Quercetin as a Precursor for the Synthesis of Novel Nanoscale Cu (II) Complex as a Catalyst for Alcohol Oxidation with High Antibacterial Activity

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the dietary flavonoids, distributed in medicinal plants, vegetables, and fruits. Quercetin has the ability to bind with several metal ions to increase its biological activities. In the last two decades, quercetin has attracted considerable attention due to the biological and pharmaceutical activities such as antioxidant, antibacterial, and anticancer. In the present study, quercetin and ethanolamine were used for the synthesis Schiff base complex, which was characterized by IR, 1H NMR, and 13C NMR spectroscopy. The Schiff base has been employed as a ligand for the synthesis of novel nanoscale Cu (II) complex. The product was characterized by FT-IR spectroscopy, FESEM, and XRD. Significantly, the product showed remarkable catalytic activity towards the oxidation of primary and secondary alcohols. The antibacterial activity of the final product was assessed against Staphylococcus aureus (Gram‐positive) and Escherichia coli (Gram‐negative) bacteria using an inhibition zone test. The synthesized nanoscale Cu (II) complex exhibited a strong antibacterial activity against both Gram-positive and Gram-negative bacteria.

Quercetin has two aromatic rings and an oxygenated heterocyclic ring containing a carbonyl group at 4-position and hydroxyl group at 3-carbon chain [39]. Functional hydroxyl groups in the flavonoids cause antioxidant activity by scavenging free radicals and chelating metal ions. e chelation of metals can prevent radical generation which damages target biomolecules. Naidu and Kinthada [40] showed that quercetin and quercetin-3-glycoside can react with thiosemicarbazide in methanol and produce thiosemicarbazone derivatives that can form stable complexes in reaction with some transition metals.
Interaction of quercetin with metal ions can change its antioxidant and biological activities due to the ability of this complex as a free radical scavenger [41,42]. Studies show that the 3′,4′-ortho-dihydroxy substitution in the B ring is critical for copper ion chelation with quercetin to increase the antioxidant activity [43].
Copper is a bio-essential element for all organisms. It is used as a metal cofactor by some enzymes, including cytochrome c oxidase (Cox) and superoxide dismutase (SOD). In the body, copper is present in Cu + and Cu 2+ forms. It acts as an intermediary for electron transfer in redox reactions. Copper is a critical element for neuronal function and oxygen transport and a cofactor for many proteins [44][45][46] and acts as a cofactor in blood for angiogenesis [41].
Copper complexes are getting more attention due to their multiple bioactivities in living organism. Copper (II) complexes play significant role in enhancing the pharmacological profile of the antimicrobial activities of some bioactive compounds [42].
To date, the complexity of nanostructures has become interesting for fundamental and practical studies. Rational design of complex nanostructures can make new desired materials with special properties [47].
In this study, we first focused on the synthesis of Schiff base from the reaction between quercetin and ethanol amine and subsequently synthesized novel nanoscale Cu (II) complex as an excellent catalyst for alcohol oxidation. We investigated the potential catalytic activity of the synthesized catalyst in primary and secondary alcohol oxidation under solvent-free conditions. ese results showed that the catalyst performs highly efficiently and due to its heterogeneous nature, it can be used several times in the chemical reactions. e experimental results also showed the synthesized nanoparticles have high antibacterial activity.

Materials and Methods
Quercetin, ethanolamine, and all solvents and reagents were purchased from Sigma-Aldrich. All chemicals were used without any further purification. e progress of the reactions and the purity of the products were monitored by TLC (thin layer chromatography). Fourier transform infrared (FT-IR) spectra were recorded with a Nicolet System 800 beam splitter in the range 400-4000 cm −1 . NMR spectra were recorded on Bruker Avance 400 Ultrashield NMR spectrometers using tetramethylsilane as an internal standard. e powder X-ray diffraction pattern (XRD) of the final product was obtained with an X'Pert Pro-MPD diffractometer between 2θ � 2°-80°. Inductively coupled plasma (ICP) atomic emission spectroscopy was carried out by using OPTIMA 7300DV. FESEM analysis was carried out by MIRA TESCAN instrument to determine the morphology of the nanoparticles.

Schiff Base Synthesis.
e synthesis of Schiff base as a ligand is shown (Scheme 1). Quercetin (0.302 g, 1 mmol) was dissolved in ethanol (7 ml). Glacial acetic acid (57 µl) was added to this solution. Ethanol amine (60.2 µl, 1 mmol) was added dropwise to the reaction flask after 30 minutes. e reaction mixture was refluxed at 60°C for 8 hours with stirring. e resulting dark red solution was concentrated and cooled, giving an orange crystalline precipitate after recrystallization from a hot solution of ethanol and dried in vacuo. e colour of the Schiff base was light orange.

Synthesis of Nanoscale Cu (II) Complex.
e nanoscale Cu (II) complex was synthesized according to a one-pot strategy. First, the ligand (0.173 g, 0.5 mmol) was dissolved by adding NaOH 10% (1 ml) in deionized water (20 ml) under magnetic stirring for 10 minutes at room temperature to obtain an orange solution. en, a solution of Cu(OAc) 2 (0.091 g, 0.5 mmol) in deionized water (10 ml) was added dropwise to the mixture under ultrasonic irradiation at room temperature and was sonicated for 30 min. e resulting brown mixture was centrifuged and placed in a vacuum oven at 80°C for 6 hours, yielding dark orange precipitation (Scheme 2).

Catalytic Procedure for Alcohol Oxidation.
e synthesized copper (II) complex was tested as a catalyst in benzyl alcohol oxidation to determine its catalytic activity. Several experiments were carried out to optimize temperature, solvent, and mol% of catalyst. e best result in the model reaction was chosen. In order to the investigation of solvent nature, the model experiment was carried out in different solvents and the best result was taken in solvent-free conditions. It seems that open catalytic sites in this condition are the reason of this observation. In the second step, the model reaction was carried out at different temperatures. e reaction yield at room temperature, 40°C and 50°C, was 98%, so the reaction was carried out at room temperature. en, the catalyst and oxidant amount were investigated. e best yield was obtained in the presence of 1 mol% catalyst and 1 mmol of TBHP. For the investigation of oxidant type, the reaction was performed with H 2 O 2 , TBHP, and NaIO 4 . With the optimized reaction conditions, the best experiment as a model was chosen. 1 mol% of catalyst, 0.10 mL (1 mmol) of benzyl alcohol, and 0.096 mL (1 mmol) of tert-butyl hydroperoxide (TBHP) as an oxidant were mixed and stirred in solvent-free condition at room temperature for half an hour. After the determination of optimum condition, the ability of the catalyst for a series of alcohols was evaluated (Table 1). e catalytic activity of the synthesized nanoparticles was compared with some other catalysis in the alcohol oxidation reaction (Table 2).

Antibacterial Activity.
e synthesized complex was tested for in vitro antibacterial activity using the agar well diffusion method. Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) were used as a model for this activity. e fresh bacteria were incubated on nutrient agar plates at 37°C for 24 hours. A nutrient agar for the plates was provided by dissolving agar powder (15.0 g L −1 ), tryptone (5.0 g L −1 ), yeast extract (2.5 g L −1 ), and glucose (1.0 g L −1 ) in deionized water. e pH of the mixture was adjusted to 7.0 ± 0.1. e diameter of the zone of inhibition was measured.
Gentamicin was used as a control substance. e antibacterial efficacy of the complex was tested against Staphylococcus aureus and Escherichia coli bacteria. e biocidal effect of the copper complex was investigated by the diffusion method. A bacterial suspension with a concentration of 5 × 10 5 cfu/mL (200 mL) was spread on the agar plates. After the agar plates were incubated at 37°C for 48 hours, the synthesized complex (1 mg) was disposed directly into the holes (7 mm diameter) which were punched over the agar plates previously. e evaluation of the antibacterial activity of the complex was carried out by the standard zone of inhibition test. e diameter of the zone of inhibition was measured in mm by the ruler.

Results and Discussion
e formation of a Schiff base between quercetin and ethanolamine was investigated by NMR spectroscopy. NMR data showed the formation of Schiff base from quercetin and ethanol amine. e binding properties and the coordination sites were investigated by using IR spectroscopy. e main peaks of quercetin, quercetin Schiff base, and quercetin Schiff base complex are shown in Table 3. Important information was obtained by comparing the quercetin with the Schiff base and the complex. It was observed that C�O stretching mode of free quercetin occurred at 1707 cm −1 , which was shifted to 1695 cm −1 in the Schiff base, and it was shifted to 1622 cm −1 by the formation of the complex. A sharp peak at 1612 cm −1 shows the formation of the imine band in Schiff base. e sharp stretching vibration at 625 cm −1 shows Cu-O chelation indicating the copper (II) is chelated to quercetin Schiff base ligand and formed the metal complex while this stretching band was absent in the FT-IR spectrum of the ligand. e broad medium intensity band at the frequency range of 3200-3450 cm −1 may be assigned to the -OH group. In the quercetin Schiff base copper complex, a broad -OH group was observed as broad vibrations around 3300 cm −1 (Figure 3).    Figure S1). e 13 C NMR spectral data of the ligand were recorded in DMSO solution.

Schiff Base
In the 13  As the ratio of metal ion and ligand is 1 : 1 and the 3hydroxy group has a more acidic proton, the 3-OH and N positions are the best site to be involved in the complexation process [29,56]. e OH group from the alcohol is not coordinated, possibly due to the distance between OH group and the center of reaction. Similar study confirms this structure [57] (Figure 6). e oxidation of alcohols mechanism may involve the generation of tBuOO • and t-BuO • radicals by the metal assisted, which behave as hydrogen atom abstractors from the alcohols. e ligand can assist proton transfer steps involved in the fundamental steps of the alcohol oxidation reaction. e mechanism is summarized in Scheme 3.
After optimizing reaction conditions, the effect of a catalyst in a series of primary and secondary alcohols including aromatic ring and electron donating and withdrawing groups was evaluated. e results are summarized in the table. In general, all substrates showed an excellent yield in alcohol oxidation reactions. Electron donating and electron withdrawing groups change the reaction yield slightly. e recyclability of the catalyst was tested in the alcohol oxidation reaction. e complex was recovered from the reaction after three times for the next reaction run by centrifugation, washing, and drying in the oven. is catalyst was reused three times without any significant change of catalytic activity. e synthesized complex was tested for the in vitro antibacterial activity against E. coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria at concentration of 100 mM. e complex was active against both of these bacteria. e results of the antibacterial activity are reported as inhibition zone diameter (mm). e antibacterial activity of the complex can be attributed to the involvement of metal ions as a candidate for bacterial growth inhibition, which could be explained by chelation theory [58].
Gram-positive bacteria have lipopolysaccharides cell wall. is wall prevents the accumulation of the complex in the cell membrane. erefore, Gram-positive bacteria are   more effective and more sensitive compared to Gramnegative bacteria. Transition metal complexes have an important place in biochemistry [38,59]. e antibacterial activity of the synthesized complex was evaluated and compared with standard (gentamicin) (Figure 7). e diameter of the zone showing complete inhibition is listed (Table 4).
is study clearly showed that synthesized complex has reasonable antibacterial activity against both Gram-negative and positive organisms. Enhanced lipophilic properties of metal ion sites caused the high antibacterial activity of the synthesized complex. e enhanced lipophilicity led to cell death by easy translocation of nanoscale Cu (II) complex.

Conclusions
In this study, formation of Schiff base between quercetin and ethanolamine is investigated. e reaction was carried out in ethanolic solution under reflux condition. Quercetin is a flavonoid with potent antioxidant activity and broad clinical effect. e biological activities of quercetin increase when it is coordinated with metal ions. is is due to the fact that after forming the complex, solubility and bioavailability of quercetin in the body are increased. In the second step, a novel nanoscale Cu (II) complex was synthesized, and its catalyst effect in oxidation reactions of alcohols was examined. Copper is a bioactive metal that plays several roles in biological processes, such as catalyzing a large number of biochemical reactions and having a key role in electron transport in mitochondria. Copper (II) complexes show a wide variety of biological activities. ey could be used as antimicrobial, anti-inflammatory, antitumor, and antiviral agents. erefore, Cu(II) complexes are synthesized as a potential drug.
e spectroscopic data showed the 3-OH group and imine groups are coordination sites with the metal ion. e alcohol oxidation reaction was performed in green condition that is in accordance with environmentally friendly protocols. e catalyst is highly stable and can be reused several times without the loss of catalytic activity in the alcohol oxidation reaction.
Furthermore, the synthesized complex indicated promising antibacterial activity against E. coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria. Staphylococcus aureus has higher antibacterial activity than E. coli due to the differences between cell structure, metabolism, and physiology of Gram-positive and Gramnegative bacteria. ese factors are influential on the sensitivity of the nanoscaled copper complex on the antibacterial activity.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.