INTRODUCTION:

Nanoparticles (NPs) are used in science and medicine due to their unique properties. They have large surface areas, specific crystal structures, and strong mechanical properties and conduct heat, absorb substances, and stay stable, making them useful for breaking down pollutant’s^1,2. Nanoparticles comes under range from 1 to 100 nanometres, due to their small in size, large surface area they are used in varied application in various sectors (drug, pharmaceutical, detergent, medicinal)^3,4,5. Polymeric nanoparticles, like Chitosan and PLA, are biodegradable and used for controlled drug release and targeted delivery⁶.

Liquid-based nanoparticles, used in cancer treatment, consist of a solid lipid core with surfactant-stabilized outer layers^7,8. Eco-friendly methods are crucial for advancing materials, including natural fibre polymers, and ensuring sustainable nanoparticle applications in diverse fields⁹.

Recent studies emphasize the importance of green synthesis for producing metal oxide nanoparticles, including zinc, iron, gold, copper, silver, nickel, and others. Green synthesis is facilitated by plants or their extracts, is gaining momentum due to its economic efficiency and environmental sustainability which is safe for industries like textiles, pharmaceuticals, and optics^6,10. Plants naturally contains chemicals that convert metal ions into nanoparticles, ensuring uniformity in size, shape, purity, and crystallinity¹¹. Green synthesis promotes the use of renewable sources and safer practices, reducing energy consumption and supporting environmentally friendly chemical processes¹².

Aegle marmelos as Bael or Bilva, which belongs to the family Rutaceae, found in regions like the Himalayas, Bengal, and Southeast Asia. Its fruits, leaves, stems, roots, and bark are utilized in Ayurvedic medicine for treating various health issues. Studies have shown that extracts from Aegle marmelos leaves act as effective reducing agents in synthesis of different nanoparticles

Zinc oxide nanoparticles (ZnO NPs) are highly valued in medicine due to their biocompatibility and low toxicity, making them safe for medical applications⁴. They exhibit efficient catalytic properties and can be recycled with minimal loss of activity. These nanoparticles possess unique characteristics such as a high excitonic binding energy and a sharp band gap of 2.57 eV, which are advantageous for applications in cancer treatment, wound healing, and bioimaging¹³. ZnO NPs find versatile use across various sectors including energy and electronics to make nanosanser due to their strong stability in harsh environments^11,14. It is being studied as new antibacterial agents because they can break down bacterial cell wall. It also used in managing wastewater pollution by removal of heavy metals like chromium by nano-adsorption process.

This study aims to synthesize zinc oxide nanoparticles (ZnONPs) (Fig 1) using an aqueous extract of Aegle marmelos as a reducing agent and study its antimicrobial activity and chromium adsorptions.

MATERIALS AND METHODS:

Plant material and extract preparation

Fresh leaves of Aegle marmelos (Bael) were collected from the campus of the School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur (C.G). The leaves sample were washed thoroughly with tap water, dried at 40-45°C in a hot air oven for 2-3 days, and then ground into a fine powder (Fig. 1). Five grams of the powder were boiled with 100 mL of distilled water (1:20) at 50-60°C for 1-2 hours on a magnetic stirrer at 150-200 rpm. The extract was filtered through Whatman filter paper no. 1. Obtained filtered solution was stored at 4°C for further use in synthesizing ZnO nanoparticles.

Preparation of zinc oxide nanoparticles

Zinc sulphate heptahydrate was prepared at a concentration of 1 mM as the metallic precursor for ZnO nanoparticles synthesis. During spectrophotometric absorption, same precursor solution was used to set blank. For the synthesis of ZnO nanoparticles, 95 mL of the freshly prepared precursor solution was heated on a magnetic stirrer for 5 minutes. Then, 5 mL of the plant extract was added dropwise to the precursor solution, maintaining a 19:1 ratio of plant extract to metallic precursor, with constant stirring for 2 hours. The solution's colour changed from yellow to dark brown (Fig. 1), indicating the formation of ZnO nanoparticles. The sample was characterized using UV-Visible spectroscopy in the range of 200-800 nm. The solution was then placed in petri dishes and dried in an oven at 45-50°C for 2-3 days. The resulting nanoparticle powder was collected from the petri dishes for antibacterial activity testing.

UV-visible spectroscopy

To measure the optical parameters of ZnO nanoparticles, spectrum scan of synthesized nanoparticles was recorded with help of a UV-visible spectrometer. The spectral analysis was performed at a resolution of 1 nm, covering wavelengths from 200 nm to 800 nm.

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy is used to examine the properties of functional groups or metabolites on the surface of nanoparticles, helping to recognize the biomolecules accountable for reducing metal ions to their neutral form. In this study, we recorded FTIR spectra over a range of 4000–400 cm⁻¹, detecting multiple functional groups present in the nanoparticles and providing insights into their surface chemistry.

Antibacterial Activity

The disc diffusion method was used to determine the antibacterial activity of ZnO nanoparticles against four bacterial strains: two gram-positive bacteria (Staphylococcus aureus MTCC 3160 and Bacillus cereus McR3) and two gram-negative bacteria (Escherichia coli MTCC 2412 and Salmonella enterica serotype typhi MTCC 733). Overnight bacterial cultures (100 µL) were spread over Nutrient Agar (NAM) plates using a sterile cotton swab. Wells were created in the smeared NAM plate. 20 microliter ZnO nanoparticles solution of different concentration (50, 100, 150, and 200 mg/mL) were poured to each well individually.The plates were incubated at 37°C for 24 hours. The diameter of the inhibition zones around each well was measured in millimeters to evaluate the bacteria-inhibiting activity of the synthesized nanoparticles. The values were represented as mean ± SE.

Adsorption of Chromium

To evaluate the adsorption rate of zinc oxide (ZnO) nanoparticles on chromium (Cr), batch adsorption studies were conducted. Potassium dichromate (K₂Cr₂O₇) was utilized to determine the chromium content. Various concentrations of dichromate solutions (containing 14, 28, 46, and 52 mg/ml of chromium) of 100 mL were prepared in Erlenmeyer flasks. Each flask received 200 mg of bare ZnO nanoparticles as the adsorbent. The mixtures were placed on an orbital shaker for approximately 2 hours to assess the adsorption rate. Following the adsorption process, the samples were centrifuged at 8000 rpm for 10 minutes to separate the nanoparticles from the liquid phase, resulting in a supernatant containing the residual chromium ions. To prepare for analysis, the supernatant was further diluted, specifically at a ratio of 1 part supernatant to 10 parts diluents. To detect the remaining Cr (VI) concentration, 1 mL of the diluted sample was mixed with 9 mL of 0.2 M H₂SO₄, followed by the addition of 0.2 mL of freshly prepared 1,5-diphenylcarbazide solution (0.25% w/v in acetone). The formation of a red-violet colour complex was measured using a UV-visible spectrometer at wavelength of 540 nm. These steps enabled the calculation of the adsorption efficiency of zinc oxide nanoparticles on chromium, based on the initial batch adsorption experiments.

Statistical analysis

The research data were examined using SPSS software. All experiments for antibacterial activity and chromium removal were conducted three times (triplicate), and the results were presented as the mean ± standard error (SE). Apart from it, statistical comparisons were made at a significance level of 5%.

RESULTS AND DISCUSSION:

Synthesis of zinc nanoparticle

ZnO nanoparticles were made using Aegle marmelos (Bael) leaf extract and zinc sulfate in an eco-friendly way. The natural chemicals in the leaf extract helps to make and stabilize these nanoparticles. When zinc sulfate mixed with the leaf extract and stirred it, the colour changed from yellowish to reddish brown. This change shows that zinc ions turned into nanoparticles (Fig. 2). The leaf extract's chemicals also help keep the nanoparticles stable by changing their surface morphology. Some recent studies shows that other plants like Saussurea lappa and Cucumis maderaspatanus can do similar things^15,16. Plant-based methods are good for synthesis of nanoparticles and due to plant based synthesis, it not cause any harm to the environment.

Fig. 2. Synthesis of ZnO nanoparticles from Aegle marmelos (Bael). (a) Precursor (b) Plant extract (c) Synthesized ZnO nanoparticle

UV-Visible Spectroscopy

The synthesis of zinc oxide nanoparticles (ZnO NPs) using Aegle marmelos (Bael) leaf extract was monitored with UV-Vis’s spectroscopy. The formation and optimization of ZnO NPs were tracked by measuring absorbance in the 200–800 nm range (Fig. 3). The absorption spectrum showed distinct peaks centered around 220-230 nm, confirming ZnO NPs formation due to the bio-reduction process facilitated by the plant extract.

The wavelengths observed for ZnO NPs in this study match with previous reported research (Table 1), confirming the successful synthesis of nanoparticles. Finding of other researchers also achieved similar spectrum scan results, it confirms that the present approach is reliable. This consistency strengthens the credibility of our findings and underscores the effectiveness of using plant extracts for nanoparticle synthesis.

Table 1: Comparison of UV-Visual Spectrophotometer peak value of pervious reports

S. No.	Nanoparticles	UV-Visible spectrum (peak value) λmax of other reports	Reference
1.	ZnO NPs	225 nm	Present Study
2.	ZnO NPs	235 nm	17
3.	ZnO NPs	320 nm	18
4.	ZnO NPs	284 nm	19

Fig. 3. UV-visible spectrophotometric analysis of synthesized zinc oxide nanoparticles

FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR):

The FT-IR spectrum of the prepared ZnO NPs was recorded from 4000 to 400 cm⁻¹. In the spectrum, the O-H stretching bands of hydroxyl groups from water vapor were noted at 3232.39 cm⁻¹, which are linked to proteins, polysaccharides, and polyphenolic groups on the ZnO surface, indicating asymmetric and symmetric stretching modes of hydroxyl compounds. A peak appeared at 1593.06 cm⁻¹, corresponding to the stretching vibrations of C=O bonds from non-ionic carboxylic groups such as carboxylic acids or their esters. Similarly, peaks were identified at 1396.19 cm⁻¹ and 1068.91 cm⁻¹, associated with the C=C and C–O vibration modes of phenols and alcohols, respectively. Strong vibrational bands below 1000 cm⁻¹ indicate the formation of metal-oxygen bonds, suggesting the presence of reducing agents that facilitate the synthesis of ZnO nanoparticles (Fig. 4). This underscores the potential of using plant extracts for the eco-friendly synthesis of ZnO nanoparticles, consistent with other studies on the green synthesis of zinc oxide nanoparticles, such as those by MuthuKathija et al. (2023)²⁰.

Fig. 4. Fourier Transform Infrared Spectrum (FITR) of ZnO nanoparticles

Antibacterial activity

The effectiveness of ZnO nanoparticles (ZnO NPs) against four bacteria (E. coli, S. aureus, and Bacillus cereus, S. enterica ser. typhi) was tested using the well diffusion method. Naturally bacterial cell walls, membranes and antibacterial resistance genes protect them against environmental threats and antimicrobial agents. This is a matter of concerns to control the bacterial infection. So, nanoparticles has a better option to control and inhibit the growth of bacteria. In current study synthesized ZnO NPs efficiently inhibit the bacterial growth. Table 2 and Fig. 5 shows how bacteria responded against ZnO NPs, with larger inhibition zones as ZnO NP concentration increased, which indicating the strong antibacterial properties. These green synthesized nanoparticles were effective against both Gram negative and positive bacteria. The zone of inhibition showed maximum in E. coli at 200 mg/mL is 25mm and against S. aureus is 27mm. As more (concentration) ZnO nanoparticles were used, they will show better inhibition properties, which indicates that they are good at fighting against bacteria. In Chennimalai's study (2021), Bacillus cereus, a Gram-positive bacterium, exhibited higher sensitivity to ZnO nanocrystals than Gram-negative pathogens²¹. Comparable results were observed in various studies where ZnO nanoparticles were synthesized using different plant extracts. The zone of inhibition observed in relation to E. coli and S. aureus in previous reports are about 22.00 mm and 12.35 mm respectively at 100 μg/mL²². Against E. coli, the zone of inhibition is 15 mm²³.

Zone of inhibition at different concentration in each bacteria followed by same alphabet do not differ significantly at 5% level by Duncan’s Multiple Range Test.

Fig. 5. Antibacterial activity of synthesized zinc oxide nanoparticles assessed against (a) Escherichia coli (b) Staphylococcus aureus (c) Salmonella enterica serotype typhi and (d) Bacillus cereus.

Adsorption of Chromium Cr (VI)

The adsorption of Cr depends on the surface area and availability of the adsorbent. A standard plot was taken in use to determine the unknown concentration of chromium using K₂Cr₂O₇. Initially, as the metal concentration increased from 14 to 56 mg/ml with the addition of ZnO NPs (Fig. 6), our synthesized ZnO NPs removed about 50% of chromium at 14 mg/ml (Fig. 6 and Fig. 7). At higher Cr (VI) concentrations, absorption is usually low.

To further enhance removal efficiency, optimizing the surface and size properties of nanoparticles of ZnO could be useful. Adding materials like activated carbon could also increase adsorption capacity. Modifying these factors can significantly enhance the effectiveness of ZnO nanoparticles in removing heavy metals and dyes from wastewater. Other researchers reported a 75% removal of chromium for synthesizing Fe₃O₄nanoparticles²⁴. According to Kumar et al. (2019), chitosan-based magnetic nanoparticles can remove 18.9% of chromium²⁵. On the other hand, Samrot et al. (2019) found that polymer-based iron oxide nanoparticles can achieved a chromium (VI) removal rate of 62-91%²⁶. Present investigation underscore the potential of optimized and modified ZnO NPs in effectively purifying contaminant wastewater.

Fig. 6. Chromium removal efficiency of FeO NPs

ANOVA: Adsoportion Concentration- F= 533.779, p≤0.001; Adsoportion Percentage- F= 11.405, p≤0 .003.

Bar and line individually followed by same alphabet do not differ significantly at 5% level by Duncan’s Multiple Range Test.

Fig. 7. Chromium adsorption assay (a) Before treatment, (b) After treatment with NPs, (c) Sample tested with 1-5 diphenylcarbazide

CONCLUSION:

Biosynthesis of metal nanoparticles is an eco-friendly method based on green chemistry principles, reducing harmful substance absorption on nanoparticle surfaces and avoiding harsh and toxic chemicals. This methodology is energy-efficient and produces biocompatible nanoparticles due to natural stabilizing and reducing agents. In the past decade, research has emphasized green synthesis employing plant extracts to control nanoparticle shape and size. Future research aims to scale up production, identify plant chemicals involved using bioinformatics, and understand how these nanoparticles inhibit harmful bacteria. Plant-based nanoparticles have potential applications in the food, pharmaceutical, and cosmetic industries. This study presented a simple, eco-friendly method for creating nanoparticles of ZnO using aqueous extracts from Aegle marmelos leaves. The shift in FTIR spectrum peaks suggested that plant metabolites acted as capping and stabilizing agents. The nanoparticles exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria, indicating their potential use as herbal medicine. Synthesized ZnO nanoparticles showed an effective result in the removal of chromium from contaminated water. So, ZnO NPs synthesized from aqueous leave extracts of Aegle marmelos have great potential in antibacterial activity and waste water treatment process of chromium metal removal.

ACKNOWLEDGEMENT

The authors acknowledge to School of Studies in Biotechnology, Pt. Ravishankar Shukla University Raipur, Chhattisgarh for providing facilities to conduct this study. Authors also acknowledge to Department of Biotechnology, National Institute of Technology, Raipur, Chhattisgarh for providing opportunity to conduct this research.

CONFLICT OF INTEREST:

Authors declare that they have no conflict of interest.

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Received on 13.06.2024 Modified on 08.08.2024

Research J. Pharm. and Tech 2024; 17(11):5417-5423.

DOI: 10.52711/0974-360X.2024.00828

Concentration of ZnO NPs Bacteria	Zone of inhibition is in mm.
Concentration of ZnO NPs Bacteria	50 mg/mL	100 mg/mL	150 mg/mL	200 mg/mL
Escherichia coli MTCC 2412	15.33± 1.76^b	16.66±0 .88^b	20.33± 0.88^ab	25.33± 3.17^a
Salmonella enterica ser. typhi MTCC 733	11.33± 0.88^c	13.00± 0.57^bc	14.33± 0.33^b	17.33± 0.66^a
Staphylococcus aureus MTCC 3160	22.33± 0.33^c	23.00± 1.00^bc	25.66± 1.20^ab	27.33± 2.33^a
Bacillus cereus McR3	15.66± 1.20^b	17.00± 0.57^b	17.33± 0.66^b	21.33± 0.33^a