Phyto-Fabrication, Structural Characterization and Antibacterial Properties of Hybanthus enneaspermus-Assisted Mn-Doped ZnO Nanocomposites

Abstract

Green synthesis of nanocomposites offers an eco-friendly and viable solution to overcome the limitations of conventional chemical and physical methods as it uses biological agents to act as reducing and stabilizing agents. The current study’s novelty is phyto-fabricated manganese (Mn)-doped zinc oxide (ZnO) nanocomposites using aqueous extract of H. enneaspermus by a biological method. Mn-doped ZnO nanocomposites were synthesized using manganese acetate and zinc acetate. The synthesized nanocomposites were characterized by XRD, FTIR, SEM, and EDX analysis. XRD shows the crystalline nature of nanocomposites with particle sizes of 30–40 nm, and FTIR reveals the presence of functional groups responsible for capping and stabilization. SEM analysis indicates spherical morphology with minor aggregation due to phytochemical interactions. EDX analysis of Mn-doped ZnO nanocomposites was used to verify the elemental composition, including Mn, Zn, O, and C. The anti-bacterial property of Mn-doped ZnO nanocomposites was assessed using the agar well-diffusion method against pathogens. The results of the anti-bacterial investigation proved that Mn-doped ZnO nanocomposites inhibit the growth of pathogens at different concentrations. The research concludes that the extract of H. enneaspermus acts as a capping and reducing agent in the synthesis process. The process can offer bio-compatible nanocomposites for new drug development against pathogens.

1. Introduction

Nanotechnology is an emerging field of pharmaceutical sciences in which the size of materials is reduced to nanometers ranging around 1–100 nm. Currently, scientists are using various physical and chemical methods to synthesize these tiny particles [1]. Nowadays, most of the nanoscale materials are synthesized using chemical methods, but most of the methods might lead to negative impacts including environmental pollution, huge energy consumption, and health risks. To overcome these problems, an effective eco-friendly method called green synthesis was introduced [2]. Green synthesis refers to the production of nanoparticles using natural sources or processes that minimize the use of harmful chemicals and energy by utilizing plants, bacteria, or algae as reducing or stabilizing agents to convert the metal ions into nanoparticles [3]. It emphasizes sustainability, cost-effectiveness, and biocompatibility. Green synthesized nanoparticles have diverse uses in various fields including biomedical applications [4], environment remediation [5], energy storage [6], fluorescent biological labeling [7], and so on. In the green synthesis methods, preparation of extracts usually involves leaves or any other part of the plant. In such extracts, phytochemicals act as reducing agents that make it easy to reduce the ions present in metals to nanoparticles. Such synthesis can also be controlled by various factors, including temperature, pH, metal ion concentrations, and reaction time, allowing further optimization for the size and morphology of nanoparticles. Characterization of green synthesized nanoparticles is also important to determine their properties and performance. UV–visible spectroscopy, FTIR, XRD, SEM, TEM, EDX, and Zeta potential were used to confirm the shape, size, and structural properties [8,9].

H. enneaspermus, commonly known as spade flower, is a tiny herb growing in the tropical regions of Africa, South Asia, and Australia. Being from the Violaceae family, it grows well on dry, rocky ground [10]. H. enneaspermus has medicinal values include aphrodisiac, antidiabetic [11], antiarthritic [12], antimicrobial [13], central nervous system [14], hypolipidemic [15], antiulcer and antisecretory [16], in vitro aldose reductase [17], and anti-infertile [18] properties. Traditional uses are supported by modern research indicating the wealth of bioactive compounds present, including tannins, flavonoids, alkaloids, saponins, and phenolic substances [19], which will eventually confirm the potential antimicrobial, antioxidant [20], and anti-inflammatory properties of the plant. This led to interest in its use in modern herbal medicines.

Various scientists have investigated the synthesis and characterization of Mn-doped ZnO nanocomposites. Hasan et al. [21] demonstrated the enhanced photocatalytic and antibacterial applications of Mn-doped ZnO synthesized through green methods when compared to traditional methods. Similarly, Djerdj et al. [22] (2007) characterized manganese oxide nanoparticles, and revealed their structural and magnetic properties, while Aadnan et al. [23] focused on the photocatalytic properties of Mn-doped ZnO in the degradation of azo dyes.

This study focused on the synthesis of Mn-doped ZnO nanocomposites with antibacterial properties using the aqueous extract of H. enneaspermus through the green chemistry method.

2. Materials and Methods

2.1. Preparation of H. enneaspermus Aqueous Extract

Healthy, young, and fresh H. enneaspermus was collected from the agricultural lands of Namakkal District, Tamil Nadu, India. The plant was washed with tap water followed by distilled water. A total of 10 g of the fresh plant was crushed into a paste using a mortar and pestle. Then, 100 mL of distilled water was added to the above paste and boiled at 80 °C for 10 min. The extract obtained was filtered with Whatman filter paper (No.1) and stored at −4 °C for further analysis [24].

2.2. Synthesis of Mn-Doped ZnO Nanocomposites

To prepare the Mn-doped ZnO nanocomposites, 0.1 M Manganese acetate and zinc acetate solutions were prepared separately and mixed in a ratio of 1:1 [21]. The solution was stirred continuously for 30 min using a magnetic stirrer. To that, 10 mL of the H. enneaspermus aqueous extract was added slowly while stirring it. pH 8 was maintained during the synthesis. The color change of the solution from pale yellow to white was observed, which confirmed the formation of the Mn-doped Zn nanocomposites [25]. The change in color of the solution was due to the activity of the phytochemicals present in the aqueous plant extract [26]. The pellet was obtained using centrifugation and dried in a hot air oven to evaporate the moisture content. The dried pellet was then collected and stored in sterile containers for future studies.

2.3. Characterization of Mn-Doped ZnO Nanocomposites

The powdered Mn-doped ZnO nanocomposites were characterized using X-ray diffraction (XRD), Fourier Transform Infra-Red Spectroscopy (FTIR), Scanning Electron Microscope (SEM), and Energy Dispersive X-ray spectrophotometer (EDX). XRD was performed for the phase identification of Mn-doped ZnO nanocomposites. EDX was carried out to analyze whether the nanocomposites contain Mn, Zn, O, and C, [27] and SEM was used to analyze the shape and size of Mn-doped ZnO nanocomposites [28]. FTIR was used to check the capping agents on the surface of the nanocomposites [29].

2.4. Determination of Anti-Bacterial Properties

The anti-bacterial properties of Mn-doped ZnO composites synthesized using H. enneaspermus extract were evaluated against Salmonella sp. and Bacillus subtilis using the agar well diffusion method. Different concentrations (40 to 100 µg/mL) of H. enneaspermus-mediated Mn-doped ZnO composites were prepared and gentamycin was used as a positive control. The method with small modification was adapted to determine the anti-bacterial properties of Mn-doped ZnO nanocomposites [30]. Three replications were maintained. The results are expressed in terms of mean ± standard deviation.

3. Results and Discussion

3.1. XRD Analysis

The X-ray diffraction pattern determines the crystalline nature of the synthesized Mn-doped ZnO nanocomposites. Figure 1 describes the XRD pattern of H. enneaspermus-mediated Mn-doped ZnO nanocomposites and it reveals that the composites were crystalline with an average size of 30–40 nm, which was calculated by Scherrer’s equation (JCPDS Card no. 00-001-1136). The XRD analysis of MnO₂ nanoparticles was carried out by Djerdj et al. [22], and they found a cubic structure and crystalline nature. Sagar Raut et al. [31] conducted research on the green synthesis of zinc oxide (ZnO) nanoparticles using Ocimum tenuiflorum leaves, and carried out XRD analysis. They demonstrated the formation of the wurtzite crystal structure of ZnO nanoparticles. XRD analysis of green synthesized W. coagulans-mediated Mn-doped ZnO nanocomposites was performed by Hasan et al. [21] and it shows clear peaks at 2θ = 31.7°, 34.3°, 36.1°, 47.2°, 56.4°, 60.7°, and 63.2°, corresponding to (100), (002), (101), (102), (110), (103), and (200).

3.2. FTIR Analysis

Fourier Transform Infrared (FTIR) Spectroscopy was essential to characterize the functional groups and chemical moiety of H. enneaspermus-mediated Mn-doped ZnO nanocomposites. Figure 2a illustrates the functional groups of H. enneaspermus extract (aqueous extract). The peaks at 3927, 3302, 2360, 1643, 694, 617, 601, 555, 462 and 416 cm⁻¹ were obtained for an aqueous extract of H. enneaspermus, indicating the presence of O-H stretching of hydroxyl groups, =C-H stretching vibrations, C≡C or C≡N stretching vibrations, C-O stretching vibrations, and C-H bending of aromatic rings and alkyl halides. Figure 2b determines the occurrence of various peaks at 3934, 3819, 3765, 3078, 2368, 1126, 1026, 771, 663, 617, 601, 524, 493 and 416 cm⁻¹. It proves that the biomolecules shifted from the extract to the surface of nanocomposites. Moreover, the peaks from 771 to 416 cm⁻¹ indicate the presence of metal oxide groups. Interestingly, the peaks at 416, 601, and 617 cm⁻¹ were observed in both H. enneaspermus- and H. enneaspermus-mediated Mn-doped ZnO nanocomposites. Similarly, the FTIR spectra of Mn-doped ZnO nanocomposites were obtained by Aadnan et al. [23], showing a strong absorption peak in the region between 425 and 540 cm⁻¹. These peaks correspond to the infrared-active modes of wurtzite ZnO. Finally, the peaks at 1440 cm⁻¹ and 1540 cm⁻¹ correspond to the residual acetate groups due to the zinc acetate precursor.

3.3. SEM Analysis

SEM analysis was performed to study the surface morphology and size of the Mn-doped ZnO nanocomposites. Figure 3A,B displays well-dispersed and perfectly spherical Mn-doped ZnO nanocomposites. The surface was smooth, with a minor aggregation due to the interaction of phytochemicals from the aqueous H. enneaspermus extract. Spherical and polydispersity castor oil-mediated Mn-doped ZnO nanocomposites were produced by Khan et al. [32] and proved by SEM analysis. Vindhya et al. [33] synthesized spherical Mn-doped ZnO nanocomposites using Annona muricata leaf extract via a green chemistry approach.

3.4. EDX Analysis

EDX analysis was performed to determine the elemental composition of H. enneaspermus-mediated Mn-doped ZnO nanocomposites. Figure 4 shows the presence of Mn, Zn, O, and C, which confirm the formation of the nanocomposites. The presence of carbon indicates the involvement of organic biomolecules from H. enneaspermus in biosynthesis as a reducing and capping agent. Similarly, Hasan et al. [21] reported the presence of oxygen, manganese, and zinc in the green synthesized Mn-doped ZnO nanocomposites through the EDX analysis.

3.5. Determination of Anti-Bacterial Properties

The antibacterial activity test was conducted using H. enneaspermus-mediated Mn-doped ZnO nanocomposites against Salmonella sp. and Bacillus subtilis. The results of the antibacterial properties of Mn-doped ZnO nanocomposites are summarized in

Table 1. Determination of antibacterial properties of H. enneaspermus-assisted Mn-doped ZnO nanocomposites.

S.NO	Pathogens	Zone of Inhibition (Diameter in mm)				Gentamicin
		40 µg/mL	60 µg/mL	80 µg/mL	100 µg/mL
1	Bacillus subtilis	10 ± 1	12 ± 1	18 ± 1	21 ± 1	11 ± 1
2	Salmonella sp.	6 ± 1	9 ± 1	11 ± 1	14 ± 1	6 ± 1

. Notably, at a concentration of 100 µg/mL, Mn-doped ZnO nanocomposites show the maximum zones of inhibition (14 mm) in Salmonella sp. as well as 21 mm for Bacillus subtilis (Figure 5a,b). Conversely, at a lower concentration of 40 µg/mL, Mn-doped ZnO nanocomposites display the minimum zones of inhibition for Salmonella sp. and Bacillus subtilis. Hasan et al. [21] found the anti-bacterial properties of Withania extract-mediated Mn-doped ZnO nanocomposites against E. coli and S. aureus. Canalli Bortolassi et al. [34] stated that Mn-doped ZnO nanocomposites disturb the cellular membrane by the interaction of metal ions with the bacterial cell membrane. It produces high amounts of reactive oxygen species that could lead to cell death [35].

4. Conclusions

Mn-doped ZnO nanocomposites were green synthesized using an aqueous extract of H. enneaspermus as effective reducing and capping agents. The characterization techniques, such as XRD, FTIR, SEM, and EDX, determine the crystalline nature of the synthesized nanocomposites and display the spherical morphology, with particle sizes of 30–40 nm. In addition, Mn-doped ZnO nanocomposites highly inhibit the growth of pathogens. This study emphasizes the role of H. enneaspermus as a biocompatible source in the synthesis of nanocomposites. Mn-doped ZnO nanocomposites may be utilized for developing new drugs in the medical field.