Introduction

Nanoparticles can be made via chemical or biological processes. Chemical production process have been associated with a number of detrimental impacts because some dangerous chemicals are present and have been absorbed on the surface. Instead of using chemical and physical methods to create nanoparticles, biological procedures using bacteria, enzymes, fungus, plants, or plant extracts are a suitable alternative¹. One important area of nanotechnology is the creation of these environmentally benign processes for generating nanoparticles, especially copper nanoparticles, which have various applications².

Some microorganisms, such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa microscopic organisms, are poisoned by copper nanoparticles, but animal cells are unaffected. Using the “green mix” method, certain odd plants and nanoparticles have been combined. A few odd plants and nanoparticles have been combined using the green union technique³. Copper nanoparticles are responsive and can surely work with other particles due to their significant surface area to volume ratio. For a very long time, colloidal copper has been used as a potent antimicrobial. Because of the use of copper nanoparticles in wound dressings and their biocidal properties, scientists have given them a lot of thought. ⁴Virulent species like Staphylococcus aureus, are susceptible to the anticipated antibacterial actions of copper nanoparticles. Due to their extensive uses in fields like mechanics, optics, biomedical sciences, hardware, sedate quality conveyance, catalytic photo electrochemical applications, and nonlinear optical devices, these nanoparticles have also caught the attention of experts.^3,5

Bryophyllum pinnatum (lam.)(BP) (equivalent word: Kalanchoepinnata) is a perpetual spice developed and utilized in folkloric medication in tropical Africa, India, China, Australia and tropical America⁶ Figure 1. In a creating nation like Nigeria, BP is delegated a weed that thrives all through the Southern piece of the nation. These plants are typically found in gardens wealthy in natural fertilizer and adequate dampness.⁷ Various dynamic mixes, including flavonoids, glycosides, steroids, bufadienolides and natural acids, have been distinguished in Bryophyllum pinnatum.⁸ In this manner, BP is utilized for medication in ethno practice treatment of injuries of the navel of recently conceived children, in ear problem, hack, looseness of the bowels, and diarrhea. It was discovered that the leaves 60% methanolic extract was efficient against several bacteria. These include Staphylococcus aureus, Shigella dysenteriae, Proteus vulgaris, Bacillus substilis, and Escherichia coli at a dosage of 25 mg/ml. The extract was found to be ineffective against Klebsiella pneumonia, Candida albicans, and Pseudomonas aeruginosa⁹.

Figure 1: Leaf of  Bryophyllum pinnatum (lam.) Click here to view figure

Polyalthia longifolia (Sonn.)  the false ashoka native to India, is an elevated evergreen tree, normally planted because of its viability in all conditions. A massive variety of bushes and trees known as Polyalthia longifolia (Sonn.)  is found across the tropics and subtropics¹⁰ Figure 2. It belongs to the Annonaceae family, which has more than 2000 species and 120 genera. Polyalthia longifolia (Sonn.)  Pendular is a tall, beautiful, evergreen tree native to Sri Lanka that has spread over the Indo-Pakistan sub-mainland. It has a straight trunk and flat branches¹⁰. The bark of Polyalthia longifolia (Sonn.)  is cited in ethno pharmacological claims to have febrifuge properties. It weakens the heart, lessens circulatory strain, and energizes breathing. It has been taken into account how Polyalthia longifolia (Sonn.)  affects fungus. It is useful in treating helminthiasis, diabetes, hypertension, and skin conditions. Additionally, it is applied to the healing of wounds.¹¹

Figure 2: Leaf of Polyalthia longifolia (Sonn.) Click here to view figure

Methanol extracts of Polyalthia longifolia (Sonn.)  var. pendula’s leaves, stems, twigs, green berries, flowers, roots, root-wood, and rootbark were tested for their antimicrobial and antifungal properties.¹² With MIC values between 7.8 and 500 g/ml, the methanol extract of leaves and berries completed bioassay-monitored isolation operations and showed good antibacterial activity.¹³ 91 clinically relevant microbial species were tested for their antibacterial potential by two dosages of various P. longifolia leaf extracts, including 1, 4-dioxan, methanol, and acetone extracts.¹⁴ All three plant  extracts are efficient against 98% of the total number of gram-positive bacterial strains at a concentration of 500 g/disc.¹⁵ While 1, 4-dioxan extract was efficacious against 18.18% of the total gram negative bacterial strains, methanolic and acetone extracts were only effective against 12.72 % of them.¹⁶

Copper has a wide scope of antimicrobial activity including bacterial species and is viewed as more powerful than costly metallic antimicrobials.¹⁷ Copper kills microorganisms by an instrument called contact executing. Copper Nanoparticles can be used as microbial development inhibitors.¹⁸ Additionally, metallic nanoparticles have a biocidal effect on both bacterial species. They are bactericidal because of their high surface to volume ratio and small size.¹⁹ They become highly intensive as a result, disrupting microbial membranes. The aqueous extract of two plant leaves and copper sulphate pentahydrate were used in this study to create copper nanoparticles (CuNP), which were subsequently analyzed utilizing cutting-edge techniques.²⁰ Bacterial and fungal species were examined for their susceptibility to the produced CuNP antimicrobial properties.²¹

 Materials and methods

Preparation to Plant extract

Bryophyllum pinnatum (lam.) and Polyalthia longifolia (Sonn.)  leaves were purchased from nursey nearby Avadi. The collected leaves were washed with running water then by distilled water and dried in shade for not more than 10 days. The shade dried leaves were gathered and squashed using mortar and chisel, followed which the leaves were taken in a receptive and 100ml of refined water is added and heated in water bath for 100˚C for 30 minutes Figure 3a and 3b. Then the extract was collected using Whatman No.1 filter paper and used in the synthesis of nanoparticles.^22,23

Figure 3: a. Leaf extract of Bryophyllum pinnatum (lam.) with CuCl2 as precursor b. Leaf extract of Polyalthia longifolia (Sonn.)  with CuSo4 as precursor. Click here to view figure

Green Synthesis of Copper nanoparticles

Copper nanoparticles were produced by 1ml of plant extract in to 30ml of copper chloride and copper sulphate solution while stirring for 30 minutes. The two different preparations were stored at darkroom overnight by wrapping in aluminum foil Figure 4a and 4b. The copper solutions original blue tint changed to an earthy green hue, visually confirming the preliminary method’s assertion that nanoparticles had formed. Both the solution were centrifuges at 10000 rpm after the change of color. After discarding the supernatant, the formatted Copper nanoparticles Figure 5a and 5b were washed with distilled water and characterization analysis is done. ²⁴

Figure 4: (a). Leaf extract of Bryohyllum pinnatum (lam.)  with CuCl2 as precursor (b). Leaf extract of Polyalthia longifolia (Sonn.)  with CuSo4 as precursor at zero hours. Click here to view figure

Figure 5: (a). Leaf extract of Bryohyllum pinnatum (lam.)  with CuCl2 as precursor (b). Leaf extract of Polyalthia longifolia (Sonn.)  with CuSo4 as precursor at 24 hours. Click here to view figure

Characteristics analysis

Shimadzu’s UV-2600 UV-visible spectrophotometer was used to capture the samples’ UV-visible absorbance and reflectance spectra. Its wavelength range is 200-800 nm. UV-DRS diffuse reflection spectroscopy, whereas transmission was utilized to analyze UV-visible spectra. The plant extract was combined with copper solution after the necessary dilution to create the Copper nanoparticle solution. An absorbance spectrum was discovered by contrasting the absorbance of a nanoparticle solution with that of a control extract solution at wavelengths ranging from 200 to 800 nm. FTIR analysis for leaf extracts of Polyalthia longifolia (Sonn.)  and Bryophyllum pinnatum (lam.) using copper sulphate and chloride, respectively, as precursors. The terms “FTIR analysis” and “FTIR spectroscopy” are frequently used to describe Fourier transform infrared spectroscopy. The analytical technique known as FTIR analysis can be used to identify organic, polymeric, and occasionally inorganic functional groups. This method uses infrared light to scan test samples and look at chemical properties ²⁵. 

Results and Discussion 

Characterization of nanoparticle

The synthesized copper oxide nanoparticles optical characteristics along with the biocomponents found in plants. Crystallinity, nanoparticle size, and shape were all examined throughout their production utilizing a variety of techniques²⁶. The produced nanoparticles were examined using FTIR (Fourier Transform Infrared Spectroscopy), UV-Visible spectroscopy, XRD analysis, and SEM examination^27. 

Fourier Transform Infrared Spectroscopy

A FTIR Spectrometer analysis was performed to determine the likely functional groups in plant extract that are involved in the synthesis, stabilization of copper oxide nanoparticles. The spectra between 650 and 4000 cm^-1 were acquired FT-IR characterization used to examine the phytocomponents from plants that are in converting cupric ions to copper oxide nanoparticles and then capping them. The peaks of the spectrum of Bryophyllum pinnatum (lam.) plant extract include 2355cm^-1, 3312cm^-1, 1640cm^-1, 3350cm^-1, 2322cm^-1, 3327cm^-1, and 776cm^-1. FTIR examination revealed the hydroxyl (-OH) group with a peak at 3312cm^-1, whereas the N-H peak is at 3350cm^-1. The peak at 2,355 cm^-1 was attributed to CN, while the peak at 3312 cm^-1 belongs to CC-H. The peak C=O stretch measurement is 1640 cm^-1. In the FTIR spectrum of copper oxide nanoparticles, the peculiar characteristic bands at 773, 974, 1074, 1097, 1119, 1153, 1339, 1383, 1633, and 3231cm^-1 correspond to the bands from the plant extract of Bryophyllum pinnatum. The N-H extension vibration is attributed to the peak at 3231cm^-1.1633 C=N by 1383, C=C by 1339, C=O by 1153, and C=O by 1119 Figure 6. This suggests that copper oxide nanoparticles were produced using biomolecules that can be found in plant extract^28.

Figure 6: FTIR Result of green synthesized CuONP with leaf extract and CuCl2 as precursor Click here to view figure

The phytochemicals from plants that are engaged in reducing copper ions to copper oxide nanoparticles and afterwards capping were also analyzed using FT-IR characterization. The peaks in the spectrum of Polyalthia longifolia (Sonn.)  leaf extract include which was evidently seen at 3354, 2922, and 1744 cm^-1, respectively, and corresponds to the O-H, C-H, and C = O stretching vibrations of polyphenol compounds. Simultaneously, the interaction between copper-based nanoparticles and polyphenols results in a broad absorption peak at 3356 and 2926 cm^-1 of O-H and C-H stretching. O-H stretching was substantially less intense as a result of the Cu ion’s oxidation process, which produced the Cu(II), Cu(I), and Cu(0) ions. The peak intensity at 1635 cm^-1 was therefore attributed to the C = O stretching from the polyphenol bond with Cu metal, leading to a decreased electron density as a result of the dispersion of electrons towards Cu ions Figure 7. The extract also functions as a stabilizing agent (which prevents re-oxidation) in addition to acting as a reducing agent^29.

Figure 7: FTIR Result of green synthesized CuONP with leaf extract and CuSO4 as precursor. Click here to view figure

XRD Analysis

Figure 8 displays the XRD result of copper oxide nanoparticles produced using Bryophyllum pinnatum (lam.) plant extract. 2 Diffraction peaks were found at 11.763°, 12.838°, 20.782°, 25.777°, 42°, and 57.06°. Strong peaks in XRD patterns indicate that the CuO nanoparticles produced by reducing Cu ions in the presence of Bryophyllum pinnatum (lam.) leaf extracts are crystalline. The JCPDS/ICDD database and published literatures were compared to the miller indices and peaks of CuO nanoparticles using the Jade software, and it was discovered that they were fairly close. The copper oxide crystal phase’s (113), (030), (036), and (436) planes are linked to the diffraction peaks at 2 values of 20.78°, 25.77°, 42°, and 57.06°, respectively. Although cuprous oxide (CuO crystalline’s) phase peaks at 11.763° and 12.83° are assigned to its (110), (110), and (220) planes, respectively, the results are in very good agreement with powder CuO from the JCPDS standards (JCPDS file no. 77-1898). These values match reported literature and the values for the cubic phase of CuO match the corresponding “JCPDS” (Joint Committee on Powder Diffraction Standards card no. 34-1354).³⁰ Both oxides are present in the crystalline phase of CuO nanoparticles, as shown by the presence of CuO and CuO-related peaks in the XRD spectrum. The average crystal size of CuONPs is 65.776 nm. XRD analysis has been used in a few publications to describe different crystalline sizes for CuONPs that were bio-produced utilizing plant extracts.³¹

Figure 8: FTIR Result of green synthesized CuONPs with leaf extract and CuCl2 as precursor  Click here to view figure

The considerable absorption of the plant extract, which was clearly detected at 3354, 2922, and 1744 cm^-1, respectively, corresponds to the O-H, C-H, and C = O stretching vibrations of the polyphenolic compound. Figure 9 illustrates this. A large absorption peak of O-H and C-H stretching is produced at 3356 and 2926 cm^-1 by the interaction of polyphenol and Cu-based NPs. O-H stretching was substantially less intense as a result of the Cu ion’s oxidation process, which produced the Cu(II), Cu(I), and Cu(0) ions. In order to explain the peak intensity at 1635 cm^-1, it was determined that the C = O stretching from the polyphenol bond with Cu metal caused a drop in electron density as a result of the reoxidation process (stabilizing agent). The polyphenol compound’s O-H bond to copper ions was further confirmed by the absorption at 1095 cm^{-1 32}

Figure 9: FTIR Result of green synthesized CuONPs with leaf extract and CuSO4 as precursor  Click here to view figure

UV-Visible Spectrophotometer

An illustration of the creation of copper oxide nanoparticles is the change in colour of the prepared formulation from blue to green completing the reactino between extract of the plant and copper ions. This result mirrors what was discovered in earlier research^33,34. Additionally, the absorbance was looked at, and it has been discovered that the highest value of copper oxide nanoparticles was seen at 434 nm Figure 10, which is consistent with earlier research. At wavelengths of 434 nm, the absorption peaks of copper oxide nanoparticles produced by Bryophyllum pinnatum (lam.) where terpenoids act as a capping agent were visible,³⁵ while copper oxide nanoparticles made from Polyalthia longifolia (Sonn.)  had peak at 730nm³⁶ Figure 11. The production of copper oxide nanoparticles is readily visible in the UV-vis spectrum between 450 and 700 nm via plasmon resonance. ³⁷

Figure 10: UV Result of green synthesized CuONPs with leaf extract and CuCl2 as precursor. Click here to view figure

Figure 11: UV Result of green synthesized CuONPs with leaf extract and CuSO4 as precursor Click here to view figure

SEM Analysis

Utilizing HR FESEM images, the morphological properties of the produced Copper nanoparticles using Bryophyllum pinnatum (lam.) and Polyalthia longifolia (Sonn.)  were identified. The produced Copper oxide nanoparticles SEM pictures showed that they are crystalline in structure and range in size from 78 to 134 nm.

Figure 12: SEM result of green synthesized CuO NPs with leaf extract and CuCl2 as precursor Click here to view figure

Figure 13: SEM result of green synthesized CuO NPs with leaf extract and CuSo4 as precursor Click here to view figure

Anti-fungal activity of copper oxide nanoparticle

Figure 14: Antifungal activity of copper oxide nanoparticle with CuCl2 as precursor Click here to view figure

An agar plate method was used to examine the antifungal activity of the copper oxide nanoparticles against Galactomyces geotrichum. After 24 hours of incubation with copper oxide nanoparticles, an 18mm and 25mm figure 14 zone of inhibition was discovered using the agar plate method, respectively. According to the outcome, a higher inhibition zone was observed. Greater inhibition zones in the agar plate test have been observed in several studies with comparable results^{38 39}.

Conclusions

Copper nanoparticles were successfully synthesized by green method, in which copper sulphate and copper chloride were used as a precursor. The green reduction of copper ions by flavonoids such as Kaempferol, terpenoids and organic acids involves change of color from copper blue to dark brown after 24 hours which preliminarily confirms the presence of copper nanoparticles. The Characterization analysis by UV-Vis Spectrophotometer, FTIR, XRD and SEM confirmed the presence of copper nanoparticles in the size of 120-150nm. The green synthesized copper nanoparticles exhibited high anti- fungal activity against Galactomyces geotrichum with the inhibition zone of 14mm for copper chloride as a precursor and 25mm for copper sulphate as a precursor. This work proposes a facile and cheap green synthesis method for the production of copper nanoparticles with high antifungal activity. Thus, the nanomaterial could be useful for controlling pathogenic fungi for various applications.

Acknowledgment

Authors are thankful to the management of Vel tech High tech Rangarajan Dr. Sakunthala Engineering College, Chennai for their constant support to complete this research paper.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding Sources

The article was entirely financed by the author themselves.           

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