High Value Utilization of Fern Extracts For Fabrication of Copper Nanoparticles: A Potent And Affordable Candidate For Water Treatment, Water Disinfection, Antioxidant Activity And Theranostic Agent.

doi:10.21203/rs.3.rs-580948/v1

This study reports a simple, green, and large–scale biosynthetic fabrication of spherical copper nanoparticles (Cu NPs), approximate 28 nm, using the aqueous extracts of Diaplazium esulentum (Retz.) Sw. fern. Characterization was carried out for morphology, size, elemental analysis, crystallinity and for identification of functionalities responsible for reduction and stabilization. Extraordinary remediation efficiency was obtained for the developed Cu NPs for the elimination of two industrially important pollutants; Methyl Violet 6B and Methylene Blue (greater than 92 % within 150 mins). A mechanism was postulated for the process using the data on the identification of NPs and also spent NPs apart from degraded products.

Furthermore, the Cu NPs revealed excellent antibacterial activity against gram positive bacteria: Staphylococcus aureus, Streptococcus pneumonia and Bacillus subtilis, as well as gram negative bacteria: Pseudomonas aeruginosa, Escherichia coli and potential action against fungal strains; Aspergillus niger and Candida albicans.

Additionally, the assay depicted that Cu NPs has fairly decent radical scavenging activity with IC₅₀ value 2.11 mM.

Moreover, the Cu NPs were found to exhibit profound anticancerous activity against two human cancer line cells.

The present research work has implications for exploitation of DE fern extract for the development of Cu NPs and their numerous applications ranging from pollutants removal to antibacterial, antifungal, antioxidant and anticancerous agents.

Environmental Chemistry

Toxicology

Phytosynthesis

Photodegradation

Antimicrobial

Antifungal

Antioxidant and Anticancer

Currently, nanostructured materials synthesized from cheap and earth abundant metals have gained substantial attention due to possibility of replacing costly noble metal catalysts and can find applications in wide ranging fields including diagnostics, sensing, electronics and surface enhanced Raman scattering (Bansal et al 2009; Cao et al 2002 and Jeong et al 2011).

Nevertheless, unlike other noble nanometals, the fabrication of pure metallic Copper Nanoparticles (Cu NPs) under aqueous phase is a cumbersome task, as Cu NPs generated in aqueous solutions are susceptible to surface oxidation because of their low reduction potential and generates a copper oxide core-shell NPs that have very less applicability (Mott et al 2007).

The synthesis protocol generally includes utilization of organic solvents, harsh reducing agents, and low concentrations apart from inert environment causing difficulty for the mass production of these technologically significant nanostructured materials (Park et al 2007).

Consequently, there is an indispensable obligation to substitute these fabrication methodologies with facile, green, rapid, economic and environment friendly methods with high quantum yield, good biocompatibility.

Therefore, biogenic fabrications of NPs employing plants extracts, microorganisms, actinomytes and fungi are intensively studied (Parikh et al 2008). Among these, plant extracts and their derivatives are more preferred for biosynthesis as this approach is practical, simple, scalable and requires less time for generation (Huang et al 2009).

Recently, there have been only few reports on the plant extract mediated fabrication of Cu NPs. Some of the plant/ plant parts that have been exploited for the synthetic protocol involves leaves of Brassica juncea (L.) Czern (Hameed et al 2008), Medicago sativa L. (Umer et al 2012), Euphorbia esula L.(Nasrollahzadeh et al 2014), Lawsonia intermis L. (Cheirmadurai et al 2014) and Phyllanthus embilica L.(Caroling et al 2015), Plantago asiatica L.(Nasrollahzadeh et al 2017), Tinospora cordifolia (Willd.) Miers (Sharma et al 2019), Celastrus paniculatus Willd. (Mali et al 2020).

Subsequently, in this context, we have utilized an edible fern, Diplazium esculentum (Retz.) Sw (DE), belonging to the family Athyriaceae for the fabrication of Cu NPs. DE comprises of various phytochemicals such as saponins, triterpenoids, high quality of steroids, lipids, alkaloids, lignin, phenolic compounds, amino acids and flavonoids (Seal 2012). It is envisaged that for the formation of the NS materials, polyphenolic compounds particularly flavonoids are involved in reduction while the proteins acted as stabilizing agents (Dwivedi and Gopal 2010).

Additionally, upon the upsurge of urbanization, industrialization and modernization, color has become an important aspect of our life and undoubtedly a lot of research have been undergoing in the production of color. Globally, more than 10,000 dyes are available commercially and 7 lakh tones of dyes are produced annually and around 15% of these dyes are released into water polluting the environment requiring proper treatment methodology (Sinha and Ahmaruzzaman 2016).

Many of these dyes are harmful to human and aqueous systems due to their carcinogenic and venomous nature (Safavi and Momeni 2012; Prado et al 2008 and Nilsson et al 1993). Therefore, removals of these dyes are essential for environmental sustainability. In the present work, we have selected two different categories of harmful dyes; MB, a basic aniline dye (responsible for urinal and haematological problems, vomiting, nausea, cardiovascular diseases); and MV6B, triphenyl methane dye, (mutagenic, carcinogenic and mitotic poison) (Wang et al 2010).

Their harmful nature demands complete removal and photo catalytic degradation using nano-based catalyst can provide effective approach in this regard.

Additionally, the problem of microbial infections is serious in many parts of the world and most the microbes have become tolerant to the antibiotics (Hoffmann et al 2011).Thus, at present, need justifies a new alternative and safe antimicrobial agents against human microbial pathogens.

Moreover, antioxidants can prevent human beings from various perilous diseases by shielding human body from free radicals and active oxygen species (Sinha et al 2017). Subsequently, for strengthening human health we should make use of antioxidants.

Cu NPs with their multifunctional characteristics can found applications in these areas for disinfections as well as antioxidant.

Moreover, cancer especially human liver and ovarian cancer has also become epidemic. So, scientific fraternities are always in search of cheap and trustful alternatives which have very negligible or no side effects and have high potentials in curing these dreadful diseases (Hare et al 2017).In this aspect, the Cu NPs were effective and was evaluated against human liver cancer Hep-G2 and human ovarian cancer SK-OV-3 cell lines.

Thus, the present research work reports a green, effortless and environmentally amicable process for the quick fabrication of Cu NPs employing the DE leaves extract and their usability as plasmonic photocatalyst for the elimination of noxious compounds (MV6B and MB) from aqueous phase. The antioxidant capabilities, antibacterial activities and anti cancerous abilities of the fabricated NPs were also portrayed in this article. Significant broad-spectrum antibiotic action was obtained against human pathogenic microbial strains compared to standard reference drugs.

Materials: Copper Sulphate (CuSO₄.5H₂O), Methylene Blue (MB) and Methyl Violet 6B (MV6B) of Analytic Reagent Grade were acquired from Sigma Aldrich and employed as obtained. All the experimentation was conducted using double distilled water.DE ferns were acquired from the local bazaar and was cleaned meticulously with double distilled water and then dried in an oven at 60⁰C till uniform mass. Employing a stainless steel grinder, then the dried samples were grounded and sieved.

Further for antimicrobial screening the following bacterial strains; Escherichia coli (MTCC 1195), Bacillus subtilis (MTCC 1427), Staphylococcus aureus (MTCC 1430), Pseudomonas aeruginosa (MTCC 1688), and Streptococcus pneumoniae (MTCC 2672) along with two fungal strains; Aspergillus niger (Lab isolates) and Candida albicans (MTCC 4748) were incorporated. The Microbial Type Culture Collection (MTCC) numbers were obtained from Institute of Microbial Technology (IMTECH), Haryana and Chandigarh, Punjab. While the Fungal strains were obtained from the Down Town Hospital, Guwahati. The stains were sub-cultured, based on requirement, by MHB (Muller Hinton Broth) and maintained at 4^oC.

In vitro cytotoxicity of Cu NPs was determined employing sulforhodamine-B (SRB) assay, on human liver cancer (Hep-G2) and human ovarian cancer cell line (SK-OV-3). In a panel of two in vitro anticancer activities were performed using standard protocols with slight modifications at Tata Memorial’s Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai, India (Vichai and Kirtikara 2006; Skehan et al 1990).

Preparation of aqueous extract of DE fern: DE fern were rinsed and cleaned meticulously with double distilled water to remove dust. It was then dried in an oven at 60⁰C till uniform mass. Employing a stainless steel grinder, then the dried samples were grounded and sieved.

In a 500 ml Erlenmeyer flask, 450 ml of double distilled water was poured and to that 10 g of finely grinded DE fern powder was added with continuous heating and stirring at 70⁰C for 20 mins. This was succeeded by centrifugation with 4000 rpm for 20 mins followed by filtration using Whatman filter paper with size 11 mm and by vacuum filtration using cellulose nitrate membrane. The extract was stored at 4⁰C and was utilized within one week.

Phytochemical Analysis of the extract of DE fern: To identify the phytochemicals present in the fern extract of the DE the standard protocols with slight modifications were carried out (Chandraker et al 2020) and the results are summarized in Table 1.

Table 1

Phytochemicals present in the extract of DE fern
S.No.	Phytochemicals	Test	Result
1.	Alkaloids	Mayer’s test, Wagner test, Dragendroff test	Positive
2.	Flavanoids	Ferric chloride test and Lead acetate test	Positive
3.	Tannins	Ferric chloride test	Positive
4.	Diterpenes	Copper acetate test	Negative
5.	Triterpenes	Salkowski test, Liberman test	Negative
6.	Steroids	Salkowski test	Positive
7.	Saponins	Foam test	Positive
8.	Phenol	Ferric chloride test	Positive

It was found that from the different tests that alkaloids, steriods, saponins, alkaloids, lipids, alkaloids, phenolic compounds and flavonoids are present in the extract which was found to be in accordance with the literature (Seal 2012).

Synthesis of Cu NPs: In a typical synthesis, an aliquot of 50 ml of 10% DE extract was added to 0.5 g of CuSO₄·5H₂O dissolved in 10 ml of distilled water maintained at pH = 9 by 0.1M NaOH solution. The mixture was then refluxed at 100°C with constant stirring and heating at different reaction times (1 h, 2 h and 3 h).The transformation of green to dark brownish color marked the formation of Cu NPs. The Cu NPs were separated by centrifugations and were washed meticulously with ethanol and distill water until the filtrate was clear. The precipitate obtained was then dried in an oven at 60⁰C for 1 day to obtain the powder Cu NPs.

Characterization of the nanoparticles: The UV–Vis absorption spectra of the synthesized nanoparticles were recorded on Cary 100 Bio spectrophotometer (λ max in nm) equipped with 1-cm quartz cell. The JEOL-JEM 2100 transmission electron microscope operated at an accelerating voltage of 200 kV was used to record the SAED pattern and the TEM, HR-TEM images. The NPs samples for TEM were made ready by putting the solution drops over carbon coated Cu grids and permitting the solvent to vaporize at room temperature. A homogenized mixture of dried sample with dehydrated KBr was made mechanically and rendered it into thin and transparent pellets of KBr by pressing. The FTIR spectra of the same were recorded with the help of Bruker Hyperion 3000 FTIR spectrometer. The EDX analyzer associated with a SEM (JSM-7600F) at an accelerating voltage of 200 kV was used to carry out the elemental analysis of NPs. The XRD pattern was witnessed by means of a Phillips X’Pert Pro Diffractometer with Cu Kα radiation of wavelength 1.5418 Å.

Evaluation of photocatalytic activity: The aqueous solutions of both the MB and MV6B were used to measure the photocatalytic activity of the synthesised Cu NPs. For this, in two different dye solutions (10^− 4 M, 200 ml), fabricated Cu NPs (10 mg) was dispersed separately. To attain adsorption-desorption equilibrium of dye on the surface of the NPs, the suspended solution was allowed to stand for 1 h in dark before solar irradiation. Thereafter, these dyes were kept under the Sun between 10:00–15:00 h at Pune city (Outdoor temperature: 32-36⁰C). 4 ml of aliquots were drawn at regular intervals and centrifuged immediately. UV-Vis Spectroscopy was used to monitor the progress of the reaction simultaneously.

Antioxidant Activity (DPPH Radical Scavenging Assay): The activity of Cu NPs to scavenge free radicals was evaluated by the method of Brand-Williams et al (Brand et al 1995). Maintaining incubation period at 30 min, the reaction was carried out in a dark area at room temperature and absorbance was ascertained at 517 nm. 3 sets of experiments were carried out on the basis of following expression:

Antimicrobial Study: Agar well dilution method with some changes was adopted to study the antimicrobial activity (Sharma et al 2019). Freshly prepared molten Potato Dextrose Agar (PDA) and Muller Hinton Agar (MHA) medium was respectively used for fungal and bacterial culture.

By spread plate method, the chosen microorganisms (1.2×10⁷ cfu/mL) were inoculated in the respective agar glass plates. After half an hour, using cork borer, 6 mm wells were punched in the seeded agar plates. For positive control of bacteria, Cephalexin, Metronidazole, Ciprofloxacin and Azithromycin were taken as reference antibacterial agent, while antibiotics; Ketoconazole and Nystatain were used for fungi. For negative control, DMSO was taken.

Cu NPs (50 µl) of different concentrations were poured into three different wells and then incubated respectively for 24 h, 37⁰C and 48 h, 25⁰C, for bacterial and fungal culture respectively.

Minimum Inhibitory Concentration (MIC): The minimum concentration of the material at which there is no growth of the microbes is the MIC, for bacteria it is MBC and for fungi it’s called MFC (Kaushik and Goyal 2008 and Qi et al 2004).

Micro dilution broth method was adopted to determine MIC values. 50 µl nutrient broths were poured into each well of the plates. To the first well of the plate, 50 µl of Cu NPs solution was added and then two fold serial dilution of every sample was done. 50 µl of standard inoculants (1.2×10⁷ cfu/mL) was added to each well except for negative control. The plates were then incubated (bacteria; 37⁰C, 24 h and fungi; 25⁰C, 48 h). When the incubation tenure was over, MBC and MFC values were recorded.

Anticancer Study: RPMI 1640 medium constituting 2mM L-glutamine and 10% fetal bovine serum were used for the cell lines growth. The cells were inoculated in 96 well micro titer plates. On completion of cell inoculation, the plates were incubated for 1 day at 37⁰C, 95% air, 5% CO₂ and 100% relative humidity. Adriamycin (ADR) was used as a positive control. ADR and Cu NPs were dissolved in DMSO (100 mg/ml) and diluted (1 mg/ml) employing water and were kept frozen before use. At the time of testing, the frozen samples were diluted to four concentrations (100, 200, 400 and 800 µg/ml). In microtiter plates comprising 90 µl medium, 10 µl of aliquots were poured which resulted in 10, 20, 40, 80 µg/ml concentration of Cu NPs and ADR.

Micrographs were taken in 20x magnification for control (untreated test samples), positive control of 80µg/ml concentration of ADR and for Cu NPs in 80µg/ml concentration.

Employing Sulforhodamine-B (SRB) assay the cell viability was measured and the absorbance was recorded on a plate reader at 540 nm while 690 nm was used as reference wavelength.

The Percentage growth inhibition is calculated as [(Ti - Tz) / Tz] × 100 (for concentrations for which Ti > / = Tz (Ti -Tz) positive or zero)

= [(T i-Tz) / (C - Tz)] × 100 (for concentrations for which Ti < Tz (Ti-Tz) negative)

Where, Tz = zero time, c = control and Ti = test growth in presence of drug at four concentration levels;

While GI₅₀ (50% Growth inhibition) is calculated as [(Ti-Tz) / (C-Tz)] × 100

GI₅₀ is the concentration resulting in a 50% reduction in the net protein increase in control cells during the test sample incubation.

The drug concentration resulting in TGI was measured from Ti = Tz.

The experimental data were obtained using linear regression methods of plots of the cell viability against the µg/mL concentration of testing sample. The values were measured for each of these parameters if the level of activity was reached; in any case, if the effect was not reached or was exceeded, the values for that parameter were expressed as greater (as more prominent) or less than the maximum or minimum concentration tested (Thosar et al 2013).

Statistical analysis: Employing SPSS software version 22.0, the statistical analysis was carried out.

Optical spectroscopy results: The optical behaviour of the synthesized Cu NPs at different reaction times (1, 2 and 3h) employing DE fern is depicted in Fig. 1. The spectra revealed absorption onset at ~ 588 nm for 1h and ~ 590 nm for 3h indicating a red shift. At higher reaction time, the peak became broad and more intense reflecting increased polydispersity and percentage population of the NPs (Patil et al 2010). The absorption thus corresponded to the spherical SPR of Cu NPs (Patil et al 2010) and illustrated that reaction time is an essential parameter for the formation of NPs.

Morphological and crystalline study: TEM spectroscopy was used for the morphological and size distribution of the Cu NPs. The TEM micrographs disclosed the formation of sphere shaped NPs with average diameter of (24 ± 2) nm and (30 ± 2) nm at 1 h and 3 h, respectively (Fig. 2a and d) corresponded to the TEM micrographs of the fabricated Cu NPs at 1h and 3h). Moreover, the particles were well detached from one another reflecting absence of any aggregation and strong capping. Thus, the particle diameter got enhanced with enhancement of reaction time. High resolution TEM images (Fig. 2b and e; with inset showing the IFFT and profile of IFFT) illustrated clean lattice fringes with fringe distancing of 0.26 nm and 0.22 nm respectively, that matched well with the (111) fcc plane of Cu (JCPDS- 71-4610).

The polycrystalline nature of the NPs was disclosed from the concentric diffraction ring arrangement obtained from the SAED results (Fig. 2c and f). The inter planer distances measured from the SAED pattern corresponded to fcc lattice of Cu NPs (JCPDS-71-4610). Thus, the TEM images demonstrated that the particle size could be varied by changing the reaction time.

Phase Study of the crystal: The XRD arrangement undoubtedly disclosed diffraction peaks at two theta values 43.4⁰, 50.5⁰ and 74.2⁰ which can be correlated with (111), (200) and (220) fcc lattice planes of Cu NPs (JCPDS-71-4610) (Fig. 2(g)) (Skehan et al 1990). Additionally, the XRD patterns were very neat with no sign of any contaminations from cupric / cuprous oxides. Employing Scherrrer’s formula the mean crystallite size was detected to be ~ 28 nm (Kumar et al 2013).

Thus, the mean crystallite size acquired from the XRD arrangement was in accordance with the the NPs size attained using TEM study (Kumar et al 2013).The XRD results thus indicated the face centered cubic nature of the particles and were in agreement with the SAED results.

Elemental Composition Analysis: To analyze the elemental composition of the fabricated Cu NPs, the EDAX study was carried out. Figure 2 (h) showed the EDAX spectrum of biosynthesized Cu NPs and depicted prominent signals of Cu at 1, 8 and 9 keV. The presence of intense peak of Cu in EDAX spectra undoubtedly revealed the presence of Cu only. Some weak signals of potassium (K), and middle range signals of sulphur (S) and carbon (C) were also detected. The S, K and C peaks originated because of biomolecules likewise enzymes, proteins and amino acids present on the surface of the biosynthesized Cu NPs (Srivastava and Mukhopadhyay 2014).

STEM-HAADF imaging and X-Ray Elemental Mapping: The STEM-HAADF illustration of the Cu NPs is depicted in Fig. 2(i). To identify further elemental dispersion of the Cu NPs, the X-Ray elemental mapping was carried out. Figure 2(j), 2 (k) and 2 (l) describes the X-ray elemental maps of Cu-K, C-K and S-K respectively. Figure 2(m) describes the superimposed X-ray elemental mapping picture (green spots describes-Cu, red spots portrays–C and blue spots depicts S). It is evident from these figures that Cu NPs is formed and it is protected by biomolecules that contain groups such as C and S which was found to be in accordance with the EDX spectra ( Sinha and Ahmaruzzaman 2015 a).

Molecular Components Identification: To access the bio molecules liable for the formation of the Cu NPs, the FTIR spectra of the DE fern extract and the synthesized Cu NPs were recorded (Fig. 2(n)). In the spectra of DE fern extract, the peak at 3452 cm^− 1 was attributed to H- bonded ν_s(O-H) while ν_s(C = O) of amide I showed up at 1601 cm^− 1 and ν_b(N-H) of amide II was observed at 1408 cm^− 1.Furthermore, the peaks at 1020 and 597 cm^− 1 was ascribed to ν_b(C-O-H) of carboxylic acid or ν_b(O-H) and ν_s(C-N)/ ν_s (C-O) /ν_b(N-H₂)/ ν_b (N − H) /ν_b(O − H) respectively (Zhou et al 2018).

Additionally, in the spectra for the synthesized Cu NPs, the wavenumbers for ν_s(O-H) and ν_b(N-H) of amide II became strong and intense and are slightly red shifted to 3343 and 1396 cm^− 1, respectively while the wavenumbers for ν_s(C = O) of amide I, ν_b(C-O-H) of carboxylic acid or ν_b(O-H) and ν_s(C-N)/ ν_s (C-O) /ν_b(N-H₂)/ ν_b (N − H) /ν_b(O − H) undergone hypsochromic shifts to 1613, 1105 and 609 cm^− 1 respectively ( The results are summarized in Table 2).

Table 2

FTIR Spectra of DE fern extract and Cu NPs.
FTIR bands (cm^− 1)
Sample	νO-H	νC = O of amide I	νN-H bending of amide II	νC − O−H bending of carboxylic acid/in plane νO − H bending	νC-N or C-O stretching vibration /νNH2/N − H wagging/out of plane νO − H bending
DE Fern extract	3452	1601	1408	1020	597
Cu NPs	3343	1613	1396	1105	609

The above mentioned data thus indicated the presence of O-H, COOH, C-N, amide C = O, and N-H moieties in the extract. These functional moieties can be allocated to the existence of proteins and polyphenolic compounds, likewise flavonoids. The peak transformations in the IR spectra of the fabricated NPs were associated to the NH₂ and OH moieties demonstrating that these functional moieties were liable for their formation and stabilization.

Probable mechanism for the formation of Cu NPs: The literature revealed that the polyphenolic compounds specially the flavonoids and proteins play a significant role in the formation of the NPs. The flavonoids can chelate with the metal ions thereby donating electrons and hydrogen atoms and ultimately reducing the metal ions while the proteins acts as a stabilizing agents due to the presence of amine and carboxylate moieties (Hamid et al 2013 and Sinha et al 2014). The presence and involvement of these moieties in the fabrication and stabilization of NPs were in accordance with the FTIR results (Fig. 2(n)). Hence, the synthesis and stabilization of Cu NPs can be visualized by the subsequent steps (Scheme 1).

(i) Complex formation of Cu metal ions and flavonoids,

(ii) Concurrent reduction of Cu metal and

(iii) Capping with proteins/oxidized polyphenols.

Therefore, the Cu NPs were formed due to the presence of proteins and polyphenols in the DE fern extract eliminating the need for commonly used reducing agents, such as hydride and sodium borohydride. This process can be aptly scaled up for large-scale production of NPs.

Evaluation of the photocatalytic Activity of the synthesized NPs: To validate the photocatalytic performance of the synthesized Cu NPs, at first a control experiment was carried out. In presence of Cu NPs but under dark conditions, both the dye solutions depicted imperceptible degradation (Supplementary information) (Fig.S1 (a and c)).

However, in presence of solar irradiation but in absence of any catalyst, no degradation was detected (Fig. S1 (b and d)). Consequently, the presence of both catalyst as well as sunlight is essential for the effective remediation of these noxious dyes.

Hence, by mixing Cu NPs (10 mg) to each aqueous dye solutions (200 ml of 10^− 4 M) under solar irradiation, the photodegradation process was monitored. The degradation procedure was administered by noticing the transformations in the UV spectrum of the reaction mixture. It was witnessed that the absorption peak analogous to each dye depreciated slowly and reached their minimum as the exposure time got increased. The absorption peaks corresponding to MV6B and MB at 580 and 664 nm displayed fast degradation and diminished completely after 150 and 135 min respectively (Fig. 3 (a and b)).

Figure 3 (c) depicted the degradation potential of the synthesized NPs for MV6B and MB, which reached to 93, and 96 %, respectively. In the current study, the degradation rates were found to be different for different dyes and it purely depended on the molecular structure of the targeted dyes. The degradation procedure was found to follow pseudo first order reaction and their kinetics can be expressed as follows (Kavitha et al 2014).

ln (C₀/C_t) = kt -(i)

where C₀ and C_t are dye concentrations at 0 and t time, respectively; k = rate constant (pseudo-1st order) and t = time (min).

Figure 3 (d) indicated the plot of ln (C₀/C_t) vs t (irradiation time) for the dyes. The plot depicted a linear relationship and therefore, slope of the line corresponded to the value of k for dye degradation. The value of k was found to be 1.73×10^− 2 and 2.2×10^− 2 min^− 1 for MV6B and MB, respectively.

Hence, it can be summarized that Cu NPs are very efficient for the abatement of toxic MV6B and MB dyes. Thus, we infer that the catalysis reaction rate increased in our present case as the electron transfer was rapid in presence of the Cu NPs (Sinha et al 2014).

Probable mechanism for the photocatalytic activity of the NPs: Photo and catalysis are the two parts and parcels of the photocatalytic mechanism. Photo is related with interaction of light material that includes absorption of photon, generation of charges, dynamics and surface trapping. While catalysis is associated with the interaction between organic pollutant, water and oxygen ie; it is correlated to radical formation and surface reactivity. Hence, particularly on crystalline nature the photocatalytic activities depends (Sinha et al 2014). Therefore, the photodegradation mechanism can be summed up as follows:

At the onset, Cu NPs absorbs the sunlight and gets photo excited which then undergoes plasmonic decomposition via three mechanisms (Sinha and Ahmaruzzaman 2015 b)

1. Elastic radiative re-emission of photons.

2. Next, a single e⁻/h⁺ pair are formed via non radiative Landau damping. Then the primary excited electron furnishes many other electrons by columbic inelastic scattering.

3. Finally, the induction of a direct electron injection into the adsorbate takes place owing to the interaction between the excited surface plasmons and the adsorbate.

Secondly, the plasmonic decay results in the formation of holes and electrons. Electrons react with oxygen to produce anionic super oxide radical (O₂⁻.) while hole react with water molecules to furnish hydroxyl radical (OH.).

Subsequently, hydro peroxyl radical (HO₂.) are produced via the protonation of superoxide ion (O₂⁻.). The protonated superoxide ion then changes to hydrogen peroxide which finally dissociates to hydroxyl radicals (OH.).

Finally, on the surface of the photocatalyst, both oxidation and reduction takes place.

Hence, the total degradation procedure can be represented by the scheme 2, and the associated reactions are illustrated in Equations ((1)-(9)).

Cu+hν → h⁺ (Cu) +e⁻ (Cu) (1)

H₂O (ads)+h⁺ → OH. + H⁺ (ads) (2)

O₂+e⁻ → O₂⁻. (ads) (3)

O₂⁻. (ads)+H⁺ ⇄ HOO.(ads) (4)

2HOO. (ads) → H₂O₂ (ads)+O₂ (5)

H₂O₂ (ads) → 2OH. (ads) (6)

Dye+OH. → CO₂+H₂O(dye intermediates) (7)

Dye+h⁺ → Oxidation products (8)

Dye+e⁻ → Reduction products (9)

Evaluation of Photostability of the Cu NPs: To explain the photostability of the Cu NPs as catalyst, recovering reactions were accomplished for the degradation of MV6B as well as MB.

In each assessment, Cu NPs were isolated from the solution, rinsed with ethanol and desiccated in vacuum (Sinha and Ahmaruzzaman 2015 c).The catalyst was found to display outstanding stability even after 3 rounds and the outcomes are portrayed in Supplementary information (S2).

Identification of the intermediate degraded products and reaction pathways of dye degradation: Employing liquid chromatography-mass spectroscopy (LC-MS) technique and by correlating the degraded intermediates with commercial standards, the fragmented ions in the mass spectra were interpreted and identified. All the recognized intermediate components of the dye degradations are depicted in supplementary information (S3).

DPPH radical scavenging assay: The assay depicts that Cu NPs has fairly decent radical scavenging activity with IC₅₀ value 2.11 mM. To analyze the results of the trial sample, a standard antioxidant; Quercetin having an IC₅₀ value of 0.025 mM was considered (Fig. 4). The radical scavenging action of Cu NPs was displayed in the Fig. 4 (a) and Fig. 4 (b).

Antimicrobial activity

A. Test for susceptibility of the standard antibiotic: On five bacteria and two fungi, an antibiotic vulnerability examination was accomplished. For bacterial screening, four antibiotics; Metronidazole, Cephalexin, Azithromycin and Ciprofloxacin was used while Ketoconazole and Nystatin antibiotics were employed for fungal studies.(Fig. 4 (c) and Fig. 4 (d)).

B. Susceptibility of the microbes to Cu NPs: The current research disclosed that Cu NPs depicted a broad-spectrum action with steady microicidal competency against all the seven microbes, presented in Fig. 4 (e) and Fig. 4 (f) where 100% of the microbes were vulnarable to the sample. Among them, the sample revealed highest action against P. aeruginosa (MTCC 1688) with all three concentrations (i.e. 0.062, 0.125 and 250 M) observed zone of inhibition (ZOI) are; 0, 8.66 ± 0.33 and 34.33 ± 0.66 mm respectively; however, lowest activity showed against S. aureus (MTCC 1430) with 0, 0 and 26.33 ± 0.88 mm ZOI. In case of antifungal activity of the sample, the lab isolated fungus A. niger showed higher efficiency with ZOI 0, 8.66 ± 0.33 and 32.88 ± 0.88 mm than that of the fungus, C. albicans (MTCC 4748) with ZOI 0, 0 and 26.66 ± 0.33 mm respectively. Figure 3 and Fig. 4 showed that at 0.125 M concentration of Cu NPs was more or less microstatic (bacteriostatic or fungistatic) to the microbes that showed activity against 71.42 % (5 out of 7) of microbes whereas, at 0.062 M showed 0 % activity to all the selected strains.

MIC, MBC and MFC of Cu NPs: As illustrated in Table 3. MIC value 0.075 ± 0.005 M and MBC value 0.105 ± 0.005 M were the lowest for the bacterium, B. subtilis (MTCC 1427), which revealed that Cu NPs has the highest activity against this bacterium at a low concentration than that of the other tested organisms. The highest MIC and MFC values were noticed for the fungus, C. albicans (MTCC 4748) with 0.180 ± 0.01 and 0.195 ± 0.005 M respectively. However, highest MBC value, 0.175 ± 0.005 M for the S. aureus (MTCC 1430) was observed. It is notable that, the MBC values for the two bacteria P. aeruginosa (MTCC 1688) and S. pneumoniae (MTCC 2672) and MFC value for the lab isolated fungus A. niger were same i.e.0.115 ± 0.005 M.

Table 3

MIC (Minimum Inhibitory Concentration), MBC (Minimum Bactericidal Concentrations) and MFC (Minimum Fungicidal Concentrations) of Cu NPs
	Microbes	MIC (M)	MBC/MFC(M)
Bacteria	Bacillus subtilis	0.075 ± 0.005	0.105 ± 0.005
	Escherichia coli	0.095 ± 0.005	0.125 ± 0.005
	Pseudomonas aeruginosa	0.090 ± 0.01	0.115 ± 0.005
	Staphylococcus aureus susp.aureus	0.155 ± 0.005	0.175 ± 0.005
	Streptococcus pneumoniae	0.085 ± 0.005	0.115 ± 0.005
Fungi	Aspergillus niger	0.095 ± 0.005	0.115 ± 0.005
	Candida albicans	0.180 ± 0.01	0.195 ± 0.005
(Key: The values are the mean of two independent replications ± SE)

The results unveiled that Cu NPs exhibited an excellent broad-spectrum antimicrobial activity against all the selected microbial strains when individual values were compared with standard antibiotics (Fig. 5(a), (b), (c) and (d)).

Plausible mechanism for antimicrobial activity: Microbial cell walls and membranes are negatively charged while the surfaces of the Cu NPs are either positively or less negatively charged which led to an electrostatic attraction between them (Praharaj et al 2004). As a result of this interaction, the NPs gets adhere to the microbes resulting in morphological alterations in the membrane structure (Roy et al 2019). It causes membrane depolarization and finally leads to cell apoptosis (Praharaj et al 2004)

Consequently, the cellular components including enzymes, proteins, metabolites, DNA, ions, and energy reservoirs seeps out in the environment. Thus, destruction of the cell wall by the NP coalescence is considered to be the principal mechanism for the antimicrobial activity.

Anticancer activity ( In vitro Sulforhodamine-B (SRB) assay against liver cancer cell line (Hep-G2) and ovarian cancer cell line (SK-OV-3): The experimentations were carried out in triplets and the average value attained were plotted as the growth curve of µg/ml concentration of Cu NPs and ADR against % control growth as shown in Fig. 6 (a) and (b). The TGI and GI₅₀ score for anticancer action against Hep-G2 and SK-OV-3 are respectively shown in Table 4. Generally, a pure compound, with GI50 value of ≤ 10µg/mL is supposed to exhibit activity (Bhosale et al 2015).

Table 4

The calculated TGI and GI₅₀ score from the graph (Fig. 6 (a) & (b))
	Test sample concentrations (µg/mL)
Hep-G2	TGI	GI₅₀
CuAV	> 80	> 80
ADR	< 10	< 10
SK-OV-3
CuAV	> 80	> 80
ADR	< 10	< 10
Yellow highlighted test values under GI₅₀ column indicate activity.

The anticancer activity farther can be established from micrographs in Fig. 6 (c), (d), (e), (f), (g) and (h). The figures portrayed that the Cu NPs with 80 µg/mL concentration, exhibited substantial anticancerous action against human ovarian cancer cell line, SK-OV-3 as compared to human liver cancer cell line, Hep-G2.This might be due to the readily metabolisable Cu metals which are frequently used as in cell proliferation; cofactors for enzymes and oxygen-carrying proteins (Turski and Thiele 2009).

In this study, we showed an environmental friendly, green, economical, additive free, non-toxic synthetic protocol for the manufacture of Cu NPs utilizing the aqueous extracts of DE fern. Flavones and proteins present in the DE fern extract are thought to be liable for the formation and stabilization of Cu NPs. The present work thus reflected an effortless shape and size controlled fabrication of Cu NPs.

These manufactured NPs were employed for the remediation of two industrial emerging pollutants (MV6B and MB) from aqueous phase and was also exploited as an antibacterial, antifungal, antioxidant and anticancerous agent. The rates for the remediation of the pollutants progressed via pseudo-first order kinetics with abatement proficiency greater than 92 % for both MV6B and MB. The degradation intermediates were accessed employing LC-MS technique and it was implicated that for MV6B and MB, respectively, initially the dye undergone demethylation of the substituent of the amine groups and elimination of methylene moieties respectively resulting in the generation of the mineralization products. The spent NPs were successfully renewed and were examined by TEM images. The regenerated NPs showed dye removal efficacy of 92.5, 91.5, 89 % for MV6B and 95.8, 95.7 and 93% for MB respectively for 1st, 2nd and 3rd rounds of successive cycles.

Further, the Cu NPs portrayed excellent antibacterial action against gram negative, Escherichia coli, Pseudomonas aeruginosa; as well as gram positive bacteria, Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae and potential action against two fungal strains; Candida albicans and Aspergillus niger. Additionally, the assay depicted that Cu NPs has fairly decent radical scavenging activity with IC₅₀ value 2.11 mM.

Moreover, the Cu NPs were found to exhibit comparative anticancerous activity against two human cancer line cells.

Thus, the present research work enlightened the scientific fraternities about the innovative exploitation of DE fern extract for the development of Cu NPs and their efficiencies for the elimination of noxious compounds and their applicability as antibacterial, antifungal, antioxidant and anticancerous agent.

Therefore, we anticipate that the Cu NPs would be a potent candidate for wastewater treatment, microbial infection, water disinfection, drug delivery, wound dressing as well as ideal therapeutic agent.

Consequently, the fabrication of Cu NPs by this process and its potential in industrial and biomedical fields are well justified.

ACKNOWLEDGEMENTS

Tanur Sinha is indebted to CSIR-NCL Pune for furnishing the lab as well as characterization facilities. TS is also thankful to DST-NPDF (Project No- PDF/2016/002925) as well as The Royal Society (Project No-NIF\R1\192613) for providing the financial assistance. Partha Pradip Adhikari is grateful to the Institutional Biotech Hub, D. K. College, Mirza, Guwahati, for furnishing lab facility and Tata Memorial’s Advanced Center for Treatment, Research and Education in Cancer (ACTREC), Mumbai, India, for cell line study.

Author contribution: Study concept, design, manuscript writing, performing experiments, drafting of manuscript- Tanur Sinha, Biological portion experimentation and drafting -Partha Pradip Adhikari, Critical discussions, corrections and Finalization- Vinay M Bhandari.

Funding: This work was financially supported by grant: (DST-NPDF (Project No- PDF/2016/002925) as well as The Royal Society (Project No-NIF\R1\192613) to Tanur Sinha.

Data Availability: “Not applicable”.

Ethical approval : This study was originally approved by CSIR-NCL Pune at India.

Consent to participate :“Not applicable” for that specific section.

Consent to publish: “Not applicable”.

Conflict of interest: The authors declare no competing interests.

Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

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GraphicalAbstract.png
SupportingInformationForCuNPs.docx
Scheme1.png
Scheme 1 Mechanism for the formation of Cu NPs.
Scheme2.png
Scheme 2. Schematic representation of the photodegradation process using Cu NPs under sunlight.

High Value Utilization of Fern Extracts For Fabrication of Copper Nanoparticles: A Potent And Affordable Candidate For Water Treatment, Water Disinfection, Antioxidant Activity And Theranostic Agent.

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Abstract

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Introduction

Experimental

Results And Discussion

Conclusion

Declarations

References

Supplementary Files

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