Abstract Synthesis of silver nanoparticles (SNP) using edible plants, namely Curcuma mangga Valeton & Zijp (P1), Momordica charantia L. (P2), Persicaria odorata (Lour.) Soják (P3), Litsea elliptica Blume (P4) and Gnetum gnemon Linn. var. tenerum Markgr. (P5), is considerably economical and safe. Plants and preparation methods can affect properties and bioactivity of SNP. The controlled temperature is suggested for synthesis. The small and spherical SNP obtained when biosynthesized at 37°C were evident. This study aimed to assess the ability of aqueous extracts (P1-P5) to form SNP at 37°C. The SNP were characterized and determined for antibacterial activity against Staphylococcus aureus ATCC 25923 (SA), Escherichia coli ATCC 25922 (EC) and Pseudomonas aeruginosa DMST 4739 (PA) and antifungal activity against Candida albicans DMST 5815 (CA) and Candida tropicalis DMST 15495 (CT). For the results, SNP formed using P1, P2 and P3 extracts had absorbance peak of 434 ± 9 nm. The SNP synthesized with P1 extract (P1-SNP) showed smallest particle sizes of 37 ± 0.92 nm. P1-SNP also showed the least in minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The same values of MIC and MBC of P1-SNP against SA, EC and PA were 6.25, 1.56 and 0.19 mg/mL, respectively. While the least in MIC and minimum fungicidal concentration (MFC) against CA and CT (12.5 and 1.56 mg/mL for both MIC and MFC) were from SNP synthesized using P3 extract. Summarily, plant-assisted antimicrobial SNP could be further utilized in pharmaceutical and medical applications dealt with antibacterial and antifungal functions.

Keywords: Silver nanoparticles, Synthesis, Antibacterial, Antimicrobial, Antifungal

Funding: The authors are grateful for the research funding provided by the Government Science and Technology Scholarship, National Science and Technology Development Agency (NSTDA).

Citation: Pradabsang, C., Sukrong, S. and  Warisnoicharoen, W. 2025. Green synthesis of antimicrobial silver nanoparticles using edible plants. Natural and Life Sciences Communications. 24(3): e2025055.

INTRODUCTION

Currently, silver nanoparticles (SNP) are extensively studied for biomedical and other applications, especially for antimicrobial purposes (Bruna et al., 2021). The ability of SNP on antimicrobial activity arrives from many mechanistic actions such as inhibition of protein synthesis, DNA damage and a breakdown of cell membrane (Fahim et al., 2024). Importantly, SNP are able to conquer the growth of antibiotic-resistant bacteria promoting them to be used in combination with antibiotic drugs for treatment (Hadi et al., 2024; Khairnar et al., 2025). Additionally, SNP can be in parts in medical devices for antimicrobial purposes such as face masks and sanitizer sprays (Kim et al., 2015; Balasubramaniam et al., 2021).

The synthesis of SNP can be achieved in several ways including physical and chemical methods (Aiad et al., 2017; Almatroudi, 2020; Ashraf et al., 2020; Widikdo et al., 2023). However, the use of natural substances for SNP synthesis is preferable in terms of friendly environment, less cost and safe. Extracts from plants consist of many phytochemicals, for example phenolic acids, flavonoids, tannin and alkaloids. The functional groups (^–OH, ^–HC=O, ^–NH₂, ^–SH) from these secondary metabolites might help reduction process of silver ion (Ag⁺) to Ag° atom and stabilize SNP (Nguyen et al., 2023). In addition, active compounds used in green synthesis of SNP promote some beneficial effects including antimicrobial, antidiabetic, anticancer and antioxidant activities (Li et al., 2024). The phytochemicals in other bioresources like seaweed also encourage the stability and bioactivity of nanoparticle system (Saravanan et al., 2025).

Edible plants are still attractive for use as a natural source for SNP synthesis. Some edible plants available in Thailand with known antimicrobial and other biological activities are Curcuma mangga Valeton & Zijp (or mango turmeric) (Pujimulyani et al., 2013; Noor et al., 2020), Momordica charantia L. (or bitter melon) (Bao et al., 2013; Pham et al., 2019), Persicaria odorata (Lour.) Soják (or Vietnamese mint) (Ridzuan et al., 2013; Sim et al., 2019), Litsea elliptica Blume (or Thammang) (Suksamerkun et al., 2013; Thongthip et al., 2017) and Gnetum gnemon Linn. var. tenerum Markgr. (or Liang) (Sae-Leaw and Benjakul, 2019; Anisong et al., 2022). The active compounds found in these plants such as alkaloids, flavonoids and phenolic compounds and their derivatives (Indis and Kurniawan, 2016; Rashid et al., 2017; Hadi et al., 2024) would be responsible not only for bioactivity but also the formation of SNP.

Although the plant-assisted synthesis of SNP was widely reported, the preparation methods seemed to be factors affecting the properties and thus biological activities of the SNP (Nahar et al., 2015; Yaqoob et al., 2020). Most of synthetic methods of SNP using plant extracts in the literature were detailed mainly using room temperature (~25°C) (Zhang et al., 2013; Poadang et al., 2017; Erci et al., 2018; Onitsuka et al., 2019; Sukkha et al., 2023). In fact, room temperature itself can vary and fluctuate during timing period, which could affect the formation of SNP. The use of controlled temperature which is not very high (i.e. 37°C) could be preferable for SNP biosynthesis (Almatroudi, 2020). The temperature at 37°C is considered not to cause any degradation of heat-labile compounds in plants. The biosynthesis of SNP using plant extract at temperature of nearly 40°C was proposed to have rapid reduction of Ag+ ions (Stavinskaya et al., 2019). In addition, the SNP produced at 37°C were reported for being smaller and more spherical nanoparticles than those produced at 50°C (Anbu et al., 2019). An increase in temperature causes rapid nucleation growth and aggregation resulting in nanoparticles with larger and less uniformity.

At present, SNP formed using aqueous extracts of P. odorata, L. elliptica and G. gnemon have not been reported yet. The synthesis of antimicrobial SNP from extracts of C. mangga and M. charantia was done mainly at room temperature (Joshi et al., 2017; Hadi et al., 2024). This study was aimed at synthesizing SNP using aqueous extracts of 5 edible plants mentioned earlier at 37°C. The SNP were characterized for morphology, size, size distribution and crystallinity. The synthesized SNP were then determined for antimicrobial ability against some bacterial and fungal strains.

MATERIAL AND METHODS

Green synthesis of silver nanoparticles

Material for nanoparticle synthesis

For the synthesis of silver nanoparticles, silver nitrate (AgNO₃, 99.9% purity) was obtained from Carlo Erba Reagents (Italy) and used without further purification. The five plant extracts were prepared for further biosynthesis of SNP. C. mangga (P1, rhizome), M. charantia (P2, fruit), P. odorata (P3, leaves), L. elliptica (P4, leaves) and G. gnemon (P5, leaves) were collected from Mueang District, Narathiwat province (Figure 1).

Figure 1. Image of edible plants (/used part); Curcuma mangga Valeton & Zijp (P1/rhizome), Momordica charantia L. (P2/fruit), Persicaria odorata (Lour.) Soják (P3/leaves), Litsea elliptica Blume (P4/leaves) and Gnetum gnemon Linn. var. tenerum Markgr. (P5/leaves).

All edible plants were examined according to their botanical characteristics to identify the right species. The dried powder of plant samples (5, 10, 20 g) in 100 mL of ultrapure water were stirred at 60°C for 10 min. After cooling down to room temperature, the aqueous extracts of plant samples were filtered through Whatman No.1 paper prior to storage at 4°C for further use.

Biosynthesis of SNP

Briefly, 10 mL of plant extracts was mixed with 90 mL of 1 mM AgNO₃ solution and then maintained at 37°C. The mixtures were stirred continuously for 10 min and further incubated at room temperature for 24 h in a light-resistant container. Prior to characterization of SNP, they were purified by centrifugation at 12,000 rpm for 15 min and the pellet was washed three times with ultrapure water.

Characterization of SNP

The UV-Vis spectrophotometer (Cary 60, Agilent) was used to measure the UV-Vis absorbance spectra of SNP. The measurement of particle diameter and polydispersity index (PI) of SNP were performed in triplicate at 25°C using a dynamic light scattering apparatus (Zetasizer Nano ZS, Malvern Inc., Worcestershire, UK) at a scattering angle of 173°. The morphology of SNP was visualized under transmission electron microscope (TEM JEM-2100, JEOL, Japan). The D2 phaser diffractometer (Bruker AXS Model D8, Germany) with Cu K_α radiation was used to analyze the X-ray diffraction pattern of SNP.

Antimicrobial activity of SNP

Media and chemicals

Nutrient broth (NB) for microorganism was obtained from Merck (India). The culture media for bacteria, Sabouraud dextrose agar (SDA) and Sabouraud dextrose broth (SDB) were purchased from Merck (India). The culture media for fungi, Mueller Hinton agar (MHA) and Mueller Hinton broth (MHB) were purchased from Difco Microbiology (USA). In this study, an antibacterial drug, ciprofloxacin was purchased from Gibco, USA while fluconazole (Sigma-Aldrich, USA) was used as an antifungal drug.

Microbial strains

Gram positive bacteria, Staphylococcus aureus ATCC 25923 (SA), and gram-negative bacteria, Escherichia coli ATCC 25922 (EC), were obtained from the American Type Culture Collection (ATCC). Gram-negative bacteria, Pseudomonas aeruginosa DMST 4739 (PA), and fungal strains, Candida albicans DMST 5815 (CA) and Candida tropicalis DMST 15495 (CT) were obtained from Department of Medical Sciences Thailand Culture Collection (DMST). The microbial suspensions were inoculated into NB, incubated at 37°C for 24 h and were then adjusted to 0.5 McFarland standard (1.5 x 10⁸ CFU/mL) using a spectrophotometer (model 22RS, Labomed, USA) at 600 nm before further antimicrobial test.

Antimicrobial assay

The antimicrobial assay was determined using broth macrodilution method following the guidelines of Clinical and Laboratory Standards Institute (CLSI, USA) (Lalitha, 2004; Balouiri et al., 2016). Briefly, serial two-fold dilution of SNP and plant extracts was made (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.3, and 0.19 mg/mL). The test samples were incubated with adjusted microbial concentration (1.5 x 10⁶ CFU/mL) in MHB for bacteria or SDB for fungi for 24 h at 37°C. Ciprofloxacin (antibacterial drug) and fluconazole (antifungal drug) were used as positive controls. Microbial suspension without any antimicrobial drug was a growth control while media without microbial inoculate and antimicrobial drug were kept as a sterile control. The minimum inhibitory concentration (MIC) is the lowest concentration of samples in which no visible growth of microbial was observed.

Minimum bactericidal concentrations (MBC) and minimum fungicidal concentrations (MFC) are the lowest concentrations of antibacterial or antifungal agent, respectively, that kill at least 99.9% of the organisms. The samples at the MIC were taken and then inoculated on sterile MHA (bacteria) and SDA (fungi) by streaking on plates. The plates were incubated at 37°C for 24 h and the concentrations with no visible growth of microbials were recorded as MBC or MFC values.

Statistical analysis

Data was displayed as means ± standard deviation of triplicate experiment. The statistical analysis (SPSS 29.0 software, IBM statistic, USA) was done using a one-way analysis of variance (ANOVA) followed by pair-wise comparisons with post-hoc Tukey HSD (Honestly Significant Difference) test at an alpha level of 0.05. An unpaired Student’s T test was used for statistical analysis of two samples.  A P-value of < 0.05 was statistically significant.

RESULTS

Characterization of silver nanoparticles

In this study, the effect of aqueous extracts of 5 edible plants (P1, P2, P3, P4 and P5) on ability to form SNP was observed. It was found that only extracts of P1, P2 and P3 could reduce silver ions to form SNP at a concentration of 100 mg/mL Ag atom. However, the concentration of P1 extract required to form SNP was higher than P2 and P3 extracts. The brown-yellow color of SNP represented the reduction of silver nitrate and surface plasmon resonance excitation of the nanoparticles. The UV absorption band of around 400 nm corresponded to surface plasmon band of spherical or roughly spherical SNP (Almatroudi, 2020). The absorption band of synthesized SNP was approximately 434 ± 9 nm. The light scattering study showed the nanometric size of SNP ranging from 37-187 nm and narrow in size distribution, PI of 0.2-0.4 (Table 1). A significant increase in particle size (P < 0.05) upon increasing extract concentration from 5 to 10 and 20 mg/mL was found in SNP prepared by M. charantia extract (P2-SNP) and P. odorata extract (P3-SNP). Moreover, a little shift of absorption band to higher wavelength (red shift) was also observed for P2-SNP and P3-SNP when extract concentration increased. Interestingly, this study was the first-time report on the ability of C. mangga extract as a reducer and a stabilizer for synthesis of SNP (P1-SNP). The sizes of P1-SNP, 37-38 nm, were smaller than P3-SNP and P2-SNP while its absorption wavelength (420 nm) was less than others. However, the sizes of P1-SNP seemed to be independent of extract concentration.

Apart from absorption spectra (Figure 2A-B) and size analysis, the size morphology studied by TEM demonstrated that P1-SNP prepared using 20 mg/mL extract (P1-SNP20) were spherical or nearly spherical in shape (Figure 2C). Their sizes were smaller than 100 nm, which corresponded to those obtained from light scattering experiment. The X-ray diffraction analysis was used to analyze the crystalline characteristics of biosynthesized SNP. The four distinct peaks were shown by the diffractogram at 38.13°, 46.37°, 64.83° and 77.25°, which corresponded to (111), (200), (220) and (311) planes of silver respectively (Figure 2D). The findings indicated that P1-SNP had crystal structure.

Table1. Absorption wavelength (λ), particle size and polydispersity index (PI) of SNP after 24 h incubation. Data represent mean ± S.D. (n = 3).

Note: Different letters indicate statistical difference when compared within the same plant extract (P < 0.05; 1-way ANOVA for P2, P3; unpaired Student’s T test for P1).

Figure 2. The appearance at different time intervals (A), absorption spectra (B), TEM image (C) and XRD spectra (D) of P1-SNP20. 

Antimicrobial activity of silver nanoparticles

The antibacterial activity against S. aureus (SA), E. coli (EC), P. aeruginosa (PA) and antifungal activity against C. albicans (CA) and C. tropicalis (CT) of biosynthesized SNP were assessed by broth macrodilution assay. It was observed that the SNP had more antimicrobial ability than their corresponding extracts as seen from about 10-fold lower in MIC of SNP (Table 2). It was noted that the results shown were from triplicate experiment and the standard deviation equaled to zero. The drugs used as positive controls had the MIC of less than the minimum concentration tested (0.19 mg/mL).

From the results, P1-SNP exhibited better antibacterial activity than P2-SNP and P3-SNP. P1-SNP20 contained the least MIC (and MBC) values of 6.25, 1.56, 0.19 mg/mL against SA, EC and PA in orderly (Table 2, Figure 3). For antifungal activity, the least in MIC (and MFC) against CA and CT of 12.5 and 1.56 mg/mL respectively were found in P3-SNP20 (Table 2, Figure 3). The antimicrobial activity of SNP synthesized using higher extract concentrations seemed better than the corresponding SNP synthesized at lower concentrations. However, P3-SNP5 showed the lower in MIC against EC and PA than those of P3-SNP10 and P3-SNP20. 

Table 2. Minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and minimum fungicidal concentrations (MFC) of SNP and plant extracts (P) against bacteria S. aureus (SA), E. coli (EC), P. aeruginosa (PA) and fungi C. albicans (CA), C. tropicalis (CT) after 24-h incubation.

 Note: NA = no activity

Figure 3. Antimicrobial assay of minimum bactericidal concentrations (MBC, mg/mL) of P1-SNP20 against S. aureus or SA (A), E. coli or EC (B), P. aeruginosa or PA (C), and of minimum fungicidal concentrations (MFC, mg/mL) of P3-SNP20 against C. albicans or CA (D), C. tropicalis or CT (E). N represents positive control of antibacterial (A-C) and antifungal (D-E) drugs while C represents growth control (no antimicrobials).

DISCUSSION

Aqueous extracts from P1 (C. mangga), P2 (M. charantia) and P3 (P. odorata) were able to biosynthesize SNP at 37°C. Additionally, the formation of SNP using P1 extract has been firstly reported here. The compounds in the extract act as reductants in the formation of SNP, adhere at the nanoparticle surface thus preventing the SNP from aggregation (Elumalai and Irfan, 2024). The phytochemical compositions found in water extracts of P1 were phenolic compounds (Indis and Kurniawan, 2016). Aqueous extract of P2 composed of phenolic, alkaloid and saponin compounds (Rashid et al., 2017) while phenolic and flavonoid compounds were mainly found in extract of P3 (Hadi et al., 2024). Previous study on P2-SNP synthesized at room temperature revealed the adsorption peak of 420-430 nm and the estimate size of 150 nm (Joshi et al., 2017).  The adsorption wavelength of P3-SNP synthesized at 25°C and the size from TEM analysis reported earlier were around 436 nm and 34 nm, in orderly (Hadi et al., 2024). The results obtained concurred with the previous reports. A rather larger size of P3-SNP obtained in this study might arise from the fact that the size measured by light scattering is hydrodynamic while the TEM measures the size in dehydrated form.

The size of plant-mediated SNP depends on the concentration of the plant extract used. It seemed that the optimum amount of extract used for synthesis might influence the occurrence of small and monodisperse SNP. An increase in concentration of extracts brings about a higher number of nanoparticles which might tend to more agglomeration (Hadi et al., 2024; Fahim et al., 2024). The larger size of P2-SNP and P3-SNP and the shift to the longer wavelength upon increasing extract concentrations implied that the excess compound molecules in the extract possibly adhered to the nanoparticle surface and caused more aggregation of SNP (Hadi et al., 2024). Unexpectedly, P1-SNP synthesized with either 10 or 20 mg/mL had small and spherical monodisperse particles of around 37-38 nm.

For the assessment of SNP on pathogenic bacteria, the results indicated that the particle size might be a factor affecting the bacterial cell survival since the bioactivity of nanoparticles depends on their interaction with the cell membrane. The smaller particles having high surface area cause greater penetration through the outer cell surface of bacteria, thus promoting apoptosis and finally cell death (Moorthy et al., 2021). Consequently, it was unsurprising that the small sized P1-SNP exhibited the lower in MIC for SA, EC and PA followed by P3-SNP (43-95 nm) and P2-SNP (99-187 nm). The findings on size dependent antimicrobial of SNP agreed with the earlier reports on plant-assisted SNP synthesis such as Rosa indica L. extract (Balu et al., 2022). The SNP synthesized using R. indica with the sizes of 12 nm showed greater antibacterial activity against S. aureus and E. coli than 18-nm SNP and the least effect was seen with the SNP sized of 770 nm. For the same plant extract used, although SNP synthesized using lower extract concentrations were smaller in size, their MIC however, were not always lower. The excess compounds in high amount of plant extract would promote the highly dispersed SNP and aid in antimicrobial activity (Khalil et al., 2014). Notably, in this study the biosynthesis of SNP was performed at different extract concentrations but the same concentration of silver atom. This could imply that antimicrobial activity was performed at similar SNP concentration. The difference in antimicrobial effect of SNP might mostly depend on the quality (i.e. sizes) of SNP obtained from types of plant extracts used. Relevant study on the effect of SNP concentration on antimicrobials indicated the higher inhibition of microbial growth with increasing SNP concentrations (Akpinar et al., 2021).

From the results, pathogenic gram-negative bacteria, EC and PA were susceptible to SNP more than gram-positive SA bacteria. In fact, the cell wall of SA has dense peptidoglycan and thicker than that of gram-negative bacteria leading to delayed penetration of nanoparticles through the cell membrane (Moorthy et al., 2021). Notably, there were few reports on the MIC of SNP synthesized from aqueous P2 and P3 extracts. The MIC of P3-SNP in this work were higher than the values reported by Lubis et al. (2022); however, this accounted for different bacterial strain used and smaller size of reported P3-SNP. Again, MIC of P2-SNP against EC and SA were higher compared to the value of 4 µg/mL observed by Moorthy and coworkers (2021). However, the P2-SNP synthesis of earlier work was done at fairly high temperature (100°C) and yielded the very small particles, 16 nm.

Unexpectedly, the P3-SNP showed greater antifungal activity especially against CT as compared to other SNP. The structure of fungal cell wall is different from bacterial cell and the outer cell membrane is composed mainly of glucan polymer (Huan et al., 2020). Hence, the interaction between SNP and fungal cell surface might differ from bacterial cell. In addition, the phytochemicals in P3 extract such as phenolic compounds, terpenoids and other organic compounds may promote a highly dispersed SNP and facilitate the penetration of the SNP into the fungal cell since the lower MIC was seen in the SNP containing higher concentrations of the extract (Khalil et al., 2014; Yanpirat and Vajrodaya, 2015).

The current study informed the use of aqueous extracts of edible C. mangga, M. charantia and P. odorata for successful synthesis of SNP at temperature of 37°C. Plant-mediated synthesis provides safe, environmentally friendly and lower cost of SNP preparation. All SNP were evaluated for their antibacterial ability against S. aureus, E. coli and P. aeruginosa with the MIC of 0.19-12.5 mg/mL and antifungal activity against C. albicans and C. tropicalis with the MIC of 1.56-12.5 mg/mL. Expectedly, the biosynthesized SNP display antibacterial and antifungal activities through various mechanisms. For antibacterial ability, SNP can adhere and disrupt the bacterial cell membrane due to electrostatic interaction between positively charged silver ions with negatively charged cell membrane. The functional compounds in plant extract which are particle stabilizers can help SNP bind to the membrane integrity. Intracellular penetration of SNP then causes cell toxicity, induces reactive oxygen species (ROS) and oxidative stress, interferes with protein synthesis and cellular function, and destroys DNA, thus ultimately leading to microbial cell death (Vanlalveni et al., 2021; Bruna et al., 2021; Fahim et al., 2024). The mechanisms of antifungal activity of the SNP are demonstrated to be the same routes of action as of antimicrobials. SNP permeate into the fungal membrane impairing cell organelles such as mitochondria, ribosome, vacuole and chromatin and cellular functions. Cell apoptosis arises from ROS induced by SNP and DNA injury (Górka and Kubiński, 2024). The possible mechanisms of antimicrobial activity of biosynthesized SNP from plant extract are illustrated in Figure 4.

Figure 4. Possible mechanisms for antibacterial and antifungal activities of biosynthesized SNP including rupture of cell membrane, generation of ROS, damage to cell structures and DNA, interfering with protein synthesis and cellular dysfunction.

CONCLUSION

The extracts from edible plants; namely C. mangga, M. charantia and P. odorata were successfully used for simply synthesis of SNP at 37°C. Alkaloids and phenolic compounds in the extracts acted as reducing and stabilizing agents for the SNP synthesis. The physicochemical properties of biosynthesized SNP were dependent on the types and concentration of the extracts. Increasing the extract concentrations possibly caused more aggregation and larger size of SNP. Among the various extracts used, the sizes of SNP seemed to be affected by the types of extracts in that the sizes of SNP formed using C. mangga (P1-SNP) were smaller (37-38 nm) than P. odorata (43-95 nm) (P3-SNP) and M. charantia (99-187 nm) (P2-SNP). For the antibacterial study against S. aureus, E. coli and P. aeruginosa, it was found that P1-SNP which were smaller having higher surface area, showed greater activity followed by P3-SNP and P2-SNP. It would probably reflect the size-dependent antibacterial activity of the SNP. An increase in extract concentration however not always resulted in higher antibacterial activity. Moreover, the antifungal action of SNP seemed to prefer on the type of extracts, P3-SNP indicated the stronger antifungal against C. tropicalis strain especially at the higher extract concentration. It would imply that some phenolic and organic compounds could facilitate the SNP permeation of the fungal membrane. Biosynthesized SNP with edible plants could be further explored for pharmaceutical and medical applications dealing with antimicrobial purposes.

ACKNOWLEDGMENTS

This research was supported by Faculty of Pharmaceutical Sciences, Chulalongkorn University and Government Science and Technology Scholarship, National Science and Technology Development Agency (NSTDA).

AUTHOR CONTRIBUTIONS

C.P. conducted all the experiments, performed the statistical analysis. C.P. and W.W. wrote the manuscript. WW. and S.S. designed the experiment. All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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