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. 2025 Oct 2;10(40):46884–46895. doi: 10.1021/acsomega.5c04977

Homeopathic Medicine: An Intrinsic Nanomaterial for Synthesis of AgNPs for Environmental and Biomedical Applications

Shalu Goyal †, Swati Rani †, Pamthingla Ragui †, Ved Prakash Meena †, Ritika Hassija Narula §, Subhash Kaushik §, Sweta Singh ‡, Sheetal Budhiraja ∥,*, Rakesh Kumar Sharma †,*
PMCID: PMC12529198  PMID: 41114231

Abstract

This study aims to furnish scientific evidence that the effectiveness of homeopathic medicine is attributable to the existence of active components in the nanoform. To serve this purpose, silver nanoparticles (AgNPs) were synthesized using the homeopathic medicine Ocimum sanctum (O. sanctum) of potency (3X) as a stabilizing and reducing agent. The successful synthesis of AgNPs utilizing O. sanctum validates the existence of bioactive compounds in homeopathic preparations, as silver ions (Ag+) would not be reduced without active components. O. sanctum active phytoconstituents were essential in stabilizing the AgNPs and converting Ag+ to Ag0, which allowed for a controlled size and shape. The successful synthesis of AgNPs was further validated by characterization using Fourier transform infrared (FTIR), ultraviolet–visible spectroscopy (UV–vis), and high-resolution transmission electron microscopy (HRTEM). The synthesized AgNPs demonstrated remarkable catalytic effectiveness under ideal conditions, following a pseudo-first-order kinetic model (PFO), degrading Rhodamine B (RhB) by 99% in 30 s. Furthermore, the nanoparticles showed strong antibacterial activity; greater inhibition zones for Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were seen, suggesting that they were more effective against Gram-positive and Gram-negative bacteria. This work not only demonstrates the potential of bioactive molecules in homeopathic medicine but also confirms their existence in the homeopathic medicine seven at ultradilution.

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1. Introduction

Homeopathic medicine is a widely used alternative treatment system based on the principles of “like cures like” and potentization. , These medicines are developed via sequential dilutions and vigorous succussion, a process believed by proponents to enhance their therapeutic efficacy. Despite its extensive use, there is ongoing debate regarding the scientific validity of homeopathic medicine. − Opponents argue that excessive dilutions make these treatments pharmacologically ineffective as the concentration of the original material is diluted to an extent that is significantly far beyond Avogadro’s number. However, new research suggests that homeopathic medicines might contain nanoparticles of the original active ingredients, potentially contributing to their biological effects. − Recently, homeopathic medicine, which is usually regarded with suspicion by scientific communities, has been recognized for its nanometric properties, establishing it as a possible application of nanomedicine. − Contrary to traditional beliefs, research indicates that homeopathic medicines, developed through serial dilution and potentization, possess nanostructures of the original active components. These ultrafine particles demonstrate increased reactivity, bioavailability, and stability, characteristics that closely resemble those of synthesized nanoparticles. − The existence of these nanostructures supports the functional efficacy of a homeopathy medicine. The synthesis of nanoparticles from homeopathic medicines indicates their enhanced potential as nanomaterials and validates the nature of homeopathic medicine, thereby expanding opportunities in nanotechnology applications. −

Among the numerous medicinal plants utilized in homeopathy, Ocimum sanctum (O. sanctum) has exhibited significant potential. Both traditional Ayurvedic medicine and homeopathy place a large emphasis on the use of O. sanctum, which is more often referred to as Holy Basil or Tulsi. Through the method of serial dilution and succussion, O. sanctum can be made in homeopathic practice either as a mother tincture or in extremely diluted forms (for example, 6C, 30C). As a result of its traditional therapeutic use, O. sanctum is frequently prescribed by homeopathic doctors for the treatment of respiratory problems such as bronchitis and asthma, as well as for conditions associated with stress and infections. The anti-inflammatory, antibacterial, and adaptogenic qualities of homeopathic O. sanctum are ascribed to its rich phytochemical content, which includes substances such as eugenol, rosmarinic acid, flavonoids, and terpenes. These phytochemicals are responsible for the therapeutic actions of O. sanctum and act as natural reducing and stabilizing agents. These bioactive components present in O. sanctum might also improve the stability and efficacy of silver nanoparticles (AgNPs). − The bioactive constituents present in O. sanctum not only facilitate the synthesis of nanoparticles but also enhance their functional efficiency, making the resulting AgNPs particularly effective for diverse biomedical and environmental applications. −

The escalating environmental pollution attributed to synthetic dyes, especially from the textile, leather, and paper sectors, has become a major global issue. , The release of these dyes into aquatic ecosystems without adequate treatment leads to significant contamination, endangering marine life and presenting health hazards to humans. The majority of industrial dyes are non-biodegradable, toxic, and environmentally persistent, rendering their removal a significant challenge. , Traditional dye removal techniques, encompassing physical and chemical treatments, frequently exhibit inefficiencies or result in the production of secondary pollutants.

AgNPs exhibit exceptional catalytic properties and environmental compatibility, rendering them highly efficient in the degradation of organic pollutants, including dyes. − In addition, AgNPs exhibit significant photocatalytic activity, enabling them to utilize light energy from ultraviolet or visible spectra to facilitate oxidative degradation processes. Besides their environmental uses, AgNPs derived from O. sanctum demonstrate significant antibacterial efficacy. − The antimicrobial properties are particularly useful in a variety of pharmaceutical and industrial applications. Although numerous studies have reported the synthesis of metal nanoparticles using plant extracts for various applications, − few reports are published specifically for the synthesis of metal nanoparticles using homeopathic medicine. − However, none of them have investigated the specific active component within the medicine responsible for nanoparticle formation. In the present study, we systematically address this gap by identifying and analyzing the active constituent present in the homeopathic medicine O. sanctum, which helps to reduce the Ag+ to Ag0 to form AgNPs.

Numerous reports published recently predominantly address two aspects: first, the recognized plant-mediated synthesis of AgNPs utilizing extracts like O. sanctum, as evidenced by Nayak, and second, the characterization of nanoparticles found in homeopathic medicines, as illustrated by Chikramane et al. in Homeopathy. Our detailed manuscript established that although numerous studies have detected nanoparticles in homeopathic medicines, there are no prior publications evidencing the active utilization of homeopathic medicines to synthesize AgNPs from silver salts and the validation of nanoparticles in homeopathic medicine in the existing literature. This work accurately distinguishes between the presence of nanoparticles in homeopathic medicine and their active application in nanoparticle synthesis.

In addition, the purpose of this study was to identify the active components found in O. sanctum homeopathic medication and show that they are retained throughout the synthesis process. This contradicts the scientific claim that no active particles remain at large dilutions. If such components were not present, the reduction of Ag+ would not take place. To investigate this, AgNPs were synthesized from the O. sanctum homeopathic medicine, which confirmed the presence of active components. Furthermore, to assess the effectiveness of the synthesized nanoparticles using homeopathic medicine, they were used in dye degradation and antibacterial studies. This study gives scientific insights into the active components of homeopathic medicines.

2. Experimental Section

2.1. Materials

Homeopathic medicine O. sanctum with potencies of 3X, 6X, and 3CH was obtained from BJAIN Pharmaceuticals Pvt. Ltd., India. Silver nitrate (AgNO3, 99% purity) was purchased from Sigma-Aldrich. A standard AgNO3 solution (1 mM) was prepared by dissolving the required amount of AgNO3 in high-performance liquid chromatography (HPLC) water and stored in an amber-colored bottle to prevent self-oxidation. Rhodamine B (RhB, 99%) and sodium borohydride (NaBH4) were sourced from Thomas Baker. All chemicals used were of analytical reagent grade and used without further purification. HPLC grade water was employed for sample preparation throughout the experiments.

2.2. Synthesis of AgNPs from Homeopathic Medicine (O. sanctum)

2.2.1. Optimization of Synthesis of AgNPs

The optimization process involved evaluating key factors, such as the ratio of the homeopathic medicine to the AgNO3 solution, the concentration of the AgNO3, and incubation time. Each parameter was systematically optimized individually while maintaining all other variables constant.

2.2.2. Optimization of AgNO3 Solution

A specific amount of AgNO3 was weighed based on the molarity of AgNO3 solution and dissolved in HPLC spectroscopic grade water to prepare solutions of different concentrations (10–1, 10–2, 10–3, and 10–4 M). The prepared AgNO3 solutions were subsequently utilized in further experimental studies.

2.2.3. Preparation of AgNPs

The synthesis of AgNPs was carried out using the homeopathic medicine, O. sanctum, at different potencies 3X, 6X, and 3CH following the previously reported method. First, 10 mL solution of different molarities (10–1, 10–2, 10–3, and 10–4 M) of AgNO3 was taken in a culture tube, and 5 mL of the homeopathic medicine was added dropwise under continuous stirring on a magnetic stirrer for 6 h at room temperature. The reaction was conducted at neutral pH (pH = 7). The color change of the solution from light green to pale brown confirmed the successful reduction of AgNO3 to AgNPs. The mixture was incubated at room temperature for 24 h. AgNPs were formed at all potencies; however, because the quantity of particles formed at very diluted potencies (6X and 3CH) was less, the pale brown color was not visible, making recording the absorption spectra impossible. In contrast, at 3X potency, a sufficient quantity of particles was produced. As a result, 3X potency was utilized to investigate the behavior of the homeopathic medicine. Furthermore, the concentration of AgNO3 was optimized by analyzing the absorption spectra with a UV–vis spectrophotometer. The details of the procedures for the synthesis of AgNPs are shown in Scheme .

1. Schematic Preparation of AgNPs.

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3. Instrumentation and Characterization

In order to identify the physical and chemical properties of the AgNPs synthesized, different characterization methods were used. In order to determine the typical surface plasmon resonance band of the nanoparticles, Ultraviolet–visible (UV–vis) spectroscopy was carried out. A quartz cuvette with a route length of 1 cm was used to analyze 0.50 mL of AgNPs that were measured with an Agilent Cary 3500 UV–visible spectrophotometer. The wavelength range for the measurements was from 200 to 800 nanometres. High-Resolution Transmission Electron Microscopy (HR-TEM) was performed using a Tecnai F20 machine in order to investigate the nanoparticles’ shape as well as their size. A drop of colloidal solution, measuring 10 μL, was put on a carbon-coated copper grid and allowed to air-dry before being used for sample preparation. In addition, to determine the size and surface charge of nanoparticles, Dynamic Light Scattering (DLS) was performed using a Malvern Zetasizer nano instrument (U.K.). This device features a 4 mW He–Ne laser, an automated laser system, and an attenuator with a transmission range of 100 to 0.0003%. The detection range for all measurements was established from 0.1 nm to 10 μm, and the temperature was calibrated to 25.0 °C to attain thermal equilibrium. Fourier Transform Infrared Spectroscopy (FT-IR) was utilized to identify the functional groups responsible for capping and stabilizing the nanoparticles. Spectra were recorded in the range of 400–4000 cm– 1 using the SHIMADZU IR Affinity-1S FT-IR spectrophotometer, providing detailed information on chemical bonding and molecular structure. Additionally, the silver concentration in the prepared nanoparticles was quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent-ICPMS (8900 Triple Quadrupole ICP-MS; Model: G3665A system) and expressed in milligrams per liter.

4. Operational Parameters for the Removal of RhB

The removal of RhB dye was monitored by using UV–visible spectroscopy. A decrease in the intensity of the characteristic peak of RhB at 554 nm indicated the successful removal of the RhB dye by AgNPs. An appreciable color change from pink to colorless served as a visual indicator of dye removal. The progressive reduction of RhB was confirmed by the decrease in absorbance values, which were evaluated every 15 s. The adsorption of dye by AgNPs was investigated in the presence of the reducing agent NaBH4. The initial dye concentration, catalyst concentration, and reaction time were all varied during the tests. The following formula was utilized to determine the RhB dye elimination effectiveness based on the absorbance values acquired throughout the experiment:

Removalpercentage(%R)ofdyeRHB=A0−AtA0×100

where A 0 and At represent the initial absorbance and absorbance at different time of dye.

4.1. Effect of the RhB Dye Concentration

In order to investigate the effect of RhB dye dosage, the dye concentration was varied from 1 to 10 mg/L under normal conditions, at a pH of 7. The degradation of the dye in each instance was assessed using a UV–vis spectrophotometer.

4.2. Effect of the Catalyst Concentration

An investigation was conducted to examine the impact of the catalyst dosage on the removal of RhB dye. This was accomplished by adjusting the concentration of AgNPs from 25 to 100 μL under ideal conditions. The parameters were measured at a dye concentration of 2 mg/50 mL and a pH of 7, respectively.

5. Media Preparation and Assessing the In Vitro Antibacterial Properties of the AgNPs

An autoclave was used to sterilize 25 g of Luria broth (from Conda) in 1 L of distilled water. The autoclave was set to 121 °C for 15 min. Then, antibacterial activity of the AgNPs was assessed using the agar disc diffusion method against both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria. A pure culture of bacteria was obtained and incubated overnight in broth at 37 °C with shaking. The bacteria were then diluted to 1 × 106 cells/mL and inoculated on an agar plate. Subsequently, 10 μL of the individual components was added on a sterile filter paper disk and the disc was placed over a bacteria-streaked plate followed by incubation for 24 h at 37 °C. After 24 h, the diameter of the clear zone of inhibition was measured around the AgNPs specimen.

6. Results and Discussion

In order to confirm the synthesis of AgNPs via using the active component of the homeopathic medicine “O. sanctum”, different techniques have been utilized, and results are discussed below.

6.1. FT-IR Spectra

The FT-IR analysis of AgNPs synthesized from the homeopathic medicine O. sanctum confirmed the participation of several functional groups in the reduction and stabilization of the nanoparticles (Figure ). A broad peak observed between 3200 and 3600 cm– 1 corresponds to O–H stretching vibrations, indicating the presence of phenolic hydroxyl groups in the O. sanctum extract (black color). Notably, the shift in this absorption band from 3343 cm– 1 in the O. sanctum to 3316 cm– 1 in the AgNPs suggests the direct participation of these hydroxyl groups in the reduction of Ag+ to Ag0 (red color). Additional peaks in the 2850–2950 cm– 1 range were attributed to aliphatic C–H stretching vibrations, likely originating from fatty acids or other organic compounds that contribute to nanoparticle stabilization. The spectrum also showed a characteristic peak at 1650–1750 cm– 1, corresponding to CO stretching vibrations from carbonyl groups in flavonoids or terpenoids, while absorptions between 1500 and 1600 cm– 1 indicated N–H bending vibrations from amide groups of proteins that may act as capping agents to prevent agglomeration. Furthermore, a peak at 1035 cm– 1 was assigned to C–O stretching vibrations from alcohols or esters, and a distinct low-frequency peak at 500–600 cm– 1 confirmed Ag–O vibrations, verifying successful nanoparticle formation. The close similarity between the FT-IR spectra of the O. sanctum-synthesized AgNPs and the original O. sanctum medicine demonstrates that the bioactive components (phenolics, proteins, and flavonoids) in the homeopathic medicine play a crucial role in the synthesis and stabilization of the nanoparticles, consistent with previous reports on plant-mediated green synthesis. Furthermore, the peaks in the range of 2850–2950 cm– 1 were ascribed to aliphatic C–H stretching, maybe originating from fatty acids or other organic compounds that contribute to the stabilization of the nanoparticles. , This further confirms the presence of active components in homeopathic medicine from the original constituent. Prior studies have also demonstrated the role of proteins and phenolic components in the plant-mediated synthesis of nanoparticles, emphasizing their potency as stabilizing and reducing agents. ,

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FT-IR spectra of homeopathic medicine and AgNPs synthesized from the homeopathic medicine O. sanctum.

6.2. Absorption Studies

The UV–vis spectra of the AgNO3 solution, the homeopathic medicine O. sanctum (3X potency), and the AgNPs that were synthesized from homeopathic medicine were monitored. Important aspects of the synthesis of AgNPs were investigated, including the concentration of AgNO3, the potency of O. sanctum, and the time duration allowed for incubation. The adjustments were made to each individual parameter while maintaining the others constant. It has been discovered that the parameters of the reaction have a considerable impact on the efficiency, as well as the form and size of the nanoparticles.

6.2.1. Effect of AgNO3 Concentration

The synthesis and stability of AgNPs were studied by varying the concentration of AgNO3 and using homeopathic medicine O. sanctum at a 3x potency as the reducing and capping agent. The synthesis of AgNPs was carried out according to the procedure mentioned in the literature. The NPs were prepared with AgNO3 concentrations of 10–1, 10–2, 10–3, and 10–4 M at ambient temperature, and the formation of nanoparticles was monitored using a UV–vis spectrophotometer. At a AgNO3 concentration of 10–1 M, a UV peak at 420 nm was observed, indicating the formation of AgNPs (Figure ). However, due to the low concentration of O. sanctum relative to the high concentration of Ag+, the nanoparticles quickly agglomerated, resulting in poor stability and inadequate capping. At 10–2 M AgNO3, the UV peak at 420 nm persisted, confirming the nanoparticle formation. Nonetheless, the solution became unstable after 3 days, leading to precipitation, which indicated weak capping and incomplete stabilization. For a AgNO3 concentration of 10–4 mM, a stable UV peak at 420 nm was observed, and precipitation was delayed for up to 15 days, suggesting improved stability compared to higher concentrations. The best results were achieved with a AgNO3 concentration of 10–3 M, where a consistent UV peak at 420 nm was observed and the solution remained colloidal for over 30 days without visible precipitation. This indicated the excellent stability of the synthesized nanoparticles. The formation of nanoparticles at all concentrations of AgNO3 shows that some active component is present even at a diluted level in the homeopathic medicine, which led to the reduction of Ag+ to Ag0. The results also highlight that the concentration of AgNO3 significantly affects the synthesis and stability of the AgNPs. At higher concentrations, the disproportionate ratio of AgNO3 to O. sanctum led to incomplete reduction and the poor stabilization of nanoparticles. Conversely, lower AgNO3 concentrations, particularly at 10–3 M, allowed for an optimal balance between reduction and capping, resulting in highly stable nanoparticles with prolonged colloidal stability. This demonstrates that careful optimization of the AgNO3 concentration is crucial for the successful synthesis of stable AgNPs and that a concentration of 10–3 M is used for further experiments.

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Absorption spectra of AgNPs from 3X potency of O. sanctum at different concentrations of AgNO3 solution.

6.2.2. Effect of Medicine-to-AgNO3 Ratio

After optimizing the concentration of AgNO3 (i.e., 10–3 M), the ratio of medicine and AgNO3 was optimized. The three different ratios (1:2, 1:1, and 2:1) of the homeopathic medicine and AgNO3 solutions were taken, and a similar procedure was followed to prepare the AgNPs at different ratios. The mixtures were stirred for 4 to 6 h at ambient temperature, and UV–vis spectra were recorded to confirm nanoparticle formation. The influence of the ratio of O. sanctum to AgNO3 on the synthesis of AgNPs was evaluated and is represented in Figure . A UV–vis absorbance peak at 420 nm was observed for the 1:2 ratio, 425 nm for the 1:1 ratio, and 422 nm for the 2:1 ratio. These distinct peaks confirmed the successful synthesis of AgNPs at all the tested ratios and show that even at a lower amount of homeopathic medicine (1:2), AgNPs are forming. These results show that even at the diluted level, the homeopathic medicine contains some active component. However, the particles formed at a 1:2 ratio were found to be stable up to 30 days. Hence, we have chosen this ratio for all further studies. A similar type of absorption spectra of AgNPs was reported by Kyaw et al. using O. sanctum leaf extract. , This confirms the presence of a bioactive component of the original material in homeopathic medicine, which helps in the reduction of Ag+ to Ag0. The concentration of AgNPs formed using the active component of the homeopathic medicine has been calculated by ICP-MS analysis and reported in the Supporting Information: Data S1. The concentration of AgNPs found through ICP-MS analysis further validates the fact that the homeopathic medicine retains its active component even at lower dilution, and that component could be in the nano range.

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Absorption spectra of AgNO3, O. sanctum and AgNPs at ratios (1:2), (1:1), and (2:1) of medicine and AgNO3 solution.

6.2.3. Effect of Incubation Time and Stability of AgNPs

The UV–vis absorbance spectra of AgNPs synthesized at different ratios of the homeopathic medicine to AgNO3 were recorded at intervals of 3, 6, 12, 24, and 48 h (Figure ). This study aimed to analyze the variations in the absorbance peak position and intensity over time, which are indicative of changes in the size, stability, and overall characteristics of the nanoparticles. For the 1:2 ratio of the homeopathic medicine to AgNO3, the absorbance peak remained constant at 420 nm throughout the 48 h incubation period. However, the peak’s intensity increased gradually over time. This increase in intensity might be due to the progressive increase in the size of the nanoparticles, which is the result of the continuous nucleation and growth processes during the incubation period. The uniform size distribution of the nanoparticles synthesized at this ratio is further supported by the sharpness of the absorbance peak at 420 nm. Conversely, the absorbance peak was observed at 422 nm throughout the 48-h period for the 1:1 ratio. The peak intensity increased over time, suggesting that the size of the nanoparticles increased, similar to the 1:2 ratio. Nevertheless, the slight redshift of the absorbance peak to 422 nm in comparison to the 1:2 ratio suggests the formation of slightly larger nanoparticles. Nevertheless, the absorbance peak for the 1:1 ratio was less pronounced, indicating a less uniform size distribution in comparison to the 1:2 ratio. The absorbance spectra for the 2:1 ratio exhibited broader curves and fluctuations in the peak position throughout the 48-h incubation period. These observations suggest that the nanoparticles synthesized at this ratio were inconsistent in terms of their size and stability. The formation of polydisperse nanoparticles is likely the result of an imbalance in the reaction kinetics and stabilization processes, as indicated by the broader and shifting absorbance peaks. Using a UV–vis spectrophotometer, the color changes and absorbance measurements of the nanoparticles that were prepared were monitored at predetermined intervals in this study. According to the data obtained, the optimal ratio for the synthesis of AgNPs is 1:2 homeopathic medicine to AgNO3. The constant absorbance peak at 420 nm and its sharpness are indicative of the nanoparticles’ consistent size and stability that were produced by this ratio. The stability at this ratio might be due to the excess amount to homeopathic medicine as a greater number of active species are available to reduce Ag+ and providing more capping tendency. In comparison, the 1:1 ratio produced slightly larger but less uniform nanoparticles, while the 2:1 ratio resulted in polydisperse and unstable nanoparticles. In conclusion, the 1:2 ratio was found to be the most effective for achieving well-defined and stable nanoparticles, making it the preferred choice for applications requiring consistent nanoparticle characteristics.

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Absorption spectra of AgNPs at different times with (a) (1:2), (b) (1:1), and (c) (2:1) medicine and AgNO3 solution for 48 h.

Further, to gain more insights about the stability of AgNPs for 1:2, the λmax vs time graph was plotted by monitoring the absorption spectra of AgNPs (Supporting Information: Figure S1). From the graph, it was found that as time increases, the wavelength also increases, but this increment in wavelength with time is less, suggesting that the active ingredient present in the medicine continues to release gradually. This slow and sustained release facilitated the complete reduction of Ag+ into AgNPs at a controlled rate. Such behavior is indicative of enhanced stability, as the progressive release of the active ingredient ensures a consistent and steady nucleation and growth process. This controlled reduction minimizes rapid aggregation or uneven nanoparticle formation, thereby contributing to the stability and uniformity of the nanoparticles over time.

6.3. TEM Analysis

The interior structure, average particle size, and shape of the AgNPs were analyzed by using HR-TEM. TEM grids were prepared by depositing the nanoparticle solution onto a copper grid. The average particle sizes of the AgNPs were determined to be approximately 22, 19, and 14 nm for the ratios of 1:2, 1:1, and 2:1, respectively (Figure ). In all cases, the nanoparticles exhibited a near-spherical shape. However, the distribution of AgNPs with 1:2 is uniform and monodisperse, but with 1:1 and 2:1, particles are found to be polydisperse. These results show that the presence of the active component of the homeopathic medicine that is also in nanorange is responsible for the synthesis and formation of nanoparticles. These size ranges align with findings reported in recent studies on AgNPs. ,,

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TEM analysis of AgNPs formed by mixing of medicine and AgNO3 solution in different ratios (a,b) (1:2); (c,d) (1:1); (e,f) (2:1).

6.4. DLS Analysis

The DLS technique was employed to determine the hydrodynamic radius and size distribution of the synthesized AgNPs (Supporting Information: Figure S2a). From the DLS data, the particle size is found to be 194 nm, which is higher than the TEM analysis. As DLS includes the hydrodynamic radius, the size obtained from DLS is greater than that obtained from TEM. The DLS analysis indicated that the AgNPs were within the nanoscale, affirming their colloidal stability and homogeneous distribution in the solution. Further, zeta potential was performed to find the surface charge of the synthesized nanoparticles, which is found to be −13.4 mV (Supporting Information: Figure S2b). The ζ-Potential signifies the surface charge of nanoparticles in a colloidal state, and results verified that the AgNPs possessed a negative surface charge, signifying excellent colloidal stability and the presence of phytochemical capping agents derived from the homeopathic drug O. sanctum utilized in the synthesis. The negative value reflects the high stability of the AgNP colloidal system using O. sanctum as reported in the literature. , These results show that the AgNPs displayed a negative zeta potential, indicating a negatively charged surface attributed to the phytochemicals from the homeopathic medicine, O. sanctum, utilized during synthesis.

7. Application of AgNPs

7.1. In Dye Degradation

To investigate the efficiency of the prepared AgNPs, RhB was used. The dye removal efficiency of the homeopathic medicine O. sanctum (3X) mediated AgNPs was investigated by monitoring absorption spectra. Degradation studies were performed by mixing 200 μL of RhB (40 mg/L in aqueous medium) with 25 μL of AgNPs and 100 μL of NaBH4, making a total volume of 3 mL. Three sets of control (without AgNPs) were also conducted: (1) Dye + O. sanctum extract (medicine), (2) Dye + AgNO3, and (3) Dye + NaBH4. The progress of the reaction was monitored using a UV–vis spectrophotometer and is presented in Supporting Information: Figure S3. The absorption peak at 554 nm is responsible for the pink color of RhB dye. − A reduction in this peak indicates successful RhB degradation. Similar types of studies have been reported in the literature using metal nanoparticles for the reduction of dye. When AgNPs alone were added to the RhB solution, the peak at 554 nm remained unchanged. Similarly, the addition of NaBH4 and homeopathic medicine alone did not result in any significant change in the RhB peak. However, when both AgNPs and NaBH4 were combined, a notable reduction in the 554 nm peak was observed. This confirms the effective degradation of the RhB dye. After the addition of AgNPs, the peak at 554 completely vanished, and the pink color of RhB disappeared. This happened because AgNPs show a surface plasmon resonance (SPR) effect.

In the presence of the reducing agent NaBH4, electron transfer occurs from the valence band to the conduction band of the AgNPs. Excited electrons interact with oxygen species (O2), which facilitate the degradation of RhB. Thus, the synergistic action of AgNPs and NaBH4 significantly enhances the dye removal process. The mechanism for the degradation of dye has been depicted in Scheme . The effective degradation of RhB in the presence of AgNPs can be explained through the electrostatic interaction between negatively charged AgNPs and cationic dye molecules that promotes effective adsorption. Moreover, numerous research studies have validated that negatively charged AgNPs are proficient in eliminating RhB from aqueous solutions. , Upon the introduction of AgNPs into the dye solution, electrostatic interactions facilitate the adsorption of RhB onto the nanoparticle surface. A larger negative zeta potential amplifies this attraction, hence augmenting the adsorption capacity. ,

2. Schematic Representation of Degradation of RhB with AgNPs.

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Further, the effect of concentration of the dye, AgNPs, and time intervals was studied as these parameters influence dye removal efficiency. The concentration of the reducing agent and dye was kept constant, while the amount of the catalyst (AgNPs) was varied from 25 to 100 μL. With an increase in the amount of the catalyst, the dye removal time decreased from 30 to 15 s, but the maximum removal efficiency also decreased from 99% to 97.5% (Figure a–e). Moreover, at concentrations of AgNPs, 25 and 75 μL, the degradation time is the same, that is, 30 s, but the removal efficiency is different. The removal efficiency is found to be maximum for 25 μL of AgNPs.

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Absorption studies for degradation of RhB using different AgNP concentrations: (a) 25 μL, (b) 50 μL, (c) 75 μL, and (d) 100 μL and (e) removal efficiency with varying amount of the catalyst.

Further experiments were performed by varying the dye concentration from 1 to 10 mg/L (Supporting Information: Figure S4). As the dye concentration increased, the maximum removal efficiency decreased due to the reduced catalytic efficiency of AgNPs to adsorb dye molecules at higher concentrations. At 5 mg/L, a maximum removal efficiency of 99% was achieved within 30 s using the reducing agent NaBH4. This phenomenon may be attributed to the fact that only a minimal amount of catalyst is required to effectively catalyze the reaction for efficient dye removal. The high removal efficiency of AgNPs for the degradation of RhB confirms the idea of an active component present in the homeopathic medicine that is able to reduce Ag+ to Ag0 and some phytochemicals. Further results have been compared with previous reported AgNPs using different methods, and it was found that AgNPs prepared from the O. sanctum have less degradation time (Supporting Information: Table S1). ,−

From the evaluation of varying concentrations of dye and catalyst, it was determined that the optimized dye concentration is 5 mg/L and the optimized catalyst amount is 25 μL.

Lastly, the degradation behavior of RhB dye using synthesized AgNPs from the homeopathic medicine was analyzed using linear kinetic models. The data obtained from absorption spectra were fitted in different kinetic models: zero order, pseudo-first order (PFO), and pseudo-second order (PSO), as presented in Supporting Information: Figure S5b–d, respectively. The degradation of RhB follows PFO as reported in the literature. Moreover, the concentration of NaBH4 was maintained at substantially greater than that of the catalyst and the RhB. As NaBH4 is used in a large amount, its concentration can be considered constant throughout the reaction. Thus, the reaction kinetics were evaluated using PFO kinetics with respect to RhB. The PFO model suggests that the process is primarily governed by the mass transfer of interfacial dye molecules (Supporting Information: Figure S5). The fundamental equation of the PFO model is

ln(Ct/C0)=−kt+C 1

Where kt is the rate constant (min– 1) of the PFO reaction, t is the adsorption time (min), Ct is the absorbance of RhB at time t, and C 0 is the initial absorbance of RhB.

A linear plot of ln (Ct /C 0) versus time (t) yielded a good linear fit with a correlation coefficient (R 2) greater than 0.93 (Supporting Information: Figure S4b). The slope of the plot provided the PFO rate constant, calculated as 0.1378 s– 1.

7.2. Antibacterial Activity

The antibacterial efficacy of AgNPs and the homeopathic medicine was tested against pathogenic strains of two bacterial species: Gram-negative E. coli and Gram-positive S. aureus, as shown in Figure a, using the agar disc diffusion method. Compared to the controls [O. sanctum 3X (1) and AgNO3 (2)], AgNP treatments at different ratios [1:2 (3), 1:1 (4), and 2:1 (5)] showed an increased zone of inhibition, as shown in Figure b, indicating enhanced antibacterial activity in comparison to homeopathic medicine alone. The values of the Inhibition zone test results for S. aureus and E. coli with AgNPs have been presented in Supporting Information: Table S2. The bar graph quantifies and compares the efficacy of these treatments. These results confirm that the AgNPs synthesized from the homeopathic medicine O. sanctum exhibited effective antibacterial activity against E. coli and S. aureus. Furthermore, the antibacterial activity of AgNPs synthesized using the homeopathic medicine O. sanctum was compared with that of AgNPs prepared from plant extract against E. coli and S. aureus (Supporting Information: Table S3). From the comparison table, it can be seen that the antibacterial activity of AgNPs prepared from Homeopathic medicine O. sanctum is found to be greater in comparison to AgNPs prepared from different plant extracts. −

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Antibacterial efficacy of AgNPs. (a) Inhibition zones against E. coli and S. aureus using the disk diffusion assay. (b) Dose-dependent inhibition zones: O. sanctum −3X and AgNO3 serve as controls, while AgNPs at ratios of 1:2, 1:1, and 2:1 represent different treatments.

8. Conclusions

This study validates the presence of active species in ultradiluted formulations by conclusively demonstrating that bioactive molecules capable of synthesizing AgNPs are retained in a very diluted homeopathic medicine of O. sanctum (3X). The absorption peak at 420 nm confirms the successful reduction of Ag+ to Ag0. This shows preserved activity of active species in homeopathic dilutions. With a hydrodynamic diameter of 194 nm and a ζ-potential of −13.4 mV, the synthesized AgNPs demonstrated stable nanoparticle formation with a negatively charged surface that was ideal for biological and catalytic applications. These nanoparticles showed dual functionality, enhanced antimicrobial activity against both Gram-positive and Gram-negative bacteria, and followed PFO kinetics (R 2 = 0.93, k = 0.1378 s– 1) in RhB dye degradation, achieving = 99% degradation within 30 s through electrostatic interactions facilitated by their surface charge of AgNPs. These findings support homeopathic principles in two ways: first, they show that homeopathic dilutions contain enough bioactive ingredients to mediate the synthesis of nanoparticles with specific physicochemical characteristics; second, they show that the resultant nanoparticles have improved biological and catalytic effects due to synergistic effects. Strong evidence that homeopathic medicine retains nanostructured active ingredients that have particular physicochemical effects can be seen in the association between the high catalytic efficiency of the nanoparticles and their properties (194 nm size and −13.4 mV charge). By proving maintained bioactivity in extremely diluted systems, our work not only overcomes the discussion related to the presence of active components in homeopathic medicine but also validates homeopathic medicine as a sustainable green method for the synthesis of nanoparticles with enhanced activity. However, the results are derived from controlled laboratory trials that were conducted with a restricted selection of bacterial strains. Further research needs to be conducted in order to properly investigate the active phytochemical present in homeopathic medicine O. sanctum and assess the stability, reusability, in vivo safety evaluations, and mechanistic pathways.

Supplementary Material

ao5c04977_si_001.pdf (600.1KB, pdf)

Acknowledgments

The authors gratefully acknowledge the Central Council for Research in Homoeopathy (CCRH), File No.-17-29/2023-24/CCRH/Tech/FR/Coll/DU-Nano-Scale/Mt in Hom/Dept. of Che/1328 for providing funding to carry out this work. The authors also thank the Institute of Eminence (IoE) University of Delhi, File no-IoE/2024-25/12/FRP, for financial support. Shalu Goyal acknowledges the University Grants Commission (UGC), India, for the Senior Research Fellowship (SRF). The authors also thank the Department of Chemistry, University of Delhi, for providing research support. Additionally, the authors acknowledge U.S.I.C., University of Delhi, and SAIF AIIMS, Delhi, for providing analytical instrumentation facilities.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04977.

  • Additional data and figures supporting the characterization of AgNPsincluding ICP-MS analysis, λmax vs time graph for silver nanoparticles, DLS and zeta potential measurements, absorbance versus time plots, RhB degradation studies, kinetic model fitting of absorption spectra, and comparative tables for RhB dye remediation, zone of inhibition for S. aureus and E. coli bacteria, and antibacterial activity of AgNPs (PDF)

The authors declare no competing financial interest.

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