Green synthesis of copper nanoparticles using leaf extract of Ageratum houstonianum Mill. and study of their photocatalytic and antibacterial activities

The novel copper nanoparticles (CuNPs) were synthesized using aqueous leaf extract of Ageratum houstonianum Mill. (AHLE). The green synthesized AH-CuNPs have a useful dye degradation property in the existence of daylight. The photocatalytic activity of AH-CuNPs was evaluated against an azo dye congo red (CR), whereas, same NPs displayed no effect on other dyes. The CR was completely degraded within 2 h, and the reaction rate was followed by pseudo-first-order kinetics, and the rate constant was recorded 3.1 × 10−4 s−1, (R2 = 0.9359). Antibacterial activity of green synthesized AH-CuNPs was studied against gram-negative bacterium Escherichia coli (MTCC no. 40), and a significant growth inhibition was recorded with 12.43 ± 0.233 mm zone of inhibition. The AH-CuNPs were characterized through UV-visible spectroscopy, XRD, SEM, FT-IR, TEM, and zeta particle size analyzer. Ageratum houstonianum mediated green synthesized copper nanoparticles (AH-CuNPs) were cubic, hexagonal, and rectangular in shape, with average size of ∼80 nm. The optical band gap was 4.5 eV, which was investigated using UV-visible spectroscopy, and the band gap value revealed that AH-CuNPs were semiconductor materials.

phytochemical analyses were purchased from CDH (Mumbai, India). All the chemicals procured were AR grade and used as received. Double distilled water (DDW) was used for the experiments. All the apparatus were washed properly with DDW and dried in the hot air oven. Ageratum houstonianum leaf extract (AHLE) was used as a reducing agent for the formation of CuNPs.

Preparation of leaf extract
The fresh leaves of A. houstonianum (figure 1) were washed repeatedly with DDW to expel the residue impurities present on the leaf surface. About 20 g of the leaves were weighed through the digital balance and cut in equal pieces. Chopped leaves were taken into the 500 ml beaker containing 200 ml DDW, boiled at 60°C for 20 min, and filtered twice through Whatman No. 42. The filtrate was stored at 4°C for further use [45].

Phytochemical analysis
The major phytochemicals present in the leaves of A. houstonianum were identified following standard protocol with slight modification [46,47]. Test for Alkaloids: • 1 ml AHLE+6-8 drops Mayer's reagentgreen precipitate.

Test for Triterpenes:
• Equal volume of Chloroform and AHLE shaken with few drops of conc. H 2 SO 4lower layer turns yellow (Salkowski test). • Equal volume of Chloroform and AHLE+Few drops CH 3 COOH+1 ml conc. H 2 SO 4deep red at the junction of two layers (Liberman test).

Test for Diterpenes:
• Equal volume of Copper acetate solution and AHLE gives green color on shaking.
Test for Saponins: • 5 ml AHLE shaken vigorously to obtain a stable froth -5-6 drops olive oil was addedformation of an emulsion.
Test for Cardiac glycoside: • 1 ml AHLE+1 ml glacial acetic acidcooled -2-3 drops ferric chloride+2 ml of conc. H 2 SO 4 along the wall of the test tubebluish-brown ring at the junction of two layers (Keller Killani test).

Green synthesis of AH-CuNPs
Eighty milliliter CuCl 2 (3 mM) was stirred in Erlenmeyer flask for 2 h, and 20 ml of AHLE was added therein. The flask was further stirred for another 24 h at room temperature until the reaction mixture turned in greenishbrown solution. The resultant solution was centrifuged at 10 000-11 000 rpm for 10 min and the pellet was separated as AH-CuNPs.

Characterization
UV-vis analysis was performed by using a Shimadzu UV-1800 spectrophotometer with 1 nm between 200 to 600 nm of resolution. The FTIR spectra were obtained in the range of 500-4500 cm −1 with Vertex 70 model, Bruker, Germany. The size and morphology of NPs were determined by using SEM (Model: EVO 18; Carl Zeiss, Germany) and TEM (Technai G20FEI). The hydrodynamic size of NPs was determined via dynamic light scattering using Zetasizer Nano ZS (Malvern Panalytical, UK). The examination of the samples was set up through covering the aqueous solution of AH-CuNPs on covered Cu lattices by moderate vanishing and afterward permitted to dry in vacuum at 25°C for medium-term. XRD studies were conducted using Bruker D8 advanced x-ray diffractometer using Cu Kα (I=1.54 A).

Photocatalytic activity
The photocatalytic activity of green synthesized AH-CuNPs was investigated against various dyes like MB, MO, Rh-B, and CR. The dye solutions were made by dissolving 1 mg powder of each dye in 100 ml DDW, and the absorption of dyes was measured through UV-visible spectrophotometer. Approximately, 10 mg of AH-CuNPs powder was added to 50 ml of each dye solution, and exposed to sunlight. A control set of experiment was also designed for the comparative study to determine the changes in color of the dye solutions in absence of AH-CuNPs. The absorption of the dye solutions was recorded at constant time intervals (every 20 min) [45]. The Langmuir-Hinshelwood kinetic model was used to determine the rate of photocatalytic reactions [48,49]. The rate of reaction was described as follows: The apparent pseudo-first order reaction rate constant, k is given as: time results in a straight line, the slope of which upon linear regression equals the apparent first-order rate constant k.

Antibacterial activity
The disc diffusion method was used to investigate the antibacterial activity of the AH-CuNPs. As a test specimen, a gram-negative bacterium E. coli (MTCC no. 40) was used. On the disc A, 50 μg ml −1 concentration of AH-CuNPs was soaked, whereas, on disk B 50 μl reference antibacterial agent ciprofloxacin was used as control. The plates were incubated at 37°C for 24 h and the zone of inhibition was measured [50].

Phytochemical analysis
The presence of alkaloids, flavonoids, tannins, triterpene, steroids, and saponins was found through the phytochemical analysis of AHEL (table 1). These compounds are supposed to act as stabilizing and capping agents for green synthesis of AH-CuNPs, and also responsible for the reduction of Cu + to Cu 0 . The plant is aromatic in nature and its essential oil was also reported to contain fifty compounds when analysed through GC-MS [51]. In a similar investigation, Zeeshan et al separated bioactive compounds from crude methanol extract of AH [43].

Green synthesis of AH-CuNPs
A. houstonianum leaves were examined to contain the phytochemicals like flavonoids, alkaloids, tannins, terpenes, steroid, and saponins, etc, which are responsible for reducing, capping and stabilization of synthesized AH-CuNPs (figure 2). The mixed solution was stirred for 24 h, and the change in colour of CuCl 2 solution from blue to gleaming greenish due to precipitation of ions confirmed the successful reduction of Cu particles into CuNPs. AH-CuNPs indicate greenish-darker shades, which is due to the surface plasmon vibrations. Color change of reacting solution from bluish brown to dark green using seed extracts of Punica granatum and leaf extract of Ocimum sanctum was also confirmed by Nazar et al and Heera et al respectively [52,53].

UV-Vis analysis
After the addition of 20 ml A. houstonianum leaf extract in 80 ml of 3 mM CuCl 2 (2:8 ratio) at room temperature, the solution was continuously stirred for 24 h. The CuCl 2 dissolves in water to give rise Cu 2+ and 2Cl − . The Cu 2+ further dissociates to give zero-valent ions (Cu 0 ) with reaction time by the activity of AHLE phytocompounds, which aggregates to form CuNPs from Cu nuclei [54]. The reaction mixture changed into gleaming greenish color due to the excitation of the surface plasmon resonance (SPR) phenomenon, which indicates the formation of CuNPs [31,32]. The SPR is the absorbance of visible electromagnetic radiation due to collective oscillation of surface electrons. The characteristic absorption maximum of phytosynthesized CuNPs was recorded with a peak at 326 nm as shown in figure 3(a). However, such peaks were neither assigned with AHLE nor CuCl 2 solution. Similar observation has been reported in previous studies showing the absorption maxima of CuNPs at 384, 350, 310 and 305 nm using the flower extract of Millettia pinnata [55], leaf extracts of Cassia arnotiana [54], Allium cepa, and Azadirachta indica [56], respectively. The complete reduction of Cu + ions to Cu 0 was found after 24 h of incubation. The hyperchromic shifts in SPR peaks of CuNPs were observed in UV-Vis spectra with increase in incubation time ( figure 3(a)).  3(b)). The unassigned peaks ( * ) appeared, are supposed to be associated with the capping agent stabilizing the nanoparticles. The crystalline nature of AH-CuNPs was  suggested by XRD and identical to simple cubic structure. The peak positions of present report are in agreement with literature standards [57]. Figure 4 showed comparative Fourier Transform Infra-red (FT-IR) spectroscopic analysis of AHLE and AH-CuNPs. It was carried out to study the functional groups of phytochemicals present on NPs as adsorbent. The main peaks of the IR spectra, their wave-number and elucidation of probable functional groups are shown in table 2. It is also apparent from the relative FTIR data that peaks of functional groups are almost similar in both the spectra. Hence, AHLE or their functional groups might be responsible for the reduction of Cu + to Cu 0 and further stabilization of AH-CuNPs.

SEM analysis
Morphology and shape of AH-CuNPs were determined by scanning electron microscopic measurements. Green synthesized AH-CuNPs were found in the maximum range of ∼200 nm, and their structures were observed in different shapes as shown in figures 5(a)-(d) at 0.5, 1, 2 and 5 μm scales, respectively. These figures interpret that AH-CuNPs are found to be highly scattered with a cubic, hexagonal, and rectangular shape in nature. Among them, few NPs are very much isolated from each other while the majority were in the agglomerated form.

TEM and DLS analysis
To determine the nanoparticle sizes, the AH-CuNPs were characterized by transmission electron microscopy under various magnifications. Figures 5(e)-(f) indicates that AH-CuNPs are agglomerated and the particles have no specific shape. The average particle size of AH-CuNPs was found to be ∼80 nm. The size of dispersed AH-CuNPs was also confirmed by DLS analysis ( figure 6) with average size in same range. In similar investigations, the extract of Aspergillus sps. synthesized larger CuNPs of 500 nm [58], whereas, a bacterial strain Pseudomonas stutzeri provided CuNPs (50-150 nm) from electroplating waste water [59]. Leaf extract of Artabotrys odoratissimus was also reported to synthesize CuNPs within 115-135 nm [60]. Figure 3(a) displays the UV visible spectrum of AH-CuNPs and band gap energy was calculated using UVspectrum following the equation of Tauc's relation [61].

Optical band gap
Where α represents the absorption coefficient, A is constant, E g shows the optical band gap energy. n is the exponent that depends on transition, and h symbolizes the Planck's constant. Figure 6(e) shows the optical band gap which was calculated using Tauc relation by plotting (αhυ) 2 versus hυ, and extrapolating the linear portion of the curve to (αhυ) 2 =0. Hence, the optical energy band gap of AH-CuNPs was 4.5 eV and it behaves as semiconductor materials and used as a photocatalyst.

Photocatalytic activity of AH-CuNPs
Since the band gap of AH-CuNPs indicates that these are semiconductor materials, it is therefore investigated the photocatalytic behaviour of AH-CuNPs in presence of different types of organic dyes (MO, MB, CR, and RB) and daylight. In this photocatalytic experiment, CR was more effectively degraded by AH-CuNPs as compared to other dyes ( figure 7(d)). The dye degradation reaction by the AH-CuNPs was performed at room temperature and observed through the UV-Visible spectrophotometer at a constant time interval. In the absence of AH-CuNPs, aqueous solutions of dyes were kept into the sunlight and after more than two hours, the color of the dyes was found unchanged. However, on the addition of the catalytic amount of AH-CuNPs to the aqueous solutions of dyes in the presence of sunlight, the color of only CR was changed from red to colorless within two hours (figures 7(a), (b)), whereas, original colour of MO, MB and RB solutions retained unchanged. The efficiency of catalyst was not changed on two fold increase of CuNPs concentration. However, CR degradation decreased by 60% on doubling the initial dye concentration. When, the solutions of CuCl 2 and AHLE were mixed separately to CR solution under sunlight, no decolourization was observed. Similarily, the reacting solution of AH-CuNPs and CR did not show any color change in dark. In similar reports, the decolorization efficiencies of CR were found to be 40 % and 70%-75% respectively, for ZnO and Aloe baradensis mediated CuO nanoparticles after 2 h [62,63]. The UV-Vis spectroscopy has shown that the absorption peak was decreased from 1 to 0.15 at 498 nm, the absoption maxima of CR ( figure 7(b)). The photocatalytic activity of AH-CuNPs against CR dye followed the pseudo-first-order kinetics. Figure 7(c) showed a good linear graph between ln C 0 /C versus time. The rate constant of CR is 3.1×10 -4 s −1 and the regression coefficient R 2 =0.9359 (slope=0.042) confirmed that the photocatalytic degradation of CR followed the Langmuir-Hinshelwood kinetic model. Therefore, the biosynthesized AH-CuNPs may act as a stable and efficient green catalyst for the degradation of CR under sunlight. The electrons of AH-CuNPs were easily transitioned from Valence Band (VB) to Conduction Band (CV) in presence of sunlight, and degrade the CR with the degradation product as shown in figure 8(a) [64]. ESI-I indicates the possible mechanism for dye degradation by AH-CuNPs against CR.

Antibacterial activity
The significant bactericidal effect of several metallic nanoparticles has been reported, and used for various therapeutic purposes [65,66]. Antibacterial activity of AH-AgNPs was investigated by the disc diffusion method. AH-AgNPs showed effective antibacterial activity against the gram-negative bacteria E. coli. Figure 8(b) showed the zone of inhibition (mm) for disks A and B, corresponding to AH-CuNPs and ciprofloxacin, respectively. Results showed that AH-CuNPs have significant antibacterial activity against E. coli with 12.43±0.233 zone of inhibition (mm±SE). However, as a positive control, ciprofloxacin showed an inhibition zone of 32.00±0.13 mm. The literature suggests different mechanisms for the antimicrobial activity of metallic NPs. Cytoplasmic membrane disturbance and the leakage of various cytoplasmic biomolecules such as protein, amino acids, and carbohydrates are the main reason for bacterial cell death due to exposure of NPs [67]. Bogdanovic et al suggested that Bacterial cell wall activity induces oxidation of CuNPs to release Cu ++ ions, further reduced to Cu + following electrostatic attraction with plasma membrane-based reductases. The Cu + ions easily move across the lipid bilayer into cytosol, producing ROS which leads to lipid lipid peroxidation and oxidation of proteins [68]. Raja et al also reported that antibacterial activity of NPs was due to the high conductivity of treated cells and the release of cellular components [69].

Conclusions
Using the advantage of the natural reducing and capping properties of plant extract, the present studies showed an easy and environment-friendly, green synthesis of AHLE-based CuNPs. The synthesis was demonstrated to be successful for the reaction time as well as stability of the synthesized NPs, which exclude external reducing agents. The absorption peak at 326 nm was confirming the formation of stable CuNPs in the reaction mixture. The shape of the NPs with average size of ∼80 nm was confirmed by SEM, DLS, and TEM. The AH-CuNPs has effective photocatalytic activity against carcinogenic azo dye CR. The antibacterial activity of AH-CuNPs was studied against gram-negative bacteria E. coli with a remarkable zone of inhibition.