Nanotechnology encompasses a broad interdisciplinary field focused on creating innovative nanostructures up to 100 nm in size, where properties such as particle size, shape and distribution play crucial roles (Sahuet al., 2023). Nanoparticles at this scale exhibit unique characteristics including electron density configuration, thermal conductivity, optical properties and magnetic behavior (Modenaet al., 2019). Extensive research has illuminated diverse applications of nanoparticles in biomedicine, pharmaceuticals, drug delivery, cosmetics and catalysis (Zahinet al., 2020). Nanotherapeutics are increasingly seen as promising solutions to address challenges in drug delivery systems, including solubility, targeting, bio-distribution, bioavailability and therapeutic efficacy (Muniyappanet al., 2021).

Recent attention has focused on Gold Nanoparticles (AuNPs) due to their distinct physicochemical properties and versatile applications (Herizchiet al., 2016). Among metallic nanoparticles, AuNPs can interact with light through Surface Plasmon Resonance (SPR) (Yoganandhamet al., 2018). However, conventional chemical synthesis methods often produce hazardous by-products that pose risks to human health (Dikshitet al., 2021), highlighting the need for eco-friendly nanoparticle synthesis approaches within nanotechnology (Kumaret al., 2021).

Utilizing plants for nanoparticle synthesis presents a sustainable alternative to chemical methods, offering scalability and environmental benefits (Sarataleet al., 2018). Plant extracts serve as efficient reducing and capping agents in nanoparticle synthesis due to their rich phytochemical content (Patil and Chandrasekaran, 2020). E. cardamomum, commonly known as Cardamom, is a fragrant spice with significant medicinal value and cultural importance (Banoet al., 2016). This study focuses on synthesizing AuNPs from different parts of cardamom seeds and pods using green synthesis methods, while evaluating their antibacterial, antioxidant and anti-inflammatory properties.

The seeds and pods of E. cardamomum were obtained from Idukki and underwent separate shade drying processes before being finely ground using a mechanical grinder. The resulting powders were stored in sealed containers. To prepare the aqueous extract, 2g of powdered samples from both pods and seeds were individually mixed with 20 mL of deionized water. This mixture was heated under controlled conditions, maintaining a temperature between 70-80ºC, for 1 hr to facilitate extraction. After this step, the solution was centrifuged at 5000 rpm for 15 min to remove any insoluble residues and obtain a clear aqueous extract. This extract, containing bioactive compounds from E. cardamomum, was then used as the bio reductant in the synthesis of AuNPs (Pattanayak and Nayak, 2013).

Initially, a 20 mL aqueous solution of HAuCl₄ was prepared with a concentration of 1 mM. Following this, 2 mL portions of the extracts from both pods and seeds were individually added to the prepared HAuCl₄ solution. These mixtures were then incubated under controlled conditions at temperatures between 40-60ºC for 15 min. Over the course of this incubation, the emergence of a noticeable violet coloration in the reaction mixtures indicated the synthesis of nanoparticles visually (Pattanayak and Nayak, 2013).

To assess the reduction of Au³⁺ salts to nanoparticles, the UV-vis absorption spectra of the extracts from seeds and pods were examined using a UV-vis Spectrophotometer from Shimadzu Corporation, Japan. This analysis was conducted across a wavelength range of 300 nm to 800 nm. Following this initial analysis, samples from the reaction mixtures containing the synthesized nanoparticles were also analyzed using UV-vis spectroscopy. The absorption spectra obtained from these analyses provided critical information about nanoparticle formation, revealing characteristic absorption peaks within the specified wavelength range. By comparing the absorption spectra of the seed and pod extracts before and after the reduction process, any shifts or changes in peak intensity indicative of nanoparticle formation.

AuNPs synthesized from extracts of both seeds and pods of E. cardamomum were characterized using XRD analysis. XRD patterns were obtained using a Rigaku Miniflex 600 X-ray diffractometer (Japan), employing Cu Kα radiation (λ=1.5406 Å). The measurements were conducted at room temperature over a 2θ range of 10-80º with a step size of 0.02º. The XRD patterns obtained were analyzed to gain insights into the crystalline structure of the synthesized nanoparticles. Furthermore, the average size of the nanoparticles was determined using the Debye-Scherrer equation: D=kλ/βcosθ.

Where D is the average size of the nanoparticles, k is a constant (typically 0.9), λ is the wavelength of the X-ray radiation, β is the Full Width at Half Maximum (FWHM) of the XRD peak and θ is the Bragg angle. This equation allowed for the calculation of nanoparticle size based on the XRD data.

FTIR analysis was performed using a SHIMADZU IR Spirit instrument (Japan). Transmittance spectra were recorded across the wave number range of 4000-600 cm^–¹ at a resolution of 16 cm^–¹. This approach facilitated a comprehensive investigation of the functional groups within samples synthesized from E. cardamomum seed and pod extracts. The FTIR analysis involved examining absorption peaks and their respective wave numbers to gain critical insights into the chemical composition and molecular structure of the extracted compounds.

SEM was employed to assess the morphological characteristics of the synthesized AuNPs. The examination was conducted using a Carl Zeiss instrument from Germany. Samples for SEM analysis were prepared using standard techniques, where a thin layer of the nanoparticle dispersion was deposited onto a suitable substrate. These prepared samples were then subjected to SEM imaging under appropriate operational conditions and magnifications. SEM analysis provided high-resolution visualization of the shape, size and distribution of the AuNPs produced from E. cardamomum seed and pod extracts.

EDX was utilized to analyze the elemental composition of AuNPs synthesized from extracts of both seeds and pods of E. cardamomum. The EDX measurement was performed using an EDS X-ray spectrophotometer integrated with the SEM instrument (ZEISS EVO) from Oxford Instruments, United Kingdom. During the EDX analysis, X-ray spectra emitted from the sample surface upon electron beam excitation were captured. This technique enabled both qualitative and quantitative assessment of the elemental constituents present within the synthesized nanoparticles.

TEM analysis was conducted to examine the morphology, size and distribution of AuNPs synthesized from extracts of E. cardamomum seeds and pods. TEM samples were prepared by depositing a drop of the nanoparticle solution onto a grid and allowing it to air dry at room temperature.

The dried grids were then analyzed using a JEOL 2000 FX-II TEM microscope from Japan at the appropriate magnification settings. This approach enabled high-resolution imaging and detailed observation of individual nanoparticles. Additionally, Selected Area Electron Diffraction (SAED) was employed during TEM analysis to investigate the crystalline structure of the nanoparticles. SAED provided valuable insights into the crystallographic orientation and atomic arrangement within the synthesized nanoparticles.

The anti-bacterial activity was carried out using three different multi-drug resistant strains such as Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. All of the test organisms were received from the Genolites Research and Development Laboratory in Coimbatore. The test organisms were kept at 4ºC in Mueller-Hinton Agar and sub cultured once a month.

The antibacterial activity of E. cardamomum seed and pod mediated nanoparticles was evaluated using standard microbiological techniques. Initially, Mueller Hinton agar plates were prepared and inoculated with an overnight culture of test organisms including Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus at a concentration of 5×10⁶ colony-forming units per milliliter (cfu/mL). Wells of 6 mm diameter were made in the agar, into which 100 µL of the respective E. cardamomum seed and pod nanoparticles were introduced. Following incubation overnight, the plates were examined for zones of inhibition around the wells and the diameters of these zones were measured in millimeters to assess the antibacterial efficacy of the nanoparticles.

Subsequently, the nanoparticles that demonstrated significant inhibition zones were selected for further comparison with standard antibiotics using the disc diffusion method. Specifically, discs containing standard antibiotics such as cefmetazole (30 µg), ampicillin (10 µg), tetracycline (30 µg) and ciprofloxacin (5 µg) were placed on Mueller-Hinton agar plates previously seeded with the respective bacterial strains. Simultaneously, 100 µL of the selected nanoparticle suspension was introduced into a separate well (6 mm) on the agar surface. After overnight incubation, the diameters of the zones of inhibition around both the nanoparticle wells and antibiotic discs were measured to compare the antibacterial effectiveness of the nanoparticles with that of standard antibiotics against the test bacterial strains (Boggulaet al., 2017).

MIC of E. cardamomum pod mediated AuNPs against various test bacterial strains was determined using the Resazurin method, following procedures outlined by Elshikhet al.,2016.

Initially, 100 μL of E. cardamomum pod mediated AuNPs were added to the first row of a sterile 96-well microtiter plate, while 50 µL of nutrient broth was added to each subsequent well to facilitate serial dilutions. This created a gradient of decreasing nanoparticle concentrations across the plate. Subsequently, 50 µL of each dilution was inoculated with the respective test organisms at a concentration of 5×10⁶ cfu/mL, followed by the addition of 10 µL of Resazurin solution to each well. The microtiter plate was then loosely covered with cling film to prevent desiccation and was placed in an incubator set at 37ºC overnight. Post-incubation, wells showing a color change from purple to pink indicated bacterial growth, with the MIC recorded as the lowest concentration of nanoparticles inhibiting this color change (Sarkeret al., 2007).

The in vitro antioxidant activity of the AuNPs synthesized using seed and pod of E. cardamomum was determined using the following methods: DPPH (2,2-Diphenyl-1-Picrylhydrazyl) scavenging method, FRAP (Ferric ion Reducing Antioxidant Power) assay, Hydrogen peroxide scavenging assay, reducing power assay and phosphomolybdenum assay. Ascorbic acid was used as standard.

The DPPH radical scavenging activity of AuNPs synthesized using aqueous extracts from seeds and pods of E. cardamomum was evaluated following the method outlined by Shimadaet al., 1992, Various concentrations (25, 50, 75, 100, 250, 500, 750 and 1000 μg/mL) of AuNPs synthesized from seed and pod extracts were prepared separately and combined with 3 mL of freshly prepared methanolic DPPH solution (0.1 mM). The mixture was then incubated at 37ºC for 30 min, after which the absorbance was measured at 517 nm using a spectrophotometer. Each sample was analyzed in triplicate to ensure reliability and accuracy of the results.

FRAP assay was employed to assess the ability of AuNPs synthesized from aqueous extracts of E. cardamomum seed and pod to reduce Fe³⁺ ions to Fe²⁺ in the presence of 2,4,6-Tripyridyl-s-Triazine (TPTZ). Different concentrations (25, 50, 75, 100, 250, 500, 750 and 1000 μg/mL) of AuNPs synthesized from seed and pod extracts were prepared separately and mixed with 3 mL of FRAP reagent. The reaction mixture was incubated at 37ºC for 10 min to facilitate the reduction reaction, after which the absorbance was measured at 593 nm using a UV-vis spectrophotometer (Guoet al., 2003).

The Hydrogen peroxide (H₂O₂) scavenging activity of AuNPs synthesized from aqueous extracts of E. cardamomum seed and pod was evaluated following the methodology described by Sivakumaret al., 2015, AuNPs at concentrations ranging from 25 to 1000 μg/mL were prepared separately and mixed with 2 mL of hydrogen peroxide solution (10 mM, pH 7.4). The mixtures were then incubated at 37ºC for 10 min to allow the scavenging reaction to occur. After incubation, the absorbance of the reaction mixture was measured at 230 nm using a spectrophotometer.

The Reducing Power Assay of AuNPs synthesized using aqueous extracts from seeds and pods of E. cardamomum was conducted following a modified protocol based on Sutharsinghet al., 2011 AuNPs at concentrations ranging from 25 to 1000 μg/mL were prepared separately and mixed with 1 mL of phosphate buffer (1%) and 1 mL of potassium ferrocyanide (1%). The mixtures were then incubated at 50ºC for 20 min to facilitate the reduction reaction. After incubation, 1 mL of trichloroacetic acid (10%) was added to each test tube, followed by centrifugation for 10 min. The supernatant (1.5 mL) was mixed with an equal volume of distilled water (1.5 mL) and 300 μL of 0.1% Ferric Chloride (FeCl₃) was added. The absorbance of the resulting solution was measured at 700 nm using a spectrophotometer to determine the reducing power of AuNPs.

The antioxidant activity of AuNPs synthesized from extracts of E. cardamomum seed and pod was assessed using the phosphomolybdenum assay as per the method outlined by Sahreenet al., 2010, A reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate was prepared. Varying concentrations (25, 50, 75, 100, 250, 500, 750 and 1000 μg/mL) of synthesized AuNPs were separately added to 1 mL of the reagent solution. The reaction mixtures were incubated in a water bath at 95ºC for 90 min and subsequently cooled to room temperature. The absorbance of each sample was measured at 765 nm using a UV-vis spectrophotometer to determine the total antioxidant capacity of the AuNPs.

The in vitro anti-inflammatory activity of the AuNPs synthesized from seed and pod of E. cardamomum was examined by the albumin denaturation method and heat-induced hemolysis method. Aspirin was used as standard.

The albumin denaturation assay was conducted following the protocol adapted from Govindappaet al., 2018, with minor changes. The reaction mixture included 3 mL of 5% aqueous bovine albumin solution and varying concentrations of AuNPs synthesized from seed and pod extracts of E. cardamomum. The test tubes were first incubated for 20 min at 37ºC, followed by heating at 50ºC for another 20 min and subsequently allowed to cool to room temperature. The turbidity of each reaction mixture was then measured spectrophotometrically at 660 nm wavelength to assess the extent of albumin denaturation inhibition by the AuNPs.

2 mL of human blood was obtained from a healthy volunteer and an erythrocyte suspension (10% v/v) was prepared using normal saline following established methods (Sakatet al., 2010; Sadique et al., 1989).

For the heat-induced hemolysis assay, varying amounts of AuNPs synthesized from seed and pod extracts of E. cardamomum were separately mixed with 3 mL of the erythrocyte suspension. The mixtures were then incubated at 55ºC for 30 min to induce hemolysis. After incubation, the reaction mixtures were centrifuged for 10 min to separate the supernatant containing hemoglobin released from lysed erythrocytes. The optical density of the supernatant was measured at 560 nm using a spectrophotometer to quantify the extent of hemolysis.

AuNPs were successfully synthesized using aqueous extracts from seeds and pods of E. cardamomum. The extracts from both seed and pod appeared pale yellow prior to synthesis. Following the reaction, a distinct dark violet coloration was observed in both reaction mixtures, indicating the reduction of Au³⁺ ions to Au nanoparticles. This color change serves as visual evidence of the effective bio-reduction capability of the seed and pod extracts, facilitating the formation of stable AuNPs through the reduction process. Thus, both extracts demonstrated their potential as efficient bio-reductants in the synthesis of gold nanoparticles Figure 1 (A) and (B).

The UV spectra of AuNPs synthesized using aqueous extracts from E. cardamomum seed and pod are illustrated in Figure 2 (A) and (B) respectively. The spectrum of pod-mediated AuNPs exhibited a prominent peak at 545 nm, whereas the seed-mediated AuNPs showed a strong absorption band centered around 550 nm. The Surface Plasmon Resonance (SPR) bands for nanoparticles mediated by seed and pod extracts were observed in the range of 530-565 nm, indicating the characteristic absorption of AuNPs. The broadening of these peaks in the UV spectra suggests the monodispersity of the synthesized nanoparticles. Notably, no significant color change or distinct absorption peaks were observed in the aqueous extracts of seed and pod alone, underscoring the role of E. cardamomum extracts in facilitating the synthesis and stabilization of gold nanoparticles with well-defined optical properties.

Both samples from this study were subjected to XRD analysis using a Rigaku Miniflex 600 X-ray diffractometer. Cu Kα radiation (λ=1.5406 Å) was employed as the X-ray source and diffraction patterns were recorded at room temperature over a 2θ range of 10-80º with a step size of 0.02º. The XRD patterns for AuNPs synthesized using E. cardamomum seed and pod extracts are presented in Figure 3 (A) and (B), respectively, facilitating structural characterization. Both diffractograms exhibited prominent peaks indicative of the presence of AuNPs. Specifically, characteristic peaks for gold (Au) at (111), (200) and (220) were observed at 2θ values of 38.29º, 44.43º and 64.68º, confirming the crystalline nature of the synthesized nanoparticles. Additional unidentified peaks were also detected in the XRD patterns, suggesting potential minor crystalline phases or impurities in the synthesized AuNPs.

FTIR spectra of E. cardamomum seed extract, pod extract and their respective mediated AuNPs are presented in Figures 4 and 5, respectively. The FTIR spectrum of E. cardamomum seed extract revealed multiple absorption peaks at 1641.09, 3162.10, 3207.85, 3253.59, 3316.49, 3333.65, 3390.83, 3413.70, 3470.88 and 3522.34 cm^-1. For seed-mediated AuNPs, characteristic IR absorption bands were observed at 1521.01, 1641.09, 3179.26, 3242.16, 3305.05, 3356.52, 3379.39, 3448.01 and 3522.34 cm^-1. Similarly, the FTIR spectrum of E. cardamomum pod extract exhibited absorption bands at 1641.09, 3173.54, 3236.44, 3299.34, 3350.80, 3390.83, 3407.98, 3442.29 and 3505.19 cm^-1, while pod-mediated AuNPs displayed peaks at 1641.09, 3179.26, 3219.28, 3236.44, 3305.05 and 3413.70 cm^-1. The spectra of both extracts and their corresponding AuNPs exhibited similar patterns with slight variations in wavenumbers, indicative of successful synthesis and functionalization of AuNPs using seed and pod extracts. Key absorption peaks at 1641.09 cm^-1 corresponded to aromatic compounds (C-H bending), 3236.44 cm^-1 represented alcohol groups (O-H stretching) for pod extract and its AuNPs and peaks at 3407.98 cm^-1 and 3413.70 cm^-1 indicated amine groups (N-H stretching) for pod extract and pod-mediated AuNPs, respectively. Additionally, the vibrational band at 3522.34 cm^-1 for seed extract and its AuNPs corresponded to strong O-H bonds (alcohol), while peaks at 3333.65 cm^-1 and 3379.39 cm^-1 indicated amine groups (N-H stretching). These findings underscore the consistent biochemical features and functional groups involved in the synthesis and stabilization of AuNPs using E. cardamomum extracts.

The morphology of AuNPs synthesized using extracts from seeds and pods of E. cardamomum was characterized using SEM. SEM images depicted in Figure 6 (A) and (B) illustrate the morphology of AuNPs synthesized from seed and pod extracts, respectively. The analysis revealed that the synthesized AuNPs exhibited an average size of approximately 20 nm and a spherical shape under room-temperature synthesis conditions. This uniformity in size and shape suggests efficient reduction and stabilization of AuNPs using E. cardamomum extracts.

EDX was employed to analyze the elemental composition of Gold Nanoparticles (AuNPs) synthesized using E. cardamomum seed and pod extracts (Figure 7 (A) and (B)). The EDX spectra of both seed and pod-based AuNPs exhibited strong absorption band peaks at 2.2 keV, characteristic of gold absorption. This indicates the presence of elemental gold in the synthesized nanoparticles. Additionally, weak signals corresponding to other elements were observed in each spectrum of AuNPs, suggesting potential trace elements or impurities present in the samples. The EDX results confirm the predominant composition of gold in both seed and pod-mediated AuNPs, corroborating their successful synthesis and purity using E. cardamomum extracts as reducing agents.

TEM analysis was conducted on all samples to investigate the morphology and size distribution of the synthesized nanoparticles. As depicted in Figure 8, the majority of the samples exhibited geometric shapes with relatively low shape homogeneity, predominantly measuring around 20 nm in size. Figure 9 illustrates a broader size distribution, with particle sizes ranging from 5 nm to 65 nm. The median diameter, representing the average particle size, was estimated to be approximately 26 nm. These TEM findings provide detailed insights into the morphology and size characteristics of the synthesized nanoparticles using E. cardamomum extracts, highlighting both the uniformity and variability in particle sizes among the samples

E. cardamomum pod-mediated AuNPs demonstrated potent bactericidal activity against the tested bacterial strains Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus (Figure 10). In contrast, AuNPs synthesized from E. cardamomum seeds did not exhibit significant antibacterial effects against these organisms. The pod-mediated AuNPs showed the highest zone of inhibition against Klebsiella pneumoniae, measuring 13 mm, followed by 12 mm for Staphylococcus aureus and 10 mm for Escherichia coli. These findings highlight the effective antimicrobial potential of E. cardamomum pod-mediated AuNPs, suggesting their promising application in combating bacterial infections caused by these pathogenic strains.

AuNPs synthesized from E. cardamomum pods exhibited exceptional antibacterial efficacy compared to standard antibiotics such as cefmetazole, ampicillin, tetracycline and ciprofloxacin, all of which showed resistance against the tested bacterial strains. None of the antibiotics produced inhibitory zones against the bacterial strains tested. Among the bacterial strains evaluated, Klebsiella pneumoniae showed the highest susceptibility to E. cardamomum pod-mediated AuNPs, with a maximum zone of inhibition measuring 13 mm. The detailed antibacterial activity of the synthesized AuNPs is summarized in Table 1.

Figure 9:
(A) Particle size analysis of E. cardamomum seed-mediated synthesis of AuNPs; (B) Particle size analysis of E. cardamomum pod-mediated synthesis of AuNPs.

MIC values of E. cardamomum pod-mediated AuNPs are presented in Figure 11. Among the tested bacterial strains, Klebsiella pneumoniae exhibited the lowest MIC value of 12.5 μg/mL, indicating its highest susceptibility to E. cardamomum pod-mediated AuNPs. Escherichia coli showed a higher MIC value of 100 μg/mL, indicating greater resistance compared to Staphylococcus aureus, which had an MIC of 50 μg/mL. These MIC values underscore the differential susceptibility of bacterial strains to E. cardamomum pod-mediated AuNPs, highlighting their potential as effective antimicrobial agents against clinically relevant pathogens, particularly Klebsiella pneumoniae.

The E. cardamomum seed and pod-mediated AuNPs exhibited concentration-dependent inhibition of DPPH radical (Figure 12). The maximum scavenging activity of pod-mediated nanoparticles was 31.94% at the concentration of 10 mg/mL, whereas for the seed-mediated nanoparticles, it was 15.87% at the same concentration. The IC₅₀ value for Pod and seed-mediated nanoparticles were 16.02 and 43.69 mg/mL, respectively. The lowest inhibition activity recorded was 9.55% and 12.67% at the concentration of 0.25 mg/mL of seed and pod-mediated AuNPs, respectively. The Pod-mediated AuNPs showed more significant DPPH radical scavenging activity than the seed-mediated nanoparticles.

The data showed the maximum FRAP value of pod and seed-mediated nanoparticles were 65.62% and 64.55% respectively at the concentration of 1000 μg/mL (Figure 13). The IC₅₀ value was found to be 3.43 μg/mL for seed-mediated AuNPs and 1.28 μg/mL for pod-mediated AuNPs. The minimum inhibition was observed at the concentration of 25 μg/mL of the seed-mediated AuNPs (43.6%) and pod-mediated AuNPs (47.85%) respectively. Hence, these findings indicated that the pod-mediated AuNPs possess higher FRAP activity than the seed-mediated AuNPs.

The data showed the maximum hydrogen peroxide scavenging value of Pod-mediated AuNPs was 38.72% and the IC₅₀ value obtained was 11.42 μg/mL. The Seed-mediated AuNPs exhibited the highest hydrogen scavenging activity of 22.39% and the IC₅₀ was found to be 19.44 μg/mL. The percentage of hydrogen peroxide scavenging activity of E. cardamomum seed and pod-mediated AuNPs was depicted in Figure 14. The hydrogen peroxide scavenging activity was observed to be increased with the increasing concentration of test samples such as seed and pod-synthesized AuNPs. The highest hydrogen scavenging activity was observed in the E. cardamomum pod-mediated AuNPs.

The Reducing power assay showed the maximum inhibition value obtained for Pod-mediated AuNPs was 74.41% (IC₅₀=4.86 μg/mL), whereas the reducing activity was not found in the case of Seed-mediated AuNPs. The minimum percentage of inhibition appeared to be 23.25% for the 25 μg/mL concentration of E. cardamomum pod-mediated AuNPs. The results of in vitro antioxidant activity analyzed using a reducing power assay were presented in Figure 15.

The Phosphomolybdenum activity of AuNPs synthesized from the E. cardamomum seed and pod was increased with elevated concentration of the extracts (Figure 16). The maximum value of pod-mediated nanoparticles was 97.05%, whereas 76.83% for the Seed-mediated nanoparticles at the concentration of 1000 μg/mL. The IC₅₀ value for Pod and Seed-mediated nanoparticles were 3.65 and 6.2 μg/mL, respectively. Hence, the highest percentage of phosphomolybdenum activity has occurred for the pod-mediated AuNPs than the Seed-mediated AuNPs.

In this current study, the in vitro anti-inflammatory activity of synthesized AuNPs was analyzed by the inhibitory activity against the denaturation of protein albumin (Figure 17). The E. cardamomum pod and Seed-mediated AuNPs inhibited the protein denaturation in a concentration-related manner. The highest inhibitory activity against albumin denaturation observed was 99.21% for pod-mediated AuNPs and 82.56% for Seed-mediated nanoparticles at the 1000 μg/mL concentration respectively. The IC₅₀ value calculated for the AuNPs synthesized from the seed and pod of E. cardamomum was 5.21 μg/mL and 1.29 μg/mL, respectively. The lowest percentage of inhibition against the denaturation of albumin was found to be 8.67% at 25 μg/mL concentration, whereas it was 85.43% for the pod-mediated AuNPs at the same concentration. Hence, these results clearly indicated that the E. cardamomum pod-mediated AuNPs have inhibited the albumin denaturation more effectively than the Seed-mediated AuNPs.

The in vitro anti-inflammatory potential of the AuNPs of E. cardamomum was determined by membrane stabilization of Red Blood Cells at various concentrations (Figure 18). All the tested concentrations of AuNPs synthesized from seed and pod of E. cardamomum respectively inhibited the heat-induced hemolysis and these results contributed additional evidence for their anti-inflammatory effect. The maximum percentage inhibition was recorded as 96.53% (IC₅₀=5.61 μg/mL) and 99.21% (IC₅₀=5.16 μg/mL) for seed and pod-mediated AuNPs. The results demonstrated that Pod-mediated AuNPs exhibited effective membrane stabilizing activity by preventing the lysis of erythrocytes induced by heat than the seed-mediated AuNPs.

Spices and aromatic plants have long played a crucial role in traditional medicine across various cultures. Spices serve not only as flavor enhancers, colorants and preservatives but also as home remedies for a range of ailments (Viuda-Martos et al., 2010). E. cardamomum is renowned for its pharmacological properties, including cytotoxic, antibacterial, anti-oncogenic and antioxidant effects (Ashokkumaret al., 2020). In this study, AuNPs were successfully synthesized using an aqueous extract of E. cardamomum seeds and pods as reducing agents. The formation of AuNPs was evidenced by a color change from pale yellow to dark violet. To our knowledge, this study represents the first attempt to compare the antibacterial, antioxidant and anti-inflammatory properties of AuNPs synthesized from E. cardamomum seeds and pods.

The AuNPs synthesized from different concentrations of E. cardamomum extracts, such as 1:1 and 1:10, exhibited characteristic absorption peaks at 530 nm and 550 nm, respectively. Previous studies have reported similar findings, noting a surface Plasmon resonance (Pattanayak and Nayak, 2013). (SPR) band at 527 nm for AuNPs synthesized using aqueous extracts of E. cardamomum seeds (Rajanet al., 2017), and a sharp peak at 526 nm for those synthesized with black cardamom extract (Singh and Srivastava, 2015). Shahet al., 2022, demonstrated an absorption peak at 535 nm for AuNPs synthesized using Sageretia thea leaves, with absorption intensity increasing proportionally with AuNP concentration in the reaction mixture.

The X-ray Diffraction (XRD) studies of AuNPs of aqueous extract prepared using Amomum villosum and E. cardamomum exhibited four distant diffraction peaks at (111), (200), (220) and (311) with 2θ values of 38.49º, 44.44º, 65.07º and 77.82º respectively (Soshnikovaet al., 2018). However, the characteristic intense peaks at 2θ=(111) 38.47º, (200) 44.53º, (220) 64.80º, (311) 77.86º and (222) 82.65 was reported for the green synthesized AuNPs of E. cardamomum seeds by Rajanet al., 2017, Moreover, the previous study carried out by Chenet al., 2019, showed that the XRD peaks were located at 2θ=38.179º (111), 44.375º (200), 64.50º (220), 77.546º (311) and 81.700º (222) for the green synthesized AuNPs of Chenopodium formosanum shell recorded using Cu Ká radiation (λ=1.54184 Å).

The FTIR analysis of Annona muricata leaf extract showed the IR absorption bands at 1635.99, 2114.93 and 3284.47cm^-1 whereas, the 1637.82, 2111.91 and 3271.14 cm^-1 for AuNPs synthesized leaf extract of Annona muricata (Folorunsoet al., 2019). The E. cardamomum seed extract yields strong bands at 1689, 1632, 1536, 1360, 1159, 1024 and 730 cm-1. A minor shift in wave numbers may be seen in the spectrum of AuNPs with a relatively identical peak (Rajanet al., 2017), which was compromised with our results. Due to the presence of NH or OH groups in the extract, Au+3 ions were converted to Au metal. The change in the peak of the carboxylic ketonic group from 1,728 to 1,635 cm1 may be an indication of the formation of AuNPs (Bawazeeret al., 2022). Likewise, the findings of Soshnikovaet al., 2018, demonstrated that the primary -OH alcohol, C=C, aliphatic -CH and alcohol C-O groups, which are responsible for the vibrational bands at 3375.45, 2928.54, 1608.89 and 1022.17 cm-1 in the FTIR spectra of dried fruits of Amomum villosum and synthesized AuNPs.

SEM analysis of Gold Nanoparticles (AuNPs) synthesized from Garcinia kola (ripe fruit) revealed the presence of spherical-shaped particles (Akinteluet al., 2021). Similarly, SEM images of AuNPs synthesized from Ziziphus zizyphus leaf extract exhibited spherical and poly-crystalline particles (Aljabaliet al., 2018) Rajanet al., 2017, reported the synthesis of 100 nm-sized AuNPs from E. cardamomum seeds, a finding consistent with the current study, which also observed spherical-shaped AuNPs of approximately 100 nm in size through SEM examination.

Bawazeeret al., 2022, found strong signals for Gold (Au) atoms at 0.4 and 2.7 keV in the EDX profile of black pepper-synthesized AuNPs, with weaker signals at 6.2, 9.8 and 11.6 keV. Similarly, Alhumaydhiet al., 2021, observed strong signals at 0.5 and 2.3 keV, with a weak signal at 9.8 keV in saffron-based nanoparticles. Bahattabet al., 2021, also confirmed the formation of AuNPs from black cumin seeds using EDX analysis, noting substantial signals at approximately 2 keV and weaker signals at 9.7, 10.2 and 11.7 keV.

Transmission Electron Microscopy (TEM) analysis by Rajanet al., 2017, showed 100 nm-sized AuNPs synthesized from E. cardamomum seeds. Salariet al., 2019, reported the synthesis of spherical-shaped AuNPs ranging from 2 to 10 nm using aerial parts of Cuminum cyminum. Particle size analysis of E. cardamomum AuNPs revealed nanoparticles with diameters averaging 432.3 nm (Pattanayak and Nayak, 2013). Sunderamet al., 2019, found an average size of 40 nm for AuNPs synthesized from Anacardium occidentale leaves.

Regarding antibacterial activity, Rajanet al., 2017, reported moderate effects of E. cardamomum seed-synthesized AuNPs against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, contrasting our results. Omprakash and Sharada, 2015, found considerable bactericidal activity of silver nanoparticles from E. cardamomum seeds against Bacillus subtilis and Klebsiella planticola. Moulai-Haceneet al., 2020, reported an MIC of 6.25 mg/mL^-1 for E. cardamomum seed extract against Carnobacterium maltoaromaticum, Bacillus cereus and Enterobacter sp. Poyil and Shamna, 2022, found MIC values of 7.8 mg for multi-drug resistant Klebsiella pneumoniae and 3.9 mg for Staphylococcus aureus using methanolic E. cardamomum pod extract.

In this study, AuNPs synthesized from E. cardamomum seeds and pods exhibited dose-dependent in vitro antioxidant activity, with pod-mediated AuNPs demonstrating significant antioxidant potential. Similar concentration-related DPPH radical scavenging activity was reported for Cardamom fruits (Soshnikovaet al., 2018). Prakashet al., 2012, found IC₅₀ values of 8.25±2.0 and 21.6±2.0 for ethanolic and aqueous extracts of large cardamom (Amomum subulatum), respectively, in the DPPH assay. Bhattiet al., 2010, reported an IC50 value of 17.26 µg/mL for methanolic E. cardamomum (70%) in the DPPH assay. Singhet al., 2008, recorded potent antioxidant activity for essential oils and oleoresins from E. cardamomum seeds and pods, supporting our findings. Aristaet al., 2023, highlighted the antioxidant potential of Cardamom (Amomum compactum) fruits, enriched with bioactive compounds. Tahiret al., 2015, found effective inhibition of free radicals by AuNPs synthesized from Nerium oleander leaves, comparable to our results.

Furthermore, this study demonstrated that E. cardamomum pod-synthesized AuNPs exhibited more pronounced in vitro anti-inflammatory effects compared to seed-synthesized AuNPs. Souissiet al., 2020, reported similar effects using seed extract. Arpithaet al., 2019, found significant reduction in inflammation with E. cardamomum seed oil and resin in a carrageenan-induced paw edema model. Kandikattuet al., 2017, observed reduced paw inflammation with intraperitoneal hexane extract of Elettaria repens (big cardamom) in the same model. This study highlights the multifaceted potential of E. cardamomum nanoparticles in combating microbial infections, oxidative stress and inflammatory conditions. The environmentally friendly synthesis process underscores the sustainable approach of utilizing natural resources for nanoparticle production.

In conclusion, AuNPs synthesized using the aqueous extracts of E. cardamomum seed and pod, characterized by their spherical morphology and average size of 20 nm. The AuNPs synthesized from E. cardamomum pods exhibited notable antioxidant properties, as evidenced by their performance in various assays including DPPH, FRAP, hydrogen peroxide scavenging, reducing power and phosphomolybdenum assay. Additionally, these nanoparticles demonstrated significant anti-inflammatory effects by effectively inhibiting albumin denaturation and heat-induced hemolysis. While exhibiting moderate antibacterial activity against tested strains, the pod-mediated nanoparticles showed superior antioxidant, antibacterial and anti-inflammatory properties compared to those synthesized from seeds.

This comparative analysis underscores the potential of E. cardamomum pods as a promising natural source for synthesizing AuNPs with robust pharmacological effects. The cost-effectiveness and widespread availability of E. cardamomum pods further enhance their suitability for large-scale nanoparticle production. Overall, this research contributes valuable insights into the medicinal applications of plant-mediated nanoparticles, highlighting the significance of exploring natural resources for synthesizing nanomaterials with therapeutic potentials.

The authors are grateful to the management as well as the faculty members in the, Department of Biochemistry, Kongunadu Arts and Science College (Autonomous), G.N. Mills P.O, Coimbatore-641029, Tamil Nadu, India for providing the support to fulfil this work.