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Article

Sustainable Fabrication of Zinc Oxide Nanoparticles Using Assam Green Tea Extract with Promising Oral Antimicrobial Potential

by
Ampa Jimtaisong
1,2,*,
Nisakorn Saewan
1,2 and
Nattakan Panyachariwat
1,2
1
School of Cosmetic Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
2
Cosmetic and Beauty Innovations for Sustainable Development (CBIS) Research Group, Mae Fah Luang University, Chiang Rai 57100, Thailand
*
Author to whom correspondence should be addressed.
Appl. Biosci. 2025, 4(3), 44; https://doi.org/10.3390/applbiosci4030044
Submission received: 9 July 2025 / Revised: 25 August 2025 / Accepted: 26 August 2025 / Published: 18 September 2025
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Abstract

In this study, zinc oxide nanoparticles (ZnO NPs) were synthesized via a green chemistry approach using aqueous extract of Camellia sinensis var. assamica (Assam green tea) as a bioreductant and stabilizing agent. Phytochemical analysis of the extract revealed high levels of phenolics (338.57 ± 3.90 mg GAE/mL) and flavonoids (123.92 ± 1.34 µg QE/mL), along with strong antioxidant and reducing activity, supporting its efficacy in nanoparticle formation. ZnO NPs were synthesized at various extract concentrations, with 25% yielding optimal characteristics based on UV–Vis spectrophotometry (λMax ≈ 390–410 nm). Structural characterization using XRD confirmed the hexagonal wurtzite phase, and SAXS indicated particle sizes of 58–60 nm. FE-SEM analysis showed semi-spherical agglomerated particles ranging from 74 to 76 nm, while EDX verified the elemental purity of Zn and O. FT-IR spectroscopy confirmed the presence of Zn–O stretching and phytochemical residues on the nanoparticle surface. Stability studies over four weeks revealed red shifts in absorbance and reduced peak intensity at ambient and elevated temperatures, suggesting nanoparticle agglomeration. Antimicrobial assays demonstrated strong antifungal activity of the ZnO NP solution against Candida albicans and, upon concentration, significant antibacterial activity against Streptococcus mutans. The synthesized ZnO NPs exhibit promising potential as eco-friendly antimicrobial agents, particularly for applications in oral healthcare.

Graphical Abstract

1. Introduction

Green synthesis of nanoparticles using plant extracts has attracted considerable interest as an eco-friendly alternative to conventional chemical methods, aiming to eliminate the use of toxic substances in the development of modern nanotechnology resources [1,2,3]. Among various metal oxide nanoparticles (NPs), titanium dioxide (TiO2) and zinc oxide (ZnO) are extensively studied due to their unique properties and broad range of applications, particularly in the pharmaceutical and cosmetic industries [3,4]. Particularly, ZnO nanoparticles (ZnO NPs) have gained significant attention for their antibacterial properties and biomedical applications, attributed to their good biocompatibility, low toxicity, and chemical and thermal stability [2,3]. The green synthesis of ZnO NPs is a bottom-up approach that employs biological entities such as plants, fungi, bacteria, and algae for nanoparticle production. These organisms act both as natural reducing agents for metal salt precursors and as stabilizers of the synthesized nanoparticles [3,5,6]. Nanosized ZnO particles have demonstrated antimicrobial activity against Streptococcus mutans (S. mutans) [7]. Moreover, they have shown efficacy against Candida albicans (C. albicans), a fungal species commonly isolated from teeth following endodontic retreatment and from root canals with persistent infections [8]. Moreover, numerous studies have also documented the use of ZnO NPs as antibacterial agents, noting that ZnO is a bio-safe material that is non-toxic to human cells [9]. Tea is arguably one of the most widely consumed beverages globally. The world’s major tea-producing countries include China, the largest producer, followed by India and Kenya. Other key producers are Sri Lanka, Vietnam, Indonesia, Japan, Turkey, Iran, and Argentina, each contributing significantly to global tea supply depending on the type and region [10]. The three primary types of tea—green, oolong, and black—are distinguished by their degree of fermentation. Green tea is unfermented, oolong tea is partially fermented, and black tea is fully fermented [11]. Tea leaves contain a wide range of chemical constituents, including carbohydrates, amino acids, proteins, alkaloids (such as caffeine, theophylline, and theobromine), volatile compounds, polyphenols, minerals, and trace elements. Among these, polyphenols—particularly flavonoids—play a significant role in the biological activities associated with tea. The predominant flavonoids in tea are flavan-3-ols (also known as flavanols or flavans), which are present in higher concentrations than in most other foods. Flavan-3-ols are classified based on their degree of polymerization, with the monomeric forms including catechins such as (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), (−)-gallocatechin (GC), and (+)-catechin (C) [12]. Among these, EGCG is the most extensively studied due to its wide range of health-promoting properties, including anti-inflammatory, antimicrobial, antitumor, antioxidant, cardioprotective, anti-obesity, and anti-aging effects [13]. Dried tea leaves—green, oolong, and black—contain different concentrations of bioactive compounds depending on their level of fermentation. Green tea, being unfermented, retains high levels of catechins such as EGCG; oolong tea, partially fermented, contains moderate amounts of catechins and theaflavins; while black tea, fully fermented, is rich in theaflavins and thearubigins but lower in catechins. These compounds contribute to the antioxidant and health-promoting properties of tea [14,15]. There are two major varieties of tea plants: Chinese tea (Camellia sinensis var. sinensis) and Assam tea (Camellia sinensis var. assamica). Green tea extract is widely recognized for its antioxidant, anti-inflammatory, and antimicrobial properties and is commonly used in cosmetic formulations. Additionally, it has been shown to contribute to oral health by preventing dental erosion, caries, and oral malodor [13,16]. Green tea mouth rinse has demonstrated comparable effectiveness to chlorhexidine in reducing S. mutans colony counts [17]. Interestingly, a separate study found that Assam black tea exhibited stronger biofilm inhibition against S. mutans compared to Chinese green tea [18]. Furthermore, green tea extract (Camellia sinensis var. sinensis) has been successfully employed in the green synthesis of ZnO NPs, which have shown antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Aspergillus niger [9].
Assam tea (Camellia sinensis var. assamica), known for its high polyphenol content, thus offers significant potential as a natural reducing agent for the green synthesis of ZnO NPs. Given the proven antimicrobial properties of both Assam tea and ZnO NPs against S. mutans, their combination presents a promising approach for the formulation of safe and effective oral care products. This study, thus, aims to systematically investigate the optimal synthesis parameters for ZnO NPs using Assam green tea (Camellia sinensis var. assamica) as a green reducing and stabilizing agent. To the best of our knowledge, this is the first report detailing the use of Assam tea extract for the green synthesis of ZnO NPs, thereby contributing novel insights into sustainable nanomaterial fabrication. The nanoparticles were characterized using UV–Visible spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) spectroscopy, Small-angle X-ray scattering (SAXS) analysis, and Field emission scanning electron microscopy (FE-SEM) [19]. Their inhibitory effects against the oral pathogens S. mutans and C. albicans were also evaluated.

2. Materials and Methods

2.1. Materials

The Assam green tea (Camellia sinensis var. assamica) samples were obtained from Chiang Rai Province, Thailand. The plant was identified and certified by comparison with the herbarium database of the Queen Sirikit Botanic Garden Herbarium, Thailand. The green tea used in this study was traditionally produced by the local community and subsequently employed for extraction. Zinc acetate dihydrate (Zn(CH3COO)2·2H2O) was purchased from Kemaus, Australia. DPPH (2,2′-diphenyl-1-picrylhydrazyl), Folin–Ciocalteu reagent, gallic acid, and L-ascorbic acid were purchased from Sigma-Aldrich, Burlington, MA, USA. Deionized water as the extraction solvent was of cosmetic grade.

2.2. Preparation of Assam Green Tea Extract

The Assam green tea was ground using an electrical grinder to obtain fine powder. The ground sample was then extracted in deionized water at a sample to solvent ratio of 1:6 w/v. The extracts were sonicated (240 W, 40 KHz, TUC100, ARI Medical, Hefei City, China) for 90 min at ambient temperatures (30–35 °C). After that, they were filtered to separate the residue and extract with filter paper. The extraction process was repeated twice, and the combined extract was used fresh for the green synthesis of ZnO NPs. The pH of the extract was measured using a pH meter.

2.3. Determination of Total Phenolic Content

The total phenolic content of the Assam green tea leaf extract was determined using the Folin–Ciocalteu colorimetric assay [20], with gallic acid as the standard. The extract solution was prepared at 10% w/v using deionized water as the solvent and added into the reaction mixture. To each sample, standard, or blank (20 μL), 1.58 mL of deionized water and 100 μL of Folin–Ciocalteu reagent were added. The mixture was incubated at room temperature for 5 min, followed by the addition of 300 μL of 10% (w/v) sodium carbonate solution. After further incubation for 90 min, the absorbance was measured at 765 nm using a visible spectrophotometer (Thermo Scientific™ GENESYS™ 30, Waltham, MA, USA). The total phenolic content was calculated from a standard calibration curve (R2 = 0.9995) and expressed as mg gallic acid equivalent (GAE)/mL extract. All the determinations were carried out in triplicate.

2.4. Determination of Total Flavonoid Content

The aluminum chloride colorimetric method was employed to determine the total flavonoid content of the Assam green tea extract [20]. The extract solution was prepared at 10% w/v using deionized water as solvent and added into the reaction mixture. Quercetin was used to construct the standard calibration curve. A volume of 1.2 mL of either standard quercetin solution or sample extract was mixed with 1.2 mL of 2% aluminum chloride solution. The mixtures were incubated at room temperature for 60 min. The absorbance was measured at 420 nm using a visible spectrophotometer (Thermo Scientific™ GENESYS™ 30, Waltham, MA, USA) against a blank. The total flavonoid content in the samples was calculated from the calibration curve (R2 = 0.9998) and expressed as µg quercetin equivalent (QE)/mL extract. All the determinations were carried out in triplicate.

2.5. DPPH Radicals Scavenging Assay

The scavenging activity of the extract was evaluated using DPPH radicals [20]. The extract solution was prepared at 0.01% w/v using deionized water as the solvent and added into the reaction mixture. Ascorbic acid was used as a standard to generate a calibration curve (R2 = 0.9998). A total of 3 mL of 0.1 mM DPPH solution in absolute ethanol was added to 1 mL of either the sample or standard. The mixtures were immediately vortexed at room temperature and then incubated at 37 °C for 30 min. The absorbance was measured at 517 nm using a spectrophotometer (Thermo Scientific™ GENESYS™ 30, Waltham, MA, USA). The DPPH radical scavenging capacity was reported as mg L-ascorbic acid equivalent (AAE)/mL extract. All the determinations were carried out in triplicate.

2.6. Determination of Reducing Power

The reducing power was determined following the previous methods [20], with modifications. The extract solution was prepared at 1% w/v using deionized water as the solvent and added into the reaction mixture. Ascorbic acid was used as a standard to generate a calibration curve (R2 = 0.9996). The Assam green tea extract solution 1 mL was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.66) and 2.5 mL of 1% potassium ferricyanide. The mixtures were incubated at 50 °C for 20 min; after that, 2.5 mL of 10% trichloroacetic acid was added. The mixture of 2.5 mL was mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride. The absorbance was measured at 700 nm. The reducing power was reported as mg L-ascorbic acid equivalent (AAE)/mL extract. All the determinations were carried out in triplicate.

2.7. Green Synthesis of Zinc Oxide Nanoparticles

Green synthesis of ZnO NPs was performed by using zinc acetate dihydrate as a zinc precursor following the previous method [21] with modifications. Zinc acetate dihydrate solution (0.2 M) 230 mL was freshly prepared and added to 100 mL of the freshly prepared Assam green tea extract (2.17 mL extract: mmol Zn2+). The reaction mixture was monitored for the formation of ZnO NPs by using a UV–Visible spectrophotometer in the 350–500 nm range. Previous studies suggested that ZnO NPs exhibit a characteristic broad absorption peak between 350 and 420 nm [2,6]. The effects of the Assam green tea extract concentration (5%, 10%, 25%, 50%, and 100% v/v) as reducing agent were studied. In addition, the effects of the reaction times (0.5 h, 1 h, 2 h, 3 h, and 4 h) and temperatures (ambient temperature and 80 °C) in green synthesis were also collected.

2.8. Characterization of Zinc Oxide Nanoparticles

The green tea extract-mediated ZnO NPs were dried in a hot air oven at 90 °C for 15 h, until they presented a thick consistency mixture due to water evaporation. The dried material was then ground into fine powder in a pestle and mortar and calcined at 600 °C for 1 h to remove any impurities [22]. The powder samples were then characterized via Fourier transform infrared (FT-IR) spectroscopy (PerkinElmer, Spectrum GX, CT, USA), X-ray diffraction (XRD) spectroscopy (Malvern PANalytical, X’Pert Pro MPD, Worcestershire, UK), Small-angle X-ray scattering (SAXS) analysis (Malvern PANalytical, X’Pert Pro MPD, Worcestershire, UK), and Field emission scanning electron microscopy (FE-SEM) (TESCAN, MIRA4, Kohoutovice, Czech Republic).

2.9. Stability of Zinc Oxide Nanoparticles

The stability of the Assam green tea leaf-mediated ZnO NPs solution (as prepared) was investigated at different conditions (ambient temperature, 4 °C, 45 °C) for 4 weeks. The presence of ZnO NPs was monitored using a UV–Vis spectrophotometer (Metertech, SP-8001, Taipei, Taiwan) at 350–420 nm [2,23].

2.10. Antimicrobial Activities of Zinc Oxide Nanoparticles

The antimicrobial activities of the Assam green tea–mediated ZnO nanoparticles were evaluated against S. mutans and C. albicans using the agar well diffusion method, following standard protocols with minor modifications [24,25]. S. mutans was cultured on Brain Heart Infusion (BHI) agar by minimizing the air in a plastic bag and then tightly closed, and C. albicans was cultured on Sabouraud Dextrose Agar (SDA) with loosely closed bags and incubated at 37 °C. Fresh overnight cultures were adjusted to 0.5 McFarland standard (~1.5 × 108 CFU/mL) using sterile saline and uniformly spread onto 90 mm agar plates. Six wells (5 mm diameter) were aseptically punched into each plate and loaded with 20 μL of Assam green tea–mediated ZnO nanoparticles solution at concentrations of 50% and 100% v/v. Negative controls (sterile distilled water), Assam green tea extract controls (25% v/v), and positive controls (0.01 mg erythromycin for S. mutans and 1 μg amphotericin B for C. albicans) were included [26]. Plates were incubated at 37 °C for 24 h, and the diameters of the zones of inhibition were measured in millimeters. The experiments were conducted in triplicate.

3. Results and Discussion

3.1. Preparation of Assam Green Tea Extract and Its Biological Properties

The aqueous extract of Assam green tea (Camellia sinensis var. assamica) was deep brown in color with a characteristic tea aroma. The extract had a pH of 5.09 ± 0.01. The bioactive compounds and biological activities of Assam green tea aqueous extract are tabulated in Table 1. The total phenolic and flavonoid contents were measured at 338.57 ± 3.90 mg GAE/mL extract and 123.92 ± 1.34 µg QE/mL extract, respectively. Polyphenolics and flavonoids are reported as important bioactives responsible for the biological activity in tea. The flavan-3-ols, e.g., catechins such as (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), and (−)-gallocatechin (GC) are the common flavonoids presented in relatively large amounts in tea [12,27,28]. The extract also exhibited strong antioxidant activity, demonstrated by a DPPH radical scavenging capacity of 10.47 ± 0.07 mg AAE/mL extract and a reducing power of 10.16 ± 0.01 mM mg AAE/mL extract. These results, expressed per milliliter of extract, are attributed to the high concentrations of phenolics and flavonoids, supporting its potential as a natural reducing agent in the green synthesis of ZnO NPs. Since the aqueous green tea extract was directly used in the biosynthesis, this approach enables a more accurate correlation between the extract’s phytochemical content and its effectiveness in nanoparticle synthesis.

3.2. Green Synthesis of Zinc Oxide Nanoparticles

3.2.1. Effects of Reducing Agent Concentration

The aqueous extract of Assam green tea was evaluated at varying concentrations (5%, 10%, 25%, 50%, and 100% v/v) to investigate its effect on the formation of ZnO NPs. Upon addition of the extract to a zinc acetate dihydrate solution, a pale-yellow coloration was observed, indicating the formation of ZnO NPs. The synthesis was further confirmed by UV–Visible spectrophotometry, which revealed characteristic absorption peaks in the range of 390–410 nm (Figure 1), corresponding to the surface plasmon resonance of ZnO NPs. The absorbance of the reaction mixture increased with rising concentrations of the reducing agent (Assam green tea extract), reaching a maximum at 25%, followed by a decline at concentrations of 50% and above. Assam green tea extract contains high levels of phenolics and flavonoids, which are known to act as reducing and stabilizing agents in nanoparticle biosynthesis. This composition may contribute to its potential as a natural reducing agent in the green synthesis of ZnO NPs [29].
Additionally, at higher extract concentrations, a red shift in the UV–Vis absorption peak was observed, suggesting the formation of larger ZnO nanoparticles. This shift is likely due to the higher levels of phytochemicals in the extract, which accelerate the reduction process, promote rapid nucleation, and enhance particle growth—ultimately producing larger particle sizes [30]. Similar trends have been reported in other plant-mediated syntheses. For example, ZnO NPs synthesized using pomegranate peel extract showed a size increase from 18.53 to 30.34 nm with higher extract concentrations, correlating with a red shift in UV–Vis spectra [31]. Likewise, a comparable spectral shift was observed when using Solanum nigrum leaf extract, which was attributed to nanoparticle agglomeration and growth [32]. The relationship between the particle size and optical properties is well established; larger ZnO NPs typically exhibit a shift toward longer wavelengths [33].

3.2.2. Effects of Reaction Times and Temperatures

Assam green tea extract at 25% concentration was selected to further investigate the effects of the reaction times (30 min, 1 h, 2 h, 3 h, 4 h) and temperatures (ambient temperature and 80 °C) on the green synthesis of ZnO NPs. The formation of pale-yellow zinc oxide (ZnO) nanoparticles was observed (Figure 2). The progress of the reaction was monitored using UV–Visible spectrophotometry (Figure 3). The absorption peak height increased with longer reaction times, indicating a higher concentration of nanoparticles. At a constant reducing agent concentration and reaction time, higher temperatures produced more intense absorption peaks, implying improved nanoparticle formation.
In the green synthesis of ZnO NPs, the reaction temperature and time are critical parameters that strongly influence the particle size, shape, crystallinity, and surface properties. Increasing the synthesis temperature generally promotes faster nucleation and crystal growth, often leading to smaller and more uniformly distributed nanoparticles when optimized; however, excessively high temperatures can induce aggregation and alter morphology [34,35]. The reaction time also plays a pivotal role, as prolonged durations may enhance crystallinity and particle stability but can cause overgrowth, resulting in larger particle sizes and possible shape transformations [36,37]. For instance, low-temperature short-time synthesis often yields quasi-spherical particles with moderate crystallinity, whereas higher temperatures or extended times can produce rod-like or hexagonal morphologies with improved crystal quality [38,39]. The interplay between these parameters must be carefully balanced, as they not only dictate the physicochemical characteristics of the ZnO NPs but also influence their photocatalytic, antimicrobial, and biological activities, which are closely tied to the particle size, surface area, and defect structure.

3.3. Characterization of Zinc Oxide Nanoparticles

The reaction mixture was poured into a clean petri plate, and the synthesized ZnO NPs were initially dried at 90 °C for 15 h to obtain a dry powder—a step commonly employed in nanoparticle preparation to remove moisture and facilitate processing (drying temperatures in the 80–100 °C range are typical)—and then calcined at 600 °C to enhance crystallinity and produce a structurally stable ZnO powder suitable for characterization [22]. The calcination temperature strongly influences the ZnO nanoparticles’ size, morphology, and crystallinity. Higher temperatures improve crystallinity but cause particle growth and aggregation [40,41]. Optimal calcination (400–600 °C) yields uniform well-crystallized NPs, while excessive heat (>700 °C) leads to agglomeration and morphology changes [42]. Controlling the temperature is key for tuning ZnO properties.

3.3.1. X-Ray Diffraction (XRD) Spectroscopy

XRD patterns of the synthesized ZnO NPs were analyzed in the range of 2 theta from 20° to 80° using a powder diffractometer. The X-ray diffraction pattern of zinc oxide nanoparticles showed definite line broadening of the X-ray diffraction peaks, indicating that the prepared particles were in the nanoscale range. The synthesized ZnO NPs showed diffraction peaks located at 31.77, 34.43, 36.27, 47.55, 56.59, 62.86, and 67.94°, which correspond to the reflection from (100), (002), (101), (102), (110), (103), and (112), respectively (Figure 4). The present diffraction peaks have been indexed as crystal planes of the hexagonal wurtzite structure of ZnO with high crystallinity. The finding showed good agreement with the other reports [43,44]. All characteristic peaks corresponded to ZnO NPs, indicating the absence of impurities in the synthesized sample. Moreover, the particle size was determined using Small-angle X-ray scattering (SAXS), in which measurements were made at very small angles, typically in the range of 0.1 degrees to 5 degrees. The average particle size of the zinc oxide nanoparticle powder is 58.74 nm at reaction at ambient temperature and 59.66 nm at reaction at 80 °C.

3.3.2. Field Emission Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-Ray (EDX) Analysis

The morphological study of the green-synthesized ZnO NPs was performed by FE-SEM (TESCAN, MIRA4, Czech Republic). The particles show a semispherical shape, and these particles are in a highly agglomerated form, as shown in Figure 5. They have various particle sizes ranging from 74.49 to 77.55 nm. The size increase was due to the overlapping of particles on each other. The particle sizes obtained from SAXS (58–60 nm) were smaller than those measured by FE-SEM. This discrepancy arises because SAXS measures nanoparticles in their dispersed state, providing the size of individual particles or small aggregates in suspension, whereas FE-SEM images represent dried samples in which particles often agglomerate due to solvent removal and the loss of stabilizing hydration layers [45,46].
Energy-dispersive X-ray spectroscopy (EDX) revealed a high signal for zinc and oxygen, which indicated the presence of zinc and oxygen at stoichiometric ratio [34]. Carbon and some other elements in the EDX spectra showed the presence of stabilizing agents, which originated from Assam green tea, as can be seen in Figure 6. It is noteworthy that a higher carbon (C) content was observed in the ZnO NPs green-synthesized at ambient temperature. This suggests an incomplete reaction and a greater amount of residual organic reducing agents remaining in the reaction mixture.

3.3.3. Fourier Transform Infrared (FT-IR) Spectroscopy

To classify functional groups in the aqueous Assam green tea extract and the synthesized ZnO NPs, FT-IR spectroscopy was performed, and the comparative spectrum in the range of 4000 to 400 cm−1 is shown in Figure 7. Assam green tea extract showed the fundamental mode of vibration at 3393 (O–H stretch), 2934, 1696 (C=O bend), 1631 (unsaturation bonds), 1450 (O–H bend), 1370 (CH3 bend), 1240 (C–O stretch), and 1068 cm−1. These characteristics involved the structure of flavonoids and phenolics namely O–H stretch, C=O bending vibrations, unsaturation bonds, phenol or tertiary alcohol (O–H bend), C–O–H deformation, and C–O stretching vibrations of aromatic ethers [47,48]. The synthesized ZnO NPs represent peaks at 3445, 2923, 2341, 1629, 1447, and 494 cm−1. The sharp peak at 494 cm−1 is attributed to the Zn–O stretching vibration, thus confirming the formation of ZnO nanoparticles [5,41]. The presence of those observed around 1629 cm−1 and 3445 cm−1, are often associated with water molecules adsorbed on the surface, specifically the O–H bending and stretching vibrations, and these peaks may also indicate the presence of a natural capping agent [49,50].

3.4. Stability Study of Zinc Oxide Nanoparticles

The stability of Assam green tea-mediated ZnO NPs solution (as prepared) was investigated at different conditions (ambient temperature, 4 °C, 45 °C) for 4 weeks. The presence of ZnO NPs was monitored using a UV–Vis spectrophotometer. The results showed a red shift in the absorption peak from approximately 399 nm at week 0 to 407 nm after 4 weeks. Moreover, the absorbance intensity decreased under ambient temperature and 45 °C storage conditions, Table 2. This suggests the nanoparticles had a little agglomeration, leading to an increase in the particle size and a reduction in the nanoparticle concentration across all storage conditions. However, the physical appearance of the Assam tea leaf-mediated green-synthesized ZnO NPs solution remained visually unchanged (Figure 8). It is noteworthy that the phenolics and flavonoids present in Assam green tea extract may also function as stabilizing agents, thereby reducing the extent of ZnO NPs’ agglomeration under various storage conditions [29,51].
Similar observations have been reported in previous studies involving green-synthesized ZnO NPs, where elevated temperatures induced aggregation and reduced the colloidal stability [21,41,43]. For instance, Irshad et al. [21] and Ramesh et al. [41] demonstrated comparable red shifts and intensity reductions during storage at room temperature and 45 °C. In contrast, refrigerated storage (4 °C) consistently preserved nanoparticle dispersion and optical properties, likely due to minimized Brownian motion and oxidative stress [21,43,44]. These findings reinforce the importance of low-temperature storage in maintaining the physicochemical integrity and functional performance of plant-mediated ZnO NPs formulations.
While UV–Vis spectroscopy provides a suitable preliminary evaluation of nanoparticle stability, more comprehensive analysis can be achieved through additional techniques such as zeta potential measurement, dynamic light scattering (DLS), and monitoring of pH changes over time [52,53]. Although not performed in the present study, this limitation is acknowledged and will be addressed in future work to strengthen the stability assessment.

3.5. Antimicrobial Activities of Zinc Oxide Nanoparticles

C. albicans and S. mutans represent two highly significant microorganisms frequently encountered in human health and disease. C. albicans is a ubiquitous dimorphic fungus, while S. mutans is a Gram-positive bacterium, both commonly residing as commensals within the human oral cavity and other anatomical sites, including the gastrointestinal tract and skin [54,55]. These two pathogens were selected to evaluate the potential application of green-synthesized ZnO NPs in oral care formulations, owing to their clinical relevance in dental and oral infections [56,57].
The Assam green tea-mediated ZnO NPs, in their as-prepared solution form, were evaluated for antimicrobial activity against S. mutans and C. albicans, using erythromycin and amphotericin B as positive controls, respectively. The zones of inhibition (mm) around the wells were measured and are presented in Table 3 and Figure 9. The as-prepared solution exhibited antimicrobial activity against C. albicans, with inhibition zones of 19.0 ± 0.0 mm and 11.5 ± 0.7 mm at 100% and 50% concentrations, respectively. However, no inhibition zone was observed against S. mutans. This lack of activity may be attributed to the low nanoparticle concentration in the solution. Therefore, the solution was concentrated by removing approximately 80% of the water using a rotary evaporator to obtain a more concentrated Assam tea leaf-mediated ZnO NPs solution. The concentrated solution demonstrated enhanced antimicrobial activity, as indicated by increased zones of inhibition against C. albicans and measurable inhibition zones against S. mutans, recorded at 12.0 ± 0.0 mm and 7.5 ± 0.7 mm for the 100% and 50% concentrations, respectively (Table 3). Assam green tea extract was a tested for comparison, and it showed no zone of inhibition. It has been reported that aqueous tea extracts produced mean inhibition zones of about 10.3–19.6 mm [58]. The lack of inhibition zone in our study can be due to the extraction method, extract concentration, and type of tea.
The antimicrobial efficacy of Assam green tea-mediated ZnO NPs against C. albicans and S. mutans in this study aligns well with earlier findings on ZnO NPs synthesized via green routes, as the non-concentrated ZnO NPs solution showed antifungal activity against C. albicans but lacked antibacterial effects against S. mutans. This scenario mirrors the outcomes in which green-synthesized ZnO NPs using plant extracts often display higher potency against fungal pathogens compared to certain Gram-positive bacteria [59]. Upon concentration of the ZnO NPs suspension, notable antibacterial activity emerged, and results are consistent with other studies reporting similar magnitudes of activity. For instance, ZnO NPs at 0.1 mg/µL produced a zone of inhibition of ~24 mm against S. mutans [60], while phyto-synthesized ZnO nanocomposites exhibited consistent ~9–16 mm zones against S. mutans [61]. The enhanced antimicrobial efficacy following concentration is likely due to the increased nanoparticle dosage and improved contact efficiency.
In this work, the as-prepared ZnO NPs suspension was concentrated by removing approximately 80% of the water using a rotary evaporator, yielding a concentrated Assam tea leaf-mediated ZnO NPs solution. This concentrated solution (20 µL) was directly loaded into the well for antimicrobial testing, with the tested concentrations presented in Table 2. Although the results cannot be directly compared with those reported in the literature due to differences in nanoparticle synthesis, concentration units, and assay conditions, they nonetheless indicate notable antimicrobial potential of the biosynthesized ZnO NPs. These findings suggest that the Assam tea-mediated synthesis route can produce ZnO NPs with promising inhibitory effects, supporting their possible application in antimicrobial formulations. Mechanistically, ZnO NPs are known to exert antimicrobial effects via multiple pathways: generation of reactive oxygen species (ROS), release of Zn2+ ions disrupting cellular processes, and disruption of microbial membranes through surface contact [9,62]. The presence of tea-derived organic capping agents may further aid in nanoparticle stability, dispersity, and membrane interaction, reinforcing the antimicrobial potency [63]. Phenolic compounds and flavonoids exert antimicrobial effects via diverse mechanisms, including disruption of cell membrane integrity, inhibition of essential enzymes, interference with nucleic acid synthesis, and suppression of biofilm formation [64,65,66]. Flavonoids such as quercetin, luteolin, and catechins often display higher efficacy than phenolic acids and can synergize with antibiotics, enhancing activity against resistant pathogens [67,68].

4. Conclusions

Assam green tea extracts effectively enabled the green synthesis of crystalline ZnO NPs with good stability and nanoscale size. The nanoparticles showed strong antifungal activity against Candida albicans and, upon concentration, antibacterial effects against Streptococcus mutans. This study highlights the potential of Assam green tea-mediated ZnO NPs as eco-friendly antimicrobial agents for biomedical applications.

Author Contributions

Conceptualization, A.J.; methodology, A.J.; validation, A.J., N.S. and N.P.; formal analysis, A.J.; investigation, A.J.; resources, A.J.; data curation, A.J.; writing—original draft preparation, A.J.; writing—review and editing, A.J., N.S. and N.P.; visualization, A.J.; supervision, A.J.; project administration, A.J.; funding acquisition, A.J., N.S. and N.P. All authors have read and agreed to the published version of the manuscript.

Funding

The research on ‘Sustainable Fabrication of Zinc Oxide Nanoparticles Using Assam Green Tea Extract with Promising Oral Antimicrobial Potential’ by Mae Fah Luang University has received funding support from the National Science, Research, and Innovation Fund (NSRF) (grant no. 672A02020).

Data Availability Statement

Data are unavailable due to privacy or ethical restrictions.

Acknowledgments

Mae Fah Luang University is acknowledged for providing space and facilities.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Shah, M.; Fawcett, D.; Sharma, S.; Tripathy, S.K.; Poinern, G.E.J. Green synthesis of metallic nanoparticles via biological entities. Materials 2015, 8, 7278–7308. [Google Scholar] [CrossRef] [PubMed]
  2. Mutukwa, D.; Taziwa, R.; Khotseng, L.E. A review of the green synthesis of ZnO nanoparticles utilising Southern African indigenous medicinal plants. Nanomaterials 2022, 12, 3456. [Google Scholar] [CrossRef] [PubMed]
  3. El-Saadony, M.T.; Fang, G.; Yan, S.; Alkafaas, S.S.; El Nasharty, M.A.; Khedr, S.A.; Hussien, A.M.; Ghosh, S.; Dladla, M.; Elkafas, S.S.; et al. Green synthesis of zinc oxide nanoparticles: Preparation, characterization, and biomedical applications—A review. Int. J. Nanomed. 2024, 19, 12889–12937. [Google Scholar] [CrossRef] [PubMed]
  4. Rathi, V.H.; Jeice, A.R. Green fabrication of titanium dioxide nanoparticles and their applications in photocatalytic dye degradation and microbial activities. Chem. Phys. Impact 2023, 6, 100197. [Google Scholar] [CrossRef]
  5. Basnet, P.; Inakhunbi Chanu, T.; Samanta, D.; Chatterjee, S. A review on bio-synthesized zinc oxide nanoparticles using plant extracts as reductants and stabilizing agents. J. Photochem. Photobiol. B 2018, 183, 201–221. [Google Scholar] [CrossRef]
  6. Villagrán, Z.; Anaya-Esparza, L.M.; Velázquez-Carriles, C.A.; Silva-Jara, J.M.; Ruvalcaba-Gómez, J.M.; Aurora-Vigo, E.F.; Rodríguez-Lafitte, E.; Rodríguez-Barajas, N.; Balderas-León, I.; Martínez-Esquivias, F. Plant-based extracts as reducing, capping, and stabilizing agents for the green synthesis of inorganic nanoparticles. Resources 2024, 13, 70. [Google Scholar] [CrossRef]
  7. Almoudi, M.M.; Hussein, A.S.; Abu Hassan, M.I.; Mohamad Zain, N. A systematic review on antibacterial activity of zinc against Streptococcus mutans. Saudi Dent. J. 2018, 30, 283–291. [Google Scholar] [CrossRef]
  8. Djearamane, S.; Xiu, L.-J.; Wong, L.-S.; Rajamani, R.; Bharathi, D.; Kayarohanam, S.; De Cruz, A.E.; Tey, L.-H.; Janakiraman, A.K.; Aminuzzaman, M.; et al. Antifungal properties of zinc oxide nanoparticles on Candida albicans. Coatings 2022, 12, 1864. [Google Scholar] [CrossRef]
  9. Sirelkhatim, A.; Mahmud, S.; Seeni, A.; Kaus, N.H.M.; Ann, L.C.; Bakhori, S.K.M.; Hasan, H.; Mohamad, D. Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Lett. 2015, 7, 219–242. [Google Scholar] [CrossRef]
  10. Food and Agriculture Organization of the United Nations (FAO). Global Tea Consumption and Production Driven by Robust Demand in China and India. 2023. Available online: https://www.fao.org/newsroom/detail/global-tea-consumption-and-production-driven-by-robust-demand-in-china-and-india/en (accessed on 12 August 2025).
  11. Bancirova, M. Comparison of the antioxidant capacity and the antimicrobial activity of black and green tea. Food Res. Int. 2010, 43, 1379–1382. [Google Scholar] [CrossRef]
  12. Cabrera, C.; Giménez, R.; López, M.C. Determination of tea components with antioxidant activity. J. Agric. Food Chem. 2003, 51, 4427–4435. [Google Scholar] [CrossRef]
  13. Bansal, S.; Choudhary, S.; Sharma, M.; Kumar, S.S.; Lohan, S.; Bhardwaj, V.; Syan, N.; Jyoti, S. Tea: A native source of antimicrobial agents. Food Res. Int. 2013, 53, 568–584. [Google Scholar] [CrossRef]
  14. Vuong, Q.V.; Stathopoulos, C.E.; Golding, J.B.; Nguyen, M.H.; Roach, P.D. Optimum conditions for the water extraction of L-theanine from green tea. J. Sep. Sci. 2011, 34, 2468–2474. [Google Scholar] [CrossRef] [PubMed]
  15. Khan, N.; Mukhtar, H. Tea and health: Studies in humans. Curr. Pharm. Des. 2013, 19, 6141–6147. [Google Scholar] [CrossRef] [PubMed]
  16. Kawarai, T.; Narisawa, N.; Yoneda, S.; Tsutsumi, Y.; Ishikawa, J.; Hoshino, Y.; Senpuku, H. Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea. Arch. Oral Biol. 2016, 68, 73–82. [Google Scholar] [CrossRef]
  17. Neturi, R.S.; Sirisha, R.; Vemuri, S.B.; Siva, S.Y.; Sushma, C.T.; Kumar, P.S. Effects of green tea on Streptococcus mutans counts—A randomised control trial. J. Clin. Diagn. Res. 2014, 8, ZC128–ZC130. [Google Scholar] [CrossRef]
  18. Subramaniam, P.; Eswara, U.; Maheshwar Reddy, K.R. Effect of different types of tea on Streptococcus mutans: An in vitro study. Indian J. Dent. Res. 2012, 23, 43–48. [Google Scholar] [CrossRef]
  19. Gharpure, S.; Yadwade, R.; Ankamwar, B. Non-antimicrobial and non-anticancer properties of ZnO nanoparticles biosynthesized using different plant parts of Bixa orellana. ACS Omega 2022, 7, 1914–1933. [Google Scholar] [CrossRef]
  20. Panyachariwat, N.; Jimtaisong, A.; Saewan, N. Antioxidative potentials of Eleutherine bulbosa bulb and its utilization in topical cosmetic emulsion. Cosmetics 2024, 11, 111. [Google Scholar] [CrossRef]
  21. Irshad, S.; Salamat, A.; Anjum, A.A.; Sana, S.; Saleem, R.S.; Naheed, A.; Iqbal, A. Green tea leaves mediated ZnO nanoparticles and its antimicrobial activity. Cogent Chem. 2018, 4, 1469207. [Google Scholar] [CrossRef]
  22. Kołodziejczak-Radzimska, A.; Jesionowski, T. Zinc oxide—From synthesis to application: A review. Materials 2014, 7, 2833–2881. [Google Scholar] [CrossRef] [PubMed]
  23. Hanif, M.A.; Lee, I.; Akter, J.; Islam, M.A.; Zahid, A.A.S.M.; Sapkota, K.P.; Hahn, J.R. Enhanced photocatalytic and antibacterial performance of ZnO nanoparticles prepared by an efficient thermolysis method. Catalysts 2019, 9, 608. [Google Scholar] [CrossRef]
  24. Bauer, A.W.; Kirby, W.M.; Sherris, J.C.; Turck, M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 1966, 45, 493–496. [Google Scholar] [CrossRef]
  25. Naeini, A.; Khonsravi, A.R.; Chitsaz, M.; Shokri, H.; Kamlnejad, M. Anti-Candida albicans activity of some Iranian plants used in traditional medicine. J. Mycol. Med. 2009, 19, 168–172. [Google Scholar] [CrossRef]
  26. Velázquez-Hernández, A.M.; Martínez-Gallegos, S.; Albiter, V.; González-Juárez, J.C.; García-Ibarra, C. Synthesis of an antimicrobial chitosan film impregnated with ZnO nanoparticles phytosynthesized with Ruta graveolens plant extract. Microb. Pathog. 2025, 200, 107268. [Google Scholar] [CrossRef]
  27. Singh, K.; Srichairatanakool, S.; Chewonarin, T.; Brennan, C.S.; Brennan, M.A.; Klangpetch, W.; Utama-ang, N. Manipulation of the phenolic quality of Assam green tea through thermal regulation and utilization of microwave and ultrasonic extraction techniques. Horticulturae 2022, 8, 338. [Google Scholar] [CrossRef]
  28. Khanongnuch, C.; Unban, K.; Kanpiengjai, A.; Saenjum, C. Recent research advances and ethno-botanical history of Miang, a traditional fermented tea (Camellia sinensis var. assamica) of Northern Thailand. J. Ethn. Foods 2017, 4, 135–141. [Google Scholar] [CrossRef]
  29. Iravani, S. Green synthesis of metal nanoparticles using plants. Green Chem. 2011, 13, 2638–2650. [Google Scholar] [CrossRef]
  30. Bekele, B.; Jule, L.T.; Saka, A. The effects of annealing temperature on size, shape, structure and optical properties of synthesized zinc oxide nanoparticles by sol-gel methods. Dig. J. Nanomater. Biostruct. 2021, 16, 471–478. [Google Scholar] [CrossRef]
  31. Al-Nehia, A.; Al-Odayni, A.-B.; Al-Sharabi, A.; Al-Hammadi, A.; Saeed, W. Pomegranate peel extract-mediated green synthesis of ZnO-NPs: Extract concentration-dependent structure, optical, and antibacterial activity. J. Chem. 2022, 2022, 9647793. [Google Scholar] [CrossRef]
  32. Muthuvel, A.; Jothibas, M.; Manoharan, C. Effect of chemically synthesis compared to biosynthesized ZnO-NPs using Solanum nigrum leaf extract and their photocatalytic, antibacterial and in-vitro antioxidant activity. J. Environ. Chem. Eng. 2020, 8, 103705. [Google Scholar] [CrossRef]
  33. Goh, E.G.; Xu, X.; McCormick, P.G. Effect of particle size on the UV absorbance of zinc oxide nanoparticles. Scr. Mater. 2014, 78–79, 49–52. [Google Scholar] [CrossRef]
  34. Bala, N.; Saha, S.; Chakraborty, M.; Maiti, M.; Das, S.; Basu, R.; Nandy, P. Green synthesis of zinc oxide nanoparticles using Hibiscus sabdariffa leaf extract: Effect of temperature on synthesis, antibacterial activity and anti-diabetic activity. RSC Adv. 2015, 5, 4993–5003. [Google Scholar] [CrossRef]
  35. Basri, H.H.; Talib, R.A.; Sukor, R.; Othman, S.H.; Arffin, H. Effect of synthesis temperature on the size of ZnO nanoparticles derived from pineapple peel extract and antibacterial activity of ZnO–starch nanocomposite films. Nanomaterials 2020, 10, 1061. [Google Scholar] [CrossRef]
  36. Sánchez-Pérez, D.M.; Flores-Loyola, E.; Márquez-Guerrero, S.Y.; Galindo-Guzman, M.; Marszalek, J.E. Green synthesis and characterization of ZnO nanoparticles using Larrea tridentata extract and their impact on the in-vitro germination and seedling growth of Capsicum annuum. Sustainability 2023, 15, 3080. [Google Scholar] [CrossRef]
  37. Batterjee, M.G.; Nabi, A.; Kamli, M.R.; Alzahrani, K.A.; Danish, E.Y.; Malik, M.A. Green hydrothermal synthesis of ZnO nanoparticles for UV-light-induced photocatalytic degradation of ciprofloxacin antibiotic in an aqueous environment. Catalysts 2022, 12, 1347. [Google Scholar] [CrossRef]
  38. Faisal, S.; Jan, H.; Shah, S.A.; Shah, S.; Khan, A.; Akbar, M.T.; Rizwan, M.; Jan, F.; Wajidullah; Akhtar, N.; et al. Green synthesis of ZnO nanoparticles using aqueous fruit extracts of Myristica fragrans: Their characterizations and biological and environmental applications. ACS Omega 2021, 6, 9709–9722. [Google Scholar] [CrossRef]
  39. Swain, M.; Mishra, D.; Sahoo, G. A review on green synthesis of ZnO nanoparticles. Discov. Appl. Sci. 2025, 7, 997. [Google Scholar] [CrossRef]
  40. Ismail, A.M.; Menazea, A.A.; Kabary, H.A.; El-Sherbiny, A.E.; Samy, A. The influence of calcination temperature on structural and antimicrobial characteristics of zinc oxide nanoparticles synthesized by sol-gel method. J. Mol. Struct. 2019, 1196, 332–337. [Google Scholar] [CrossRef]
  41. Ramesh, M.; Anbuvannan, M.; Viruthagiri, G. Green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract and their antibacterial activity. Spectrochim. Acta Part A 2015, 136, 864–870. [Google Scholar] [CrossRef]
  42. Zahra, S.; Qaisa, S.; Sheikh, A.; Bukhari, H.; Amin, C.A. Effect of calcination temperature on the structure and morphology of zinc oxide nanoparticles synthesized by base-catalyzed aqueous sol-gel process. Eur. J. Chem. 2022, 13, 162–167. [Google Scholar] [CrossRef]
  43. Sangeetha, G.; Rajeshwari, S.; Venckatesh, R. Green synthesis of zinc oxide nanoparticles by Aloe barbadensis Miller leaf extract: Structure and optical properties. Mater. Res. Bull. 2011, 46, 2560–2566. [Google Scholar] [CrossRef]
  44. Jayachandran, A.; Aswathy, T.R.; Nair, A.S. Green synthesis and characterization of zinc oxide nanoparticles using Cayratia pedata leaf extract. Biochem. Biophys. Rep. 2021, 26, 100995. [Google Scholar] [CrossRef] [PubMed]
  45. Appel, C.; Kuttich, B.; Kraus, T.; Stühn, B. In-situ investigation of temperature induced agglomeration in non-polar magnetic nanoparticle dispersions by small angle X-ray scattering. Nanoscale 2021, 13, 6916–6920. [Google Scholar] [CrossRef]
  46. Honecker, D.; Bersweiler, M.; Erokhin, S.; Berkov, D.; Chesnel, K.; Venero, D.A.; Qdemat, A.; Disch, S.; Jochum, J.K.; Michels, A.; et al. Using small-angle scattering to guide functional magnetic nanoparticle design. Nanoscale Adv. 2022, 4, 1026–1059. [Google Scholar] [CrossRef]
  47. Nunes Oliveira, R.; Cordeiro Mancini, M.; Oliveira, F.; Passos, T.; Quilty, B.; Thiré, R.; McGuinness, G. FTIR analysis and quantification of phenols and flavonoids of five commercially available plant extracts used in wound healing. Rev. Mater. 2016, 21, 767–779. [Google Scholar] [CrossRef]
  48. Thummajitsakul, S.; Silprasit, K. Analysis of FTIR spectra, flavonoid content and anti-tyrosinase activity of extracts and lotion from Garcinia schomburgkiana by multivariate method. Trends Sci. 2022, 19, 5780. [Google Scholar] [CrossRef]
  49. Hoseinpour, V.; Souri, M.; Ghaemi, N.; Shakeri, A. Optimization of green synthesis of ZnO nanoparticles by Dittrichia graveolens (L.) aqueous extract. Health Biotechnol. Biopharma 2017, 1, 39–49. [Google Scholar] [CrossRef]
  50. Elumalai, K.; Velmurugan, S.; Ravi, S.; Kathiravan, V.; Adaikala Raj, G. Bio-approach: Plant mediated synthesis of ZnO nanoparticles and their catalytic reduction of methylene blue and antimicrobial activity. Adv. Powder Technol. 2015, 26, 1639–1651. [Google Scholar] [CrossRef]
  51. Ahmed, S.; Ahmad, M.; Swami, B.L.; Ikram, S. A review on plant extract mediated synthesis of silver nanoparticles for antimicrobial applications: A green expertise. J. Adv. Res. 2016, 7, 17–28. [Google Scholar] [CrossRef]
  52. Ray, T.R.; Lettiere, B.; de Rutte, J.; Pennathur, S. Quantitative characterization of the colloidal stability of metallic nanoparticles using UV–vis absorbance spectroscopy. Langmuir 2015, 31, 3577–3586. [Google Scholar] [CrossRef]
  53. Tomaszewska, E.; Soliwoda, K.; Kadziola, K.; Tkacz-Szczesna, B.; Celichowski, G.; Cichomski, M.; Szmaja, W.; Grobelny, J. Detection limits of DLS and UV-vis spectroscopy in characterization of polydisperse nanoparticle colloids. J. Nanomater. 2013, 2013, 313081. [Google Scholar] [CrossRef]
  54. Macias-Paz, I.U.; Pérez-Hernández, S.; Tavera-Tapia, A.; Luna-Arias, J.P.; Guerra-Cárdenas, J.E.; Reyna-Beltrán, E. Candida albicans, the main opportunistic pathogenic fungus in humans. Rev. Argent. Microbiol. 2023, 55, 189–198. [Google Scholar] [CrossRef] [PubMed]
  55. Lemos, J.A.; Palmer, S.R.; Zeng, L.; Wen, Z.T.; Kajfasz, J.K.; Freires, I.A.; Abranches, J.; Brady, L.J. The biology of Streptococcus mutans. Microbiol. Spectr. 2019, 7. [Google Scholar] [CrossRef] [PubMed]
  56. Krzyściak, W.; Jurczak, A.; Kościelniak, D.; Bystrowska, B.; Skalniak, A. The virulence of Streptococcus mutans and the ability to form biofilms. Eur. J. Clin. Microbiol. Infect. Dis. 2014, 33, 499–515. [Google Scholar] [CrossRef]
  57. Kashyap, B.; Padala, S.R.; Kaur, G.; Kullaa, A. Candida albicans induces oral microbial dysbiosis and promotes oral diseases. Microorganisms 2024, 12, 2138. [Google Scholar] [CrossRef]
  58. George, D.E.; Shetty, R.; Shetty, P.J.; Gomes, L.A. An in vitro study to compare the effect of different types of tea with chlorhexidine on Streptococcus mutans. J. Clin. Diagn. Res. 2017, 11, ZC05–ZC07. [Google Scholar] [CrossRef]
  59. Varghese, R.M.; Kumar, A.; Shanmugam, R. Antimicrobial activity of zinc oxide nanoparticles synthesized using Ocimum tenuiflorum and Ocimum gratissimum herbal formulation against oral pathogens. Cureus 2024, 16, e53562. [Google Scholar] [CrossRef]
  60. Barma, M.D.; Muthupandiyan, I.; Samuel, S.R.; Amaechi, B.T. Inhibition of Streptococcus mutans, antioxidant property and cytotoxicity of novel nano-zinc oxide varnish. Arch. Oral Biol. 2021, 126, 105132. [Google Scholar] [CrossRef]
  61. Harshitha, V.R.S.; Ilangovar, I.G.K.; Suresh, V.; Pitchiah, S. Synthesis of zinc oxide nanoparticles from Cymodocea serrulata leaf extract and their biological activities. Cureus 2024, 16, e55521. [Google Scholar] [CrossRef]
  62. Caron, A.J.; Ali, I.J.; Delgado, M.J.; Johnson, D.; Reeks, J.M.; Strzhemechny, Y.M.; McGillivray, S.M. Zinc oxide nanoparticles mediate bacterial toxicity in Mueller-Hinton broth via Zn2+. Front. Microbiol. 2024, 15, 1394078. [Google Scholar] [CrossRef]
  63. El-Fallal, A.A.; Elfayoumy, R.A.; El-Zahed, M.M. Antibacterial activity of biosynthesized zinc oxide nanoparticles using Kombucha extract. SN Appl. Sci. 2023, 5, 332. [Google Scholar] [CrossRef]
  64. Cushnie, T.P.T.; Lamb, A.J. Recent advances in understanding the antibacterial properties of flavonoids. Int. J. Antimicrob. Agents 2011, 38, 99–107. [Google Scholar] [CrossRef]
  65. Daglia, M. Polyphenols as antimicrobial agents. Curr. Opin. Biotechnol. 2012, 23, 174–181. [Google Scholar] [CrossRef] [PubMed]
  66. Xie, Y.; Yang, W.; Tang, F.; Chen, X.; Ren, L. Antibacterial activities of flavonoids: Structure–activity relationship and mechanism. Curr. Med. Chem. 2015, 22, 132–149. [Google Scholar] [CrossRef]
  67. Stapleton, P.D.; Shah, S.; Anderson, J.C.; Hara, Y.; Hamilton-Miller, J.M.T.; Taylor, P.W. Modulation of β-lactam resistance in Staphylococcus aureus by catechins and gallates. Int. J. Antimicrob. Agents 2004, 23, 462–467. [Google Scholar] [CrossRef]
  68. Xu, S.; Kang, A.; Tian, Y.; Li, X.; Qin, S.; Yang, R.; Guo, Y. Plant flavonoids with antimicrobial activity against methicillin-resistant staphylococcus aureus (MRSA). ACS Infect. Dis. 2024, 10, 3086–3097. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Absorption spectra of ZnO NPs using Assam green tea as reducing agent at different concentrations.
Figure 1. Absorption spectra of ZnO NPs using Assam green tea as reducing agent at different concentrations.
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Figure 2. The appearance of the reaction mixture in the green synthesis of ZnO NPs at different times and temperatures.
Figure 2. The appearance of the reaction mixture in the green synthesis of ZnO NPs at different times and temperatures.
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Figure 3. Absorption spectra of reaction mixture in green synthesis of ZnO NPs at different times and temperatures.
Figure 3. Absorption spectra of reaction mixture in green synthesis of ZnO NPs at different times and temperatures.
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Figure 4. X-ray diffraction pattern of green-synthesized ZnO NPs at (a) ambient temperature and (b) 80 °C.
Figure 4. X-ray diffraction pattern of green-synthesized ZnO NPs at (a) ambient temperature and (b) 80 °C.
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Figure 5. Field emission scanning electron microscope (FE-SEM) images of green-synthesized ZnO NPs and its corresponding particle size distribution curve at (a) ambient temperature and (b) 80 °C.
Figure 5. Field emission scanning electron microscope (FE-SEM) images of green-synthesized ZnO NPs and its corresponding particle size distribution curve at (a) ambient temperature and (b) 80 °C.
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Figure 6. EDX analysis of green-synthesized ZnO NPs at (a) ambient temperature and (b) 80 °C.
Figure 6. EDX analysis of green-synthesized ZnO NPs at (a) ambient temperature and (b) 80 °C.
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Figure 7. FT-IR spectra of (a) Assam green tea extract and (b) green-synthesized ZnO NPs.
Figure 7. FT-IR spectra of (a) Assam green tea extract and (b) green-synthesized ZnO NPs.
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Figure 8. UV–Vis Absorption spectra of Assam tea leaf-mediated green-synthesized ZnO NPs solution and its physical appearance after 1 month of storage under different conditions.
Figure 8. UV–Vis Absorption spectra of Assam tea leaf-mediated green-synthesized ZnO NPs solution and its physical appearance after 1 month of storage under different conditions.
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Figure 9. Antimicrobial activities of green-synthesized ZnO NPs against C. albicans and S. mutan, where (Aa,Ba) Assam green tea-mediated ZnO NPs (100%), (Ab,Bb) Assam green tea-mediated ZnO NPs (50%), (Ac,Bc) concentrated Assam green tea-mediated ZnO NPs (100%), and (Ad,Bd) concentrated Assam green tea-mediated ZnO NPs (50%).
Figure 9. Antimicrobial activities of green-synthesized ZnO NPs against C. albicans and S. mutan, where (Aa,Ba) Assam green tea-mediated ZnO NPs (100%), (Ab,Bb) Assam green tea-mediated ZnO NPs (50%), (Ac,Bc) concentrated Assam green tea-mediated ZnO NPs (100%), and (Ad,Bd) concentrated Assam green tea-mediated ZnO NPs (50%).
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Table 1. Bioactive compounds and biological activities of Assam green tea aqueous extract.
Table 1. Bioactive compounds and biological activities of Assam green tea aqueous extract.
Bioactive Compounds and Biological ActivitiesResults
Total phenolic content (mgGAE/mL extract)338.57 ± 3.90
Total flavonoids (µgQE/mL extract)123.92 ± 1.34
DPPH (mgAAE/mL extract)10.47 ± 0.07
Reducing power (mgAAE/mL extract)10.16 ± 0.01
Table 2. λMax and absorbance values of Assam green tea-mediated ZnO NPs solution after 1 month of storage under different conditions.
Table 2. λMax and absorbance values of Assam green tea-mediated ZnO NPs solution after 1 month of storage under different conditions.
ConditionλMax (nm)Absorbance
Week 03990.859
Week 4 at 4 °C4070.851
Week 4 at ambient temperature4070.822
Week 4 at 45 °C4070.781
Table 3. Zone of inhibition of the synthesized ZnO NPs against C. albicans and S. mutans.
Table 3. Zone of inhibition of the synthesized ZnO NPs against C. albicans and S. mutans.
Sample TypeZone of Inhibition (mm)
C. albicansS. mutans
Assam green tea-mediated ZnO NPs (100%)19.0 ± 0.00
Assam green tea-mediated ZnO NPs (50%)11.5 ± 0.70
Concentrated Assam green tea-mediated ZnO NPs (100%)27.0 ± 0.012.0 ± 0.0
Concentrated Assam green tea-mediated ZnO NPs (50%)21.5 ± 0.77.5 ± 0.7
Assam green tea extract00
Amphotericin B (1 µg)14.7 ± 0.6-
Erythromycin (0.01 mg)-29.3 ± 0.6
Water00
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Jimtaisong, A.; Saewan, N.; Panyachariwat, N. Sustainable Fabrication of Zinc Oxide Nanoparticles Using Assam Green Tea Extract with Promising Oral Antimicrobial Potential. Appl. Biosci. 2025, 4, 44. https://doi.org/10.3390/applbiosci4030044

AMA Style

Jimtaisong A, Saewan N, Panyachariwat N. Sustainable Fabrication of Zinc Oxide Nanoparticles Using Assam Green Tea Extract with Promising Oral Antimicrobial Potential. Applied Biosciences. 2025; 4(3):44. https://doi.org/10.3390/applbiosci4030044

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Jimtaisong, Ampa, Nisakorn Saewan, and Nattakan Panyachariwat. 2025. "Sustainable Fabrication of Zinc Oxide Nanoparticles Using Assam Green Tea Extract with Promising Oral Antimicrobial Potential" Applied Biosciences 4, no. 3: 44. https://doi.org/10.3390/applbiosci4030044

APA Style

Jimtaisong, A., Saewan, N., & Panyachariwat, N. (2025). Sustainable Fabrication of Zinc Oxide Nanoparticles Using Assam Green Tea Extract with Promising Oral Antimicrobial Potential. Applied Biosciences, 4(3), 44. https://doi.org/10.3390/applbiosci4030044

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