae, fungi, yeast, etc., which are composed of biomolecules. These naturally occurring biomolecules have been identified to play an active role in the formation of nanoparticles. Methods: This research work mainly aims to investigate the anti-oxidant (diphenylpicrylhydrazyl assay) and anti-cancer (Michigan cancer foundation-7 cell line) capacities of biologically prepared copper oxide mediated from the hydroalcoholic extract of Justicia glauca by simple precipitation and also to identify the phytochemicals in the extract by qualitatively. Results: On screening test, the extract shows the presence of carbohydrate, phenolics, alkaloids, and terpenoids saponins which are chiefly act as a reducing, stabilizing, and capping agents in nanomaterial preparations. The medicinal plant Justicia glauca extract-mediated copper oxide materials were synthesized by lost cost, simple, effective, and eco-friendly precipitation method. The prepared copper nanomaterials were characterized by ultraviolet–visible, Fourier-transform infrared spectroscopy, energy dispersive X-ray analysis, X-ray diffraction, and scanning electron microscope. The obtained spectral results reveal that the prepared particles were found to be elliptical flat shapes of copper oxide with the average size of 19.72 nm with 51.11% of copper and 48.89% of oxygen elements. Especially, on anti-oxidant and anti-cancer activities the prepared Justicia glauca extract-mediated copper oxide revealed excellent potent while comparing the other green synthesized copper oxide particles. Conclusion: Overall results evidenced that the aqueous extract of Justicia glauca is a very good bioreductant for the synthesis of copper oxide nanoparticles.

Keywords: Anti-cancer activity, anti-oxidant activity, copper (II) sulfate pentahydrate, copper oxide nanoparticles, Justicia glauca extract


How to cite this article:
Agila A, Vimala JD, Bharathy MS, Dayana Jeyaleela G, Sheela SA. Anti-Oxidant and anti-cancer activities of biogenic synthesized copper oxide nanoparticles. Biomed Biotechnol Res J 2022;6:341-8

How to cite this URL:
Agila A, Vimala JD, Bharathy MS, Dayana Jeyaleela G, Sheela SA. Anti-Oxidant and anti-cancer activities of biogenic synthesized copper oxide nanoparticles. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Dec 8];6:341-8. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/341/356144




  Introduction Top


Nanoscience/nanotechnology has been applied extensively in pharmacological activities (drug delivery, biological applications) to develop and improve the therapeutic outcomes of several diseases. Nanostructured materials of semiconductor acquire much more attention in the recent years owing different properties and wide range of applications in numerous fields such as sensors, drug discovery, catalysts, pharmacology, highly effective and functional devices, and photoelectron devices, etc.[1],[2],[3] Metal oxides especially applicable in catalytic reaction because of their high catalytic activity with the large surface area. The properties, behavior, and applications of metal oxides are depending on the size and surface morphology of the materials. This size and morphology is directly controlled by the reducing and capping agents which was used in the synthesis.[4],[5] Nanoparticles were also widely used in daily used commercial products, such as plastics, food packaging, soaps, pastes, food, and textiles, which has increased their market value to a great extent. Plenty of physical, chemical, and biological methods were reported in the nanomaterial synthesis among all the biological methods is the low-cost, eco-friendly, and most efficient compared to others.[6],[7]

Biosynthesis or green synthesis of nanoparticles is an alternative approach to the synthesis of nanoparticles using plant extracts, fungi, bacteria, yeast, algae, etc.[8],[9],[10] Plant extracts-mediated nanoparticles approach is most biocompatible, effective, safe, and low-cost process than other methods. Furthermore, plant extract also acts as stabilizing and reducing agents in nanomaterials synthesis.[11] Literature states that plant extracts are successfully used to synthesize nanoparticles such as zinc, copper, silver, gold, cobalt, platinum, palladium, and magnetite.[12],[13] Plant extracts-mediated zinc oxide nanomaterials have an effective remedy for cancer, hepatitis, malaria, and other acute diseases. Nowadays, nanoparticles were increasingly used as alternative antibiotics against various bacteria and bacterial infectious diseases. Copper oxide nanoparticles are utilized to prevent infection, control bacterial infections, and also used to treat infectious diseases. Other than antibiotic effects copper oxide have reported numerous therapeutic outcomes such that, antioxidant effects, immunomodulatory, sunscreen and anticancer, etc.[14],[15] In plant extract-mediated nanomaterials excess of phytocompounds were involved in the synthesis which can affect the shape, size, and property of the resulted copper oxide nanomaterials.[1],[16]

The plant Justicia glauca also known as Galucous Justica and are belongs to the acanthaceae family. It is as undershrub mostly found in the foothills of peninsular India. From the literature studies in the plant, Justicia glauca exhibited medicinal uses such as anticancer, antirheumatic, diuretic, laxative and sedative, antimalarial, cardio protective, antisyphilitic, antitubercular, antifungal, and antibacterial effects.[17] It clearly showed the pharmacological potential of the plant Justicia glauca and this triggers us to study the usage of whole plant extract as a reducing agent for copper oxide nanomaterial preparations. The utilization of medicinal plant extracts may reduce the toxicity of the nanomaterials and thus enhance the medicinal applications in pharmacological fields.[18],[19],[20],[21] Main advantage of practicing plants is the unnecessary usage of capping and stabilizing agents. The plant extracts act as all three reagent's roles because of the presence of many phytocompounds present in the single extract. Thus, the present study aimed to biosynthesis and characterize the Justicia glauca leaf extract-mediated CuONPs and their antibacterial and antifungal activities.


  Methods Top


Collection and extraction of Justicia glauca

Plant material Justicia glauca were collected from the Alangudi area located at Pudukottai. The taxonomy of the plant is identified by Dr. S. John Britto, Director, the Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph's College (Autonomous) Trichy. About 500 g of disease-free leaves of Justicia glauca were chopped and grind. Then transfer into the 500 ml beaker to that, 500 ml of 70% ethanol is mixed. Maintain the solvent level in the beaker at 2 cm above than the plant material, kept for 5–8 h on heating mantle for the complete extraction of all the phytoconstituents from the leaves. The final extract was first filtered by normal filter paper then, followed by Whatman No-1 filter paper. The filtrate was stored in the refrigerator for further qualitative screening and nanoparticle preparations.

Qualitative analysis of hydro alcoholic extract of Justicia glauca

The different qualitative chemical tests can be performed to establish or screen the phytochemical profile of extract of Justicia glauca. It is an essential test for detecting bioactive compounds (plant products) present in the different extracts of plants. Standard screening tests and procedures were carried out which is performed on the extract to detect various phytocompounds present in them.[22]

Biogenic synthesis of CuO nanoparticles from the aqueous extract of Justicia glauca

Fifty ml of 0.1 M copper sulfate solution was taken in a 250 ml beaker, and placed on the magnetic stirrer. To this, 10 ml of the filtered hydroalcoholic extract of Justicia glauca was slowly added at every 30 min intervals. After completion of 50 ml extract addition, the reaction mixture was kept overnight at room temperature. Finally, Justicia glauca extract mediated copper oxide particles are denoted as JG-CuONPs. The synthesized nanomaterials were deposited at the beaker bottom and it was collected by centrifuging at 6000 rpm. To achieve the maximum purity of JG-CuONPs, the collected nanomaterials were washed with D. D water, followed by ethanol. Solvent-free JG-CuONPs was dried in an oven for complete conversion of Cu(OH)2 into CuO. Synthesized JG-CuONPs were confirmed with the help of ultraviolet-visible (UV-Visible) spectroscopy, Fourier-transform infrared spectroscopy (FT-IR) spectral analysis, X-ray diffraction (XRD), scanning electron microscope (SEM), and energy dispersive X-ray analysis (EDAX) analysis.[23],[24],[25]

Anti-oxidant activity of synthesized JG-CuONPs

The antioxidant activity of JG-CuONPs was assessed by diphenylpicrylhydrazyl (DPPH) free radical assay. 5, 10, 20, 40, and 80 μl/ml of synthesized nanoparticles JG-CuONPs were mixed with 2 ml of 0.3 mm DPPH reagent and kept at the dark place then allowed to reacting at room temperature. After 30 min, the absorbance was recorded at 517 nm in UV-visible spectrophotometry and the percentage of radical scavenging activity, i e., anti-oxidant activity was calculated by following standard formulae.[26],[27],[28]



Ab of control = Control Absorbance

Ab of test = Test solution Absorbance

Anti-cancer activity of synthesized JG-CuONPs

The JG-CuONPs sample was tested for in vitro cytotoxicity, using Michigan Cancer Foundation-7 (MCF-7) cells by MTT assay. The cultured MCF-7 cells were harvested by trypsinization, and pooled in a 15 ml tube. Then, the cells were plated at a density of 1 × 105 cells/ml cells/well (200 μL) into the 96-well tissue culture plate in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% FBS and 1% antibiotic solution for 24–48 h at 37°C. The wells were washed with sterile phosphate-buffered saline (PBS) and treated with various concentrations of the JG-CuONPs sample in a serum-free DMEM medium. Each sample was replicated three times and the cells were incubated at 37°C in a humidified 5% CO2 incubator for 24 h. After the incubation period, MTT (20 μL of 5 mg/ml) was added into each well and the cells were incubated for another 2–4 h until purple precipitates were clearly visible under an inverted microscope. Finally, the medium together with MTT (220 μL) was aspirated off the wells and washed with 1X PBS (200 μl). Furthermore, to dissolve formazan crystals, dimethyl sulfoxide (100 μL) was added and the plate was shaken for 5 min. The absorbance for each well was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, USA) and the percentage cell viability and IC50 value were calculated using Graph Pad Prism 8.0 software (USA).[29],[30],[31]


  Results Top


Phytochemical screening results of hydro alcoholic extract of Justicia glauca

The phytochemical components present in the leaves extract of Justicia glauca was investigated by standard phytochemical screening procedure. Results showed [Figure 1] the presence of carbohydrates, glycosides, alkaloids, saponin, phenolics, alkaloids, terpenoids, xanthoproteins, etc., in the plant extract (as reducing agents) which plays synergistic effect in the reduction of copper in copper sulfate.
Figure 1: Phytochemical Screening results of hydro alcoholic extract of Justicia glauca

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Ultraviolet-visible results of Justicia glauca-mediated copper oxide nanoparticles

UV-visible spectra of Justicia glauca extract show [Figure 2] the maximum absorbance at 218 nm, and 391 nm which is due to unsaturated groups or hetero atoms (C = C, S, N, O) in the extract and are mainly flavonoids, phenolics, alkaloids, etc., the absorbances at 413, 535, and 663 is because of chlorophylls (organic chromophores) in the plant extract. In the JG-CuONPs UV spectrum, the maximum absorption peak is observed at 434 nm which confirms the formation of CuONPs (due to the surface plasmon resonance effect) and the peak at 670 nm indicates the unreacted copper in the reaction mixture [Figure 3].[27]
Figure 2: UV-visible of hydro alcohoic extract of Justicia glauca. UV-visible-Ultraviolet–visible

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Figure 3: UV-Visible Spectrum of Justicia glauca-mediated CuONPs. UV-visible-Ultraviolet–visible

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Fourier-transform infrared spectroscopy results of Justicia glauca-mediated copper oxide nanoparticles

FT-IR spectra of plant extracts reveal the functionalities of OH, C = O, C = C, C-O which indicates the presence of phyto-molecules [Figure 4]. Usually, the metal-oxygen (M-O) bond showed FT-IR frequencies at below 600 cm−1. The FT-IR spectrum of JG-CuONPs nanoparticles [Figure 5] absorbed broadband at 638 cm−1 which strongly confirms the metal-oxygen bond in the prepared copper oxide.[28]
Figure 4: FT-IR Spectrum of hydro alcoholic extract of Justicia glauca. FT-IR-Fourier-transform infrared spectroscopy

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Figure 5: FT-IR Spectrum of Justicia glauca-mediated CuONPs. FT-IR-Fourier-transform infrared spectroscopy

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X-ray diffraction and energy dispersive X-ray analysis results of Justicia glauca-mediated copper oxide nanoparticles

XRD was used to determine the crystal structure of a material system. The 2Θ values of synthesized copper oxide nanoparticles [Figure 6] were found to be 32.55°(−111), 35.32°(002), 38.70°(111), 48.71°(−202), 58.14°(202), 66.09°(−311), 68.07°(113), and 75.0°(400). The peaks of the graph were in good agreement with the literature report (JCPDS File no: 45–0937). Using the Debye Scherrer equation (Dp = [0.94*λ]/[β*CosΘ]), the average size of the nanoparticles was determined. The average crystalline size of the formed CuONPs was found to be 19.72 nm with the monoclinic phase of JG-CuONPs. XRD patterns obtained in this study are similar to XRD patterns obtained in the other biologically synthesized CuO nanoparticles. SEM analysis of JG-CuONPs is displayed in [Figure 7] and [Table 1] which is used to identify the morphology, size, and shape of the synthesized nanoparticles. The resulted SEM images also revealed the elliptical flat-shaped Justicia glauca-mediated CuONPs (JG-CuONps).[29],[30],[31],[32]
Figure 6: XRD Pattern of Justicia glauca-mediated CuONPs. XRD-X-ray diffraction

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Figure 7: SEM Images of Justicia glauca-mediated CuONPs. SEM-Scanning electron microscope

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Table 1: X-ray diffraction pattern of Justicia glauca-mediated copper oxide nanoparticles

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The energy-dispersive X-ray EDAX investigation was done to check the elemental compositions of the synthesized Justicia glauca-mediated CuONPs. The EDAX spectrum showed the highest percentages of copper and oxygen with the elemental composition of 51.11% and 48.89%, respectively, which is shown in [Figure 8].[33],[34],[35],[36]
Figure 8: EDAX Image of Justicia glauca-mediated ZnONPs. EDAX-Energy dispersive X-ray analysis

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Anti-oxidant activity results of Justicia glauca-mediated CuONPs

The antioxidant capacities of standard drug ascorbic acid and synthesized JG-CuONPs were evaluated and their results are shown in [Table 2]. From the results, it is found that when increasing the concentration of the JG-CuONPs the percentage of scavenging effects also increased. The IC50 of ascorbic acid is 72.33 μg/ml, while JG-CuONPs is 37.31 μg/ml when comparing both the results it is found that, the Justicia glauca-mediated CuONPs were showed an excellent scavenging effect than the ascorbic acid.
Table 2: Anti-oxidant activity results of Justicia glauca-mediated copper oxide nanoparticles

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Anti-cancer activity results of Justicia glauca-mediated CuONPs

The anticancer capacities of Justicia glauca-mediated copper oxide nanomaterials were (JG-CuONPs) evaluated and their results are shown in [Table 3] and their images are shown [Figure 9]. From the results, concentration depended activity is found, at a higher concentration of 500 μg/ml around 74.03% of breast cancer cells (MCF-7) are destroyed, i.e., only 25.97% of MCF-7 cells were alive. The IC50 of JG-CuONPs is 28.72 μg/ml.
Figure 9: Images of anti-cancer activity results of Justicia glauca-mediated CuONPs against MCF-7 Cell line. MCF-7-Michigan cancer foundation-7

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Table 3: Anti-cancer activity results of Justicia glauca-mediated copper oxide nanoparticles against Michigan Cancer Foundation-7 cell line

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  Discussion Top


From literature studies, it understood that plant extract can be the well know and successful reducing agents and their properties and applications varies from plant extracts to plant extracts which is due to the phytoconstituents present in the extracts. In this study, the free radical scavenging efficacies of copper oxide nanomaterials were examined and it shows IC50 of 37.31 μg/ml which is higher than the reported anti-oxidant activities of standard ascorbic acid as well as the other reported works. Ijaz et al. also reported the antioxidant effect of Abutilon indicum-mediated CuONPs which is possessed the IC50 of 120 μg/ml.[37] The Desmosdium gangeticum root extract treated CuONps were shown the radical scavenging ability with the IC50 value of 0.03 mg/ml which was recorded by Guin et al.,[38] similarly, Prunus africana and Camellia sinensis-based copper oxide synthesis and their anti-oxidant effect was studied which shows the minimal inhibition of 23.1% and 23.4%, respectively, even at higher concentration of 300 μg/ml.[39] Rehana et al. also extensively studied the free radical scavenging behavior of copper oxide nanomaterials synthesized from the medicinally important five plants Hibiscus rosa-sinensis, Moringa oleifera, Azadirachta indica, Tamarindus indica, and Murraya koenigii and their IC50 values are between the ranges of 20–38 μg/ml.[40] Similarly, numerous researches were recorded on anti-cancer potentials of greener copper oxide nanomaterials. The Eucalyptus globulus leaf-derived copper nanoparticle shows the IC50 of >100 μg/ml,[41] likewise Cammellia sinenesis is mediated CuONPs is 50.3 μg/ml,[42] Manilkara zapota extract-based copper nanoparticles is 53.89 μg/ml, Nilgirianthus ciliates extract-mediated shows 85.58 μg/ml and Annona muricata copper oxide exhibits 35 μg/ml of IC50 ranges.[43] Peculiarly, the Azadirachta indica leave extract mediated copper oxide nanomaterial lowest IC50 of 21.5 μg/ml.[44] The anti-cancer activities of green synthesized copper oxide nanoparticles were varied by its sizes, shapes, and also the phytochemicals involves in the reduction process.


  Conclusion Top


Green synthesis of nanoparticles used in the research work was found to be eco-friendly, nontoxic, and less usage of chemicals than the physical and chemical methods. The phytochemicals present in the leaf extract itself help in the synthesis of metal oxide nanoparticles. The presence of functional groups that induce the nanoparticle synthesis were alcohols and phenols that are widely seen in secondary metabolite flavonoids. From the literature, it is understood that, 70% ethanol extract possess flavonoid compounds (major phyto-molecule) which is also confirmed by qualitative and UV-visible. Further, the XRD analysis proved the monoclinic crystalline nature of the copper oxide nanomaterial with an average size of 1972 nm. The anti-oxidant and anti-cancer activities of Justicia glauca-mediated copper oxide nanomaterials were excellently evidenced its pharmacological applications and also from the research the hydro alcoholic extract of Justicia glauca can act as a very good bioreductant for the synthesis of copper oxide nanoparticles.

Limitation of the study

The above research findings support the green synthesis of copper oxide nanoparticles which are found to be facile and feasible. However, this synthetic root has shortcomings and challenging issues such as concentration of the Justicia glauca extract, stoichiometric ratios of the extract and precursor, yield and stability of the copper nanoparticles, and other optimization factors like pH, time, and temperature. All the limitations and shortcomings are can be rectified by advanced research in nanomaterial preparations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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