Capsicum annuum Fruit Extract: A Novel Reducing Agent for the Green Synthesis of ZnO Nanoparticles and Their Multifunctional Applications

A simple, efficient and convenient method for the preparation of zinc oxide (ZnO) nanoparticle was described. Several parameters like size and morphology of the prepared nanoparticles were characterized thorough a variety of analytical techniques such as XRD, FT-IR, UV-Vis, SEM, and EDX. The prepared ZnO nanoparticles were successfully used as catalyst for the formylation of amino acid esters and biodiesel synthesis. Further, the synthesized formamide esters were well characterized through HRMS, 1H NMR and 13C NMR analysis and subjected for the in vitro antibacterial and antifungal tests and the results indicated that some of them showed promising activity against targeted bacterial pathogens.


Introduction
Zinc oxide is a multifunctional material with its unique physical and chemical properties, such as high chemical stability, high electrochemical coupling co-efficient, high range of radiation absorption and high photostability. 1 ZnO as a catalyst with high specific surface area 2 finds the potential use in electronics, optoelectronics, laser technology and is made possible because of broad energy band (3.37a eV), high bond energy (60 MeV), thermal and mechanical stability at room temperature. 3 ZnO NPs have fascinated the research world through its significant applications in pigment electronics, spintronics and piezoelectricity fields. 4 Nano structured material ZnO has the ability to generate power, which also finds an extensive application in self-power generating devices for medical, wireless technologies and sensor applications. 5 ZnO shows diverse group of growth morphologies such as nano rings, nano springs, nano combs, nanowires and nano cages. 6 These have been synthesized using solid-vapour phase thermal sublimation technique 7 under specific growth condition. ZnO nanoparticles have drawn attention due to their antimicrobial activity; this finds application in food packaging. 8 Biocompatibility and biodegradability makes it a material of interest for biomedicine and in pro-ecological systems. 9 Nano ZnO plays a vital role as a semiconductor photo catalyst for UV-induced degradation of methylene blue 10 and doped ZnO also exhibits good optical and electrical properties.
Dhiman et al. reported the synthesis of Fe-doped ZnO nanoparticles by solution combustion method. 11 Recently, there are several attempts for the green biosynthesis approach for the preparation of ZnO, SnO 2 , silver and reduced graphene oxide-silver (RGO-Ag) nanocomposites using leaf extracts of Plectranthus amboinicus and Corymbia citriodora as a reducing agent at different temperatures. The synthesized NPs showed a superior photocatalytic, catalytic activity towards dye molecules degradation and also investigated the electrochemical properties of ZnO, RGO-Ag. [12][13][14][15][16] It has been widely studied that, ZnO is a highly efficient catalyst for a plethora of organic reactions, such as Friedel-Crafts acylation, Beckmann rearrangements, synthesis of cyclic ureas from diamines, N-alkylation of imidazoles and ring-opening of epoxides. 17 Nehal and others synthesized the ZnO nanoparticles and studied the effect of annealing temperature on its particle size. 18 The prepared ZnO NPs were reported to be employed in various functional group transformations. Among the transformations, protection of reactive amino groups is commonly required in organic synthesis using formyl group. 19 Formylation of amine is one of the important protocol in organic synthesis and medicinal chemistry. Various formylating reagents such as formic acid-DCC, formic acid-EDCI, KF-alumina, and ammonium formate were employed. [20][21][22][23] The obtained formamides are the main class of organic intermediates, which act as Lewis bases. 24 Synthesis of formamide was also achieved by the reaction of isocyanate and formic acid in the presence of DMAP, 25 acetic formic anhydride, 26 and metallic zinc. 27 Literature survey reveals that, many catalysts were used for the N-formylation of amines with formic acid, such as amberlite IR-120, 28 TiO 2 -P25 or sulfated titania, 29 and HEU zeolite. 30 N-formylation of amines using hydroxylamine hydrochloride as a catalyst under neat condition was reported by Deepali agarwal and co-workers. 31 Using amine and formic acid in the presence of a catalytic amount of thiamine hydrochloride, formamide derivatives have been synthesized in excellent yields. 32 Nano MgO, ZnO, and CeO 2 were also used as catalysts in the synthesis of formamides. [33][34][35] Chandra shekhar et al. reported the facile N-formylation of amines using Lewis acids such as, FeCl 3 , AlCl 3 , and NiCl 2 as novel catalysts. 36 There are various methods to prepare nano structured particles. Some of the methods include chemical vapour deposition, 37 hydrothermal, 38 precipitation, 39 and sol-gel method. 40 The conventional physical and chemical methods available for the synthesis of NPs have adverse effects like critical temperature conditions and pressure, expensive chemicals, toxic byproducts etc.
Herein, green synthesis of zinc oxide nanoparticles using eco-friendly and non-toxic Capsicum annuum extract was reported. Capsaicin is an alkaloid found mainly in the fruit of the Capsicum genus, which provides spicy flavour andhas pharmacological effects to determine specific applications, such as for weight-loss and as an analgesic. 41 Literature survey confirms that the capsaicin has anti-bacterial and anti-diabetic properties. 42 Capsicum annuum is a rich source of ascorbic acid generally known as vitamin C, a very essential antioxidant for human nutrition. 43 This method has several benefits such as simple procedure, inexpensive reagents and good stability of nanoparticles. The solid catalyst is of great importance because of its advantages such as non-hazardous nature, requirement in small proportions and easier reaction workup.
Biodiesel is recognized as an alternative fuel due to its similar bearing with diesel fuel. Existing days, research has been intensive to the alternative sources of energy. 44,45 Commonly, biodiesel is a mixture of fatty acid methyl ester (FAME), which is synthesized from vegetable oil, waste oils and animal fat through transesterification reaction. Homogeneous catalysts such as KOH, NaOH, CH 3 OK, and CH 3 ONa show a favorable catalytic efficiency with various drawbacks such as generation of waste water, corrosion of equipment. 46 In display, solid heterogeneous catalysts are favorable for biodiesel synthesis because of environmentally friendly, easy separation, and could be reused many times. 47 Many solid acid catalysts like zeolite, 48 WO 3 /ZnO 2 , 49 and sulphated zirconia 50 were suitable for esterification reaction under 60-75 o C. In fact, solid acid catalysts (phosphotungstic acid, 12-tungstophosphoric acid, and ionic liquids) are used for the esterification and transesterification through one pot method. Meanwhile, solid base catalysts such as Ca(OCH 3 ) 2 , 51 CaO 52 , and KOH/Al 2 O 3 were used in transesterification reaction at mild condition. Among the transition metal oxides, zinc oxide was reported the best catalyst for transesterification due to its minimum weight loss and high activity in the reaction. 53 Currently, application of nano catalysts for biodiesel synthesis has drawn much attention, as a result of easy separation of products, less pollution, higher catalytic activity and reusability. 54 Recently, nano catalysts such as CaO, 55 Ti(SO 4 )O, 56 KF/CaO-Fe 3 O 4 , 57 Ag/ZnO 58 , and mixed oxide TiO 2 -ZnO 59 were used for biodiesel production. Presently, non-edible oils are used for biodiesel production to reduce edible oil conflict among food and fuel purpose. In this study, Buteamonosperma oil (non-edible) is used for biodiesel production and this plant belongs to a fabaceae family which is native to Indian subcontinent and seeds contain 23% of oil. 60,61 In the present work, we report the synthesis of ZnO NPs through solution combustion by using Capsicum annuum extract as the combustible fuel and the obtained ZnO is employed as catalyst for the N-formylation of amino acid esters and biodiesel production. Further, the synthesized formamide derivatives were subjected for biological activities.

1. General
All chemicals were purchased from Sigma-aldrich and Merck and used without purification. The pathogenic bacterial strains were procured from National chemical laboratory Pune, India. The Capsicum annuum fruits were collected from local market, Tumakuru district, Karnataka, India. TLC analysis was carried out using pre-coated silica gel F 254 . The phase identity and crystalline size of ZnO NPs were characterized through shimadzu powder X-ray diffractometer (PXRD-7000). IR spectra were re-Lalithamba et al.: Capsicum annuum Fruit Extract: ... corded on Bruker Alpha-T FT-IR spectrometer (KBr windows, 2 cm -1 resolution), SEM analysis on Hitachi-7000 Scanning Electron Microscopy and elemental analysis was obtained from energy dispersive X-ray diffraction (EDX). UV-Vis diffused reflectance spectra were analyzed through Lambda-35 (Parkin Elmer) spectrophotometer. Mass spectra were recorded on a Micromass Q-ToF Micro Mass Spectrometer. Melting points were taken on open capillaries, 1 H NMR, and 13 C NMR spectra of the formamide derivatives were done on a Bruker AMX 400 MHz spectrometer using Me 4 Si (tetramethylsilane) as an internal standard and CDCl 3 (deuterated chloroform) as a solvent.

Synthesis of Nano Zinc Oxide Particles Through Solution Combustion Method
The Capsicum annuum fruit was collected and washed with distilled water. The whole mass was grinded to get the powder and then mixed with distilled water and boiled at 80 o C. After cooling to room temperature, the mixture was filtered using a Whatman filter paper no. 1 to obtain chilli extract. Zinc nitrate as precursor and Capsicum annuum fruit extract as fuel in the ratio of 4:1 were used for the synthesis of ZnO NPs through solution combustion method. 62 The solution was heated to 450 o C for 30 min and then dried in hot air oven for 4-5 h to obtain ZnO NPs in good yield.

3. General Procedure for the Synthesis of Formamide Esters Using ZnO NPs
The prepared amino acid ester (1.0 mmol) was dissolved in dry DCM (dichloromethane) and neutralized with NMM (N-methyl morpholine) (1.5 mmol). To this solution, formic acid (2.0 mmol) was added at room temperature, followed by the addition of nano ZnO (0.5 mmol). The reaction mixture was stirred for 2 to 3 hours. The product was extracted into DCM and the organic layer was washed with hydrochloric acid solution (10 mL), sodium carbonate solution (15mL), water (15 mL) and brine (15 mL). It was dried over anhydrous sodium sulphate and concentrated under reduced pressure.

4. Synthesis of Biodiesel Using ZnO NPs as a Catalyst
Transesterification reaction was carried out in a three necked round bottom flask equipped with reflux condenser on the middle neck, thermometer on the side neck and placed on the plate of the magnetic stirrer. In the beginning, 100 ml of B. monosperma oil was pre heated at 70 o C then a mixture of 2% wt. ZnO and 9:1 molar ratio of methanol to oil was added. The entire reaction was carried out at 65 o C for duration of 2 h. After the completion of reaction, the mixture was allowable to phase separation, the biodiesel (top layer), glycerine (middle layer) and catalyst (bottom layer) phase were separated. Then, the catalyst and glycerine were drained out and unreacted methanol was recovered from biodiesel. The obtained biodiesel was filtered to remove any dissolved zinc oxide catalyst.
The average crystallite size of prepared sample was calculated by Debye-Scherrer's 64 formula i.e.
(1) where, D is crystalline size, λ is X-ray wavelength (0.154 nm), β is full-width at half-maximum and θ is Bragg's angle. The average crystallite size of the ZnO NP was found to be 33.26 nm. Figure 2(a) and 2(b) show DRS spectra and band gap plot of ZnO nanoparticles respectively. DRS spectrum reveals that the absorption is at ~ 400 nm. The Kubelka -Munk function 65,66 was utilized to determine the band gap energy (E g ) of ZnO NPs. The intercepts of the tangents to the plots of [F(R ∞ ) hν] 1/2 versus photon energy (hν) were shown in Figure 2(a) and 2(b). The Kubelka-Munk function F (R ∞ ) and photon energy (hν) can be calculated by following Equations (2), (3) and (4): where R ∞ ; reflection coefficient of the sample, A; the absorbance intensity of ZnO nanoparticles and λ; the absorption wavelength. The energy band gap value was found to 3.10 eV. Figure 3 shows the SEM images of as prepared zinc oxide nano particles. Figure 3c is the enlarged part of 3b and 3a, which clearly shows that the particles are agglomerated cluster to form spongy cave like structures. The sizes of the particles are found to be in the 500 nm to 1 micrometer.
The Energy Dispersive X-ray Diffractive study was carried out for the prepared ZnO NPs to know about the elemental composition. The EDX spectrum confirms the   Figure 5 is the FT-IR spectrum of ZnO NPs and the band in the region of 680-400 cm -1 is the characteristic peak of ZnO NPs. Thus, the formation of pure ZnO NPs at 535 cm -1 is evidenced by FT-IR 67 and the peak at 1122 cm -1 in FTIR spectrum is due to C-O stretching mode. 68 The peaks observed at 1628 cm -1 and 3500 cm -1 were due to the presence of -OH stretching and bending vibrations respectively assigned to the H 2 O adsorption on the surface of metal. 69 Figure 6(a), (b) show the photoluminescence (PL) spectra of ZnO NPs recorded at room temperature. The recombination of photo generated free charge carriers leads to photoluminescence emission in semiconductor materials. The spectrum was recorded under UV excitation (375 nm) using Xenon lamp as source. The result obtained reveals that nano ZnO shows the strong emission peak at 600 nm ( Figure 6(a)). The emission spectrum was monitored at 375 nm showed a broad emission at 600 nm was shown in Figure 6(b). The broad 600 nm peak was due to the transition between single charged oxygen vacancies. The correlated color temperature was one of the essential parameter to know the color appearance of the light emitted by a light source with respect to a reference light source   when heated up to a specific temperature. The color clarity of any luminescent material was expressed in terms of chromaticity coordinates, called Commission International De I'Eclairage (CIE).

2. Application of ZnO NPs as a Catalyst for the Formylation of Amino Acid Esters
In the current years, the use of metaloxides as a catalysts and reaction media has received considerable tremendous interest because of their high level of environmental compatibility, chemo selectivity and availability at low cost. Therefore, we explored the application of nano ZnO powder as an inorganic catalyst to carry out N-formylation of amino acid ester (1 mmol) in DCM with aq. 98% formic acid (1.5 mmol) at room temperature. Formamide derivative of amino acid ester was obtained in trace amount in the absence of ZnO catalyst, while good results were obtained with use of 0.5 mmol ZnO catalyst after same reaction conditions as mentioned. But less than 0.5 mmol considerably decreased the percentage of formamide esters and may took longer time for the completion of reaction. Using more than 0.5 mmol of ZnO has less effect on the final yield of the products. Thus, we found that 0.5 mmol of ZnO could efficiently catalyze the reaction for preparation of the desired products and observed that the excellent yield obtained using 1.5 mmol of formic acid. In literature survey Suresh babu et al. achieved successful synthesis of formamide derivatives of protected amino acids by the reaction of isocyanate with aqueous formic acid using DMAP as an organo catalyst. 70 However, this procedure suffers from the difficulties such as expensive reagents, highly toxic and may also require special care. Hence, it is necessary of convenient reagent for the synthesis of stable formamides in terms of economic via-bility and operational simplicity. Therefore, it is necessary to study this reaction using nano ZnO with a variety of amino acid esters as starting materials which were subjected to formylation reaction and the results were presented in Table 1. For the synthesis of titled compounds (2a-h), amino acid ester containing different aryl/alkyl groups prepared from thionyl chloride was dissolved in dry DCM and neutralized with NMM, to which aqueous formic acid was added followed by the addition of nano ZnO. The reaction mixture was stirred till the completion of the reaction (as monitored by TLC). After the simple work-up, desired products were obtained in good yield (Scheme 1). Using this procedure several formamide esters were synthesized from amino acid esters and characterized by their 1 H NMR, 13 C NMR and mass spectral studies.

Spectral Data of the Synthesized
Compounds:

4. Antibacterial and Antifungal Activity of Formamide Derivatives
The synthesized compounds were evaluated for their antibacterial activity against E. coli (MTCC 443) and S. Aureus (MTCC 5823). Lack of activity of the tested substances against Gram +ve bacteria could be explained by the differences in the structure of cell walls of Gram +ve and Gram -ve microorganisms. In most Gram +ve bacteria, the cell wall consists of many layers of peptidoglycan, forming a thick, rigid structure. Whereas, the cell walls of Gram -ve bacteria consist of one or a very few layers of peptidoglycan and a lipid-rich outer membrane. 71 Similar type of results employing different type of compounds such as Schiff 's bases and amine derived from alkyl 2-(2-formyl-4-nitrophenoxy) alkanoates was recorded by Goszczyn´ska. 72 A contrasting difference in antibacterial activity employing Gram +ve and Gram -ve strains representing better susceptibility by E. coli than S. aureus employing novel benzothienopyrimidines compounds was shown.
In this study, the antibacterial screening indicated quite varied results among the tested samples as depicted in Table 2, 3, and 4 exhibiting antibacterial and antifungal activities respectively. For E. coli (MTCC 443), formamide ester derivatives of Ala, Tyr, Phe have shown good susceptibility over a wide volume range taken for a fixed concentration of samples (Table 2). Serine derivative has shown best result in comparison to others at higher volume of fixed concentration of the sample tested. Rest of the samples were found to be resistant against the bacterial strain. A different result has been observed in terms of susceptibility pattern tested against S. aureus (MTCC 5823). Derivatives of Ala, Ser,  OHCHN-Ala-OMe ++++ -- OHCHN-Tyr-OMe ++++ --- OHCHN-Phe-OMe ++++ -- OHCHN  Table 4. Derivatives of Ala, Tyr, Leu, Pro and Val have shown some activity in comparison to standard. While the derivatives of Phe, Ser, and Met have also shown good activity over a wide range of volume of samples (50-300 µl) tested for a fixed concentration. We are also measured the equivalent zones of inhibition (in mm) at varying dilutions and depicted in the photographs of the culture plates for the best cases ( Figure 8).

4. Application of ZnO NPs as a Catalyst for the Biodiesel Production
After transesterification of Buteamonosperma oil, the yield of biodiesel was found to be 82.7%. In order to evaluate the quality of biodiesel, the fuel properties of the biodiesel were determined according to ASTM D6751 standards as shown in the Table 5. The fuel properties such as kinematic viscosity (4.3 cSt), flash point (151 o C), acid value (0.2 mg KOH/g), calorific value (37790 kJ/kg), density (880 kg/m 3 ) and copper strip corrosion (1a) were within the range of ASTM standard. Schematic diagram of ZnO NPs catalyzed transesterification for the production of biodiesel is shown in the Figure 9.

Conclusions
Multifunctional ZnO nanoparticle has been synthesized via a simple solution combustion method using Capsicum annuum extract as a new fuel. The prepared NPs were characterized by UV-Vis DRS, XRD, SEM, and EDX and also evaluated its photoluminescence property. The method is environmental friendly and overcome the demerits of conventional physical and chemical methods of synthesis. In the presence of nano ZnO catalyst excellent yield of formamide esters and biodiesel have been obtained. The synthesized formamide esters were successfully characterized by 1 H NMR, 13 C NMR, and mass spectroscopy analysis. Finally, the formamide esters were subject to biological activities against bacterial pathogens and few of the molecules exhibited considerable biological activities.

Acknowledgements
We thank the Principal and Director of Siddaganga Institute of Technology, Tumakuru, Karnataka, for the research facilities. One of the authors (HSL) is thankful to the Vision Group of Science and Technology, Department of Information Technology, Biotechnology and Science & Technology, Government of Karnataka for providing funds under CISEE programme (GRD No. 472) to carry out the present research work by means of a sponsored project.