High-yield biogenic fabrication and phytochemical screening of silver nanomaterials (AgNMs) from Kaempferia parviflora rhizome extract

Nanotechnology is one of the promising scientific advancements that has captured widespread interest across various industries, notably in medicine. The utilization of plants for the synthesis of silver nanomaterials (AgNMs) has emerged as a promising and eco-friendly approach, offering cost-effective solutions for potential biomedical applications. The study aims to optimize the efficiency of biogenic AgNMs fabrication by employing Kaempferia parviflora aqueous extraction (KP-AE) as both a reducing and encapsulating agent, thus optimizing the yield of AgNMs. Phytochemical screening was conducted to identify the phytochemical compounds present in KP-AE. Various parameters were optimized, including pH, temperature, and the ratio of KP extract to AgNO 3 , over different incubation periods. The synthesized AgNMs were analyzed spectroscopically and microscopically using UV-Vis and SEM techniques. At varying concentrations of KP and AgNO 3 , the KP-AE AgNMs were successfully biogenic fabricated, but the yields varied. As the concentrations of AgNO 3 increased, a greater yield of KP-AE AgNMs was achieved. Phytochemical screening KP-AE demonstrated the presence of potential phytochemicals such as alkaloids, saponins, tannins, flavonoids, terpenoids, glycosides, and oils that assisted in the biogenic fabrication of AgNMs. This study established an efficient, affordable, and ecologically sustainable approach for fabricating stable AgNMs using KP-AE AgNMs. Synergistically, reducing and capping potential has been achieved by combining the plant extract in plant-mediated biogenic fabrication, producing stabilized NMs compared to those produced individually. The AgNMs derived from KP-AE exhibit robust antioxidant properties, showcasing promise for further exploration in pharmaceutical applications. Additional research is needed to investigate the biological potential and pharmacological properties of the biogenic fabricated KP-AE AgNMs.


INTRODUCTION
Over recent decades, significant advancements have been made in developing a reproducible technique for biogenic production of nanomaterials.Concurrently, there has been a growing market for chemical -free, eco-friendly manufacturing processes.Nanomaterials, owing to their extensive exploration, versatility, and captivating utility, offer myriad solutions across diverse industries such as medicine, nutrition, and energy.The term  Table 1 Phytochemicals constituents of Kaempferia parviflora rhizomes

METHODOLOGY Plant material
The rhizome of Kaempferia parviflora, originally sourced from Thailand, was procured from Kelantan, Malaysia.A voucher specimen (KM0071/23) was authenticated at the Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM).The stems of the freshly obtained rhizomes underwent meticulous cleaning, peeling, and subsequent freeze-drying to produce Kaempferia parviflora rhizome powder.This powder was finely ground using a grinder (Retsch Ultra Centrifugal Mill ZM200, Haan, Germany).The resulting finely ground material was carefully stored in a glass jar, ensuring airtightness, and placed in a chiller maintained at a temperature ranging between 4 and 8 °C for future use.Kaempferia parviflora (AgNMs) was fabricated using silver nitrate (AgNO3) and optimized at the Research and Diagnostic Laboratory at Management and Science University, Malaysia.Preparation of plant material The aqueous deionized water was used to extract Kaempferia parviflora (KP-AE) from the dried rhizomes following a combined method from Numat et al. (2022) and Khalir et al. (2022).The magnet stirrer was used when mixing 50 mL of distilled water with 2.5 grams of rhizome powder.The mixture was heated up for 15 minutes at 60 °C.The extraction solution was then centrifuged for 10 minutes at 9000 rpm, filtered through Whatman filter paper No. 1, and used to reduce Ag + ions into biogenic production of KP-AE AgNMs.The extract aliquots were used as a capping and reductive material and stored between 4 and 8 °C, Numan et al. (2022) and Khalir et al., (2022).

Biogenic fabrication of silver nanomaterial
In a titration flask, 90 mL of 2 mM (AgNO3) and 10 mL of KP-AE were mixed.The mixture is then consistently stirred at 400 rpm with a magnetic stirrer until the temperature reaches 60°C.While reducing silver nanomaterial from Ag + to Ag 0 ions, aluminium foil was wrapped around the flask to keep the light away.The mixture was left to sit in the dark to complete the biogenic reduction.Control AgNO3 was kept throughout the time without mixing with KP-AE.The initial noticeable reduction of Ag + ions to Ag 0 silver nanomaterial was observed from the colour changes from clear to brown.From observing the absorbance peak with UV-visible spectroscopy, KP-AE AgNMs was produced effectively using biogenic manufacturing (Liaqat et al., 2022).

Collection biogenic fabrication of silver nanomaterial
Visual observation of the colour produced was used to determine the reduction of silver nanomaterial Ag + to Ag 0 .The transformation from a light purple colour to a brownish-dark brown coloration can be visually observed (Iravani et al., 2014).The unconverted Ag + ions and KP-AE residue were separated from the biogenic fabrication of KP-AE AgNMs by centrifugation at 6000 rpm for 30 minutes (Sorvall ST 16R, Thermo Scientific, Waltham, MA, USA).The KP-AE AgNMs was retrieved after double rinsing and washing and then redistributed in double-distilled water.Soon after re-dispersion, the KP-AE was freeze-dried using a tabletop lyophilizer (ScanVac Coolsafe 110-4).The KP-AE-AgNMs were obtained and stored at 4°C in a light-protected environment.Analysis of the biogenic fabrication KP-AE AgNMs using a UV-Vis spectrophotometer (Multiskan go, Thermo Scientific, Waltham, MA, USA) revealed a peak at approximately 428 nm, which is associated with the establishment and development of AgNMs.

Stability analysis
The optimized fabricated KP-AE AgNMs solutions were kept in the dark for 30 days, and the stability of synthesized AgNMs was determined using UV-Vis spectral analysis.

Percentage yield
To determine the percent yield accurately (Table 3), the initial step involves determining the anticipated quantity of the product through stoichiometry.This value is termed the theoretical yield, representing the maximum achievable amount of product based on the given reactant quantities.On the other hand, the actual yield signifies the quantity of product obtained through practical execution of the reaction in a laboratory context.The percent yield, expressed as a percentage, is the ratio of the actual yield to the theoretical yield, providing insight into the efficiency of the reaction.

Qualitative analysis of phytochemicals
Phytochemical analysis for the qualitative detection of biologically important compounds, including alkaloids, saponins, tannins, flavonoids, phenols, terpenoids, glycosides in Kaempferia parviflora, were examined according to the method with slight modifications by (Thakor et al., 2023); (Kancherla et al., 2019); (Akullo et al., 2023;Muhammad Abdulrazak et al., 2016).These phytochemicals in Kaempferia parviflora are responsible for the biogenic fabrication of Ag + to Ag 0 that clings onto nanomaterials and increases their biological characteristics and stability of KP-AE AgNMs.

Alkaloids screening (Hager's test)
2 mL of Hager's reagent will be added into 2 mL of 1% aqueous black ginger extract.The presence of alkaloids will be observed by the formation of yellow precipitates.Saponin screening 2 mL of 1% aqueous black ginger extract will be added into 2 mL of distilled water.The mixture is then stirred in a test tube for 15 minutes until it is homogenous.The presence of saponin will be observed by the formation of a 1 cm layer of frothing foam.

Tannin screening
A solution containing 2 mL of 1% aqueous black ginger extract will be combined with 2 mL of ferric chloride (FeCl3) solution with a concentration of 5%.The presence of dark shade bluish or greenish black indicates the presence of tannin.

Flavonoids screening
A solution containing 1 mL of 2N NaOH will be combined with 2 mL of black ginger extract with the concentration of 1% in water.The presence of flavonoids will be observed by the appearance of yellow colour.

Terpenoids screening (Salkowski test)
A solution containing 1 mL of black ginger extract with a concentration of 1% in water was combined into 2 mL of chloroform.Subsequently, a layer will be formed by cautiously adding 3 mL of concentrated sulfuric acid.The presence of terpenoid will be observed by a reddish-brown hue on the interface of the solution.

Glycoside screening (Benedict's test)
5 mL of Benedict's reagent will be combined with 8-10 drops of black ginger extract, and the mixture will be subsequently heated for 5 minutes using a magnetic stirrer and a hot plate.The presence of carbohydrates will be confirmed by the formation of a dark crimson precipitate.

Visual evaluation
Visual inspection will be conducted to observe the colour changes in the reaction medium of KP-AE AgNMs, which will indicate the reduction of Ag + to Ag 0 .

Scanning electron microscopy analysis
The morphology and size of KP-AE AgNMs were examined by scanning electron microscope (SEM) (Quanta 250 FEG), which was used in high-vacuum mode and supplied with a 15 kV acceleration voltage.

Phytochemical analysis
The phytochemical analysis was conducted to identify the chemical constituents present in KP-AE.The results, summarized in Table 2, indicate the presence of various phytochemicals such as alkaloids, saponins, tannins, flavonoids, terpenoids, glycosides, and oils.These compounds were detected using standard qualitative methods, which confirmed their presence in significant quantities.Each of these phytochemicals is known for its distinct bioactive properties, suggesting that KP-AE may possess a broad spectrum of biological activities.

Visual confirmation of KP-AE AgNMs synthesis
As shown in Figure 2, the KP-AE solution, initially purple, turned brownish-red upon the addition of AgNO3.This colour change confirms the successful biogenic synthesis of KP-AE AgNMs, indicating the reduction of Ag+ ions to Ag0.

Characterization of silver nanomaterials UV-visible spectra analysis
The UV-Vis spectral analysis, illustrated in Figure 3, demonstrated the optimal conditions for the conversion of Ag+ to Ag0 during the synthesis of KP-AE AgNMs.The best parameters were a 5:4 mL ratio of AgNO3 to KP-AE, a 2 mM concentration of AgNO3, a pH of 11, and a temperature of 75 °C, with a 24-hour incubation period.Under these conditions, the SPR band exhibited maximum absorbance peaks at 474 nm, confirming the formation of KP-AE AgNMs.Additionally, the optimization experiments revealed sharp SPR peaks within the 410-480 nm range, indicating successful nanoparticle synthesis.

Field emission scanning electron microscopy (FE-SEM)
The FE-SEM analysis, as depicted in Figure 4, revealed that the KP-AE AgNMs ranged in size from approximately 19 nm to 40 nm.The nanoparticles exhibited a uniform distribution and a predominantly spherical morphology after 24 hours of interaction time.The high-magnification micrographs further demonstrated the surface deposition of the KP-AE AgNMs, confirming that they were well-dispersed with no significant agglomeration observed.

Field emission scanning electron microscopy (FE-SEM)
The SEM images revealed KP-AE AgNMs ranging from 19 nm to 40 nm, exhibiting uniformity and a spherical morphology after 24 hours of interaction time.Notably, the high-magnification micrographs clearly illustrate the surface deposition of KP-AE AgNMs.As stated by Rose et al. (2019), the presence of bio-organic substances strongly supports our hypothesis that secondary materials derived from KP-AE molecules may serve a critical role as binding agents, effectively coating and encapsulating the nanomaterials.

DISCUSSION
The identification of alkaloids, saponins, tannins, flavonoids, terpenoids, glycosides, and oils in KP-AE underscores the extract's potential as a source of bioactive compounds.Alkaloids and flavonoids, in particular, are well-documented for their antioxidant and anti-inflammatory properties, which may contribute to the therapeutic potential of KP-AE.The presence of these secondary metabolites is not only indicative of the plant's medicinal value but also suggests their possible involvement in the green synthesis of nanomaterials.
According to Varghese et al. (2021), secondary metabolites in plant extracts can facilitate the reduction of metal ions, such as Ag+, to their corresponding nanoparticles (Ag0).In this context, the phytochemicals identified in KP-AE likely play a crucial role in both the reduction process and the stabilization of the synthesized AgNMs.The encapsulation of these nanoparticles by the phytochemicals may also enhance their bioavailability and therapeutic efficacy.Future studies should focus on elucidating the exact mechanisms by which these phytochemicals contribute to the biogenic synthesis of AgNMs and exploring their potential applications in various biomedical fields.
The observed colour change from purple to brownish-red upon the addition of AgNO3 to KP-AE is attributed to surface plasmon resonance (SPR), a phenomenon that occurs when the phytochemical constituents in Kaempferia parviflora facilitate the biogenic reduction and synthesis of Ag+ ions to Ag0.SPR is a characteristic feature of metal nanoparticles, and its occurrence in this study suggests successful synthesis of AgNMs.The factors influencing SPR, including the shape, morphology, size, dielectric environment, and composition of the synthesized nanomaterials, are crucial in determining the optical properties of the nanoparticles.
In this study, the presence of a single SPR peak suggests that the synthesized KP-AE AgNMs likely possess a spherical shape.The role of the phytochemicals in driving this process highlights the potential of KP-AE as a reducing and stabilizing agent in the green synthesis of nanomaterials.Further characterization studies are necessary to confirm the shape and size distribution of these AgNMs and to explore their potential applications.
The UV-Vis analysis confirms that the synthesized KP-AE AgNMs exhibit characteristic surface plasmon resonance (SPR) bands, particularly with maximum absorbance at 474 nm.These results suggest that the parameters used in this study are effective for achieving high yields of AgNMs, aligning with findings from previous research (Melkamu et al., 2021;Saxena et al., 2016;Revathi et al., 2022;Liaqat et al., 2022).The optimal conditions-specifically the 2 mM AgNO3 concentration and a pH of 11-contributed significantly to the stability and size uniformity of the synthesized AgNMs, as evidenced by the sharp SPR peaks.Further analysis shows that increasing AgNO3 concentration, pH levels, temperature, and incubation time progressively improved the yield and stability of the AgNMs (Table 3).The observed increase in SPR intensity at 75 °C and under alkaline conditions indicates that these parameters are critical for efficient nanoparticle synthesis.These findings corroborate previous studies (Aramwit et al., 2014;Balakumaran et al., 2015;Singh et al., 2020), which have also reported that higher temperatures and alkaline pH values favor the formation of stable and uniform silver nanoparticles.
Moreover, the gradual color change from purple to dark brown over time serves as a visual confirmation of the efficient biogenic reduction process.The progressive increase in SPR intensity with extended reaction time further suggests that prolonged incubation enhances nanoparticle formation and stability (Bhatt et al., 2018;Yusuf et al., 2020).The optimal absorbance observed at 419 nm after 24 hours of incubation signifies that the reaction conditions employed in this study are suitable for producing stable and well-formed KP-AE AgNMs.
The SEM analysis confirms that the KP-AE AgNMs exhibit a consistent size distribution and spherical shape, ranging from 19 nm to 40 nm.This uniformity and morphology suggest effective synthesis conditions.The highmagnification images further indicate that the nanoparticles are well-deposited on the surface, which aligns with the hypothesis that bio-organic substances from KP-AE may play a crucial role in binding, coating, and encapsulating the nanomaterials.These findings are consistent with previous research by Rose et al. (2019), which also highlighted the role of plant-derived secondary metabolites in stabilizing and capping silver nanoparticles.
The FE-SEM analysis supports the effectiveness of the biogenic synthesis method used in this study, as evidenced by the uniform size and spherical morphology of the KP-AE AgNMs.The size range observed (19 nm to 40 nm) is consistent with what is typically expected for silver nanoparticles synthesized using plant extracts, which often results in well-defined and stable nanoparticles.The uniformity in size distribution and morphology suggests that the phytochemicals present in KP-AE played a crucial role in controlling the nucleation and growth of the nanoparticles, leading to the formation of consistently sized AgNMs.Additionally, the high-magnification image illustrates the surface deposition of KP-AE AgNMs, which indicates that the bio-organic substances from the KP-AE extract may act as effective stabilizing agents.This finding is in agreement with the hypothesis proposed by Rose et al. (2019), suggesting that the secondary metabolites in KP-AE could serve as capping and stabilizing agents, preventing agglomeration and enhancing the dispersion of the nanoparticles.The lack of significant agglomeration further highlights the effectiveness of KP-AE in producing stable and well-dispersed silver nanomaterials.

CONCLUSION
The phytochemical components of Kaempferia parviflora aqueous extracts were used for this study employing a sustainable, green methodology, yielding spherical shapes with a range size of 19 nm to 40 nm.The most successful approach to biogenic manufacturing has been effectively proven by synthesizing at pH (11), temperature (75°C), incubation time (24 hours), and 2 mM AgNO3 with high product yields.The biogenic fabrication of KP-AE AgNMs is rapid, economical, and non-toxic.However, additional studies are required to demonstrate numerous biological features (such as antifungal, antidiabetic, anti-inflammatory, and cytotoxic potential) as a first step toward pharmaceutical implementation of these biogenic fabrics to produce AgNMs.

AUTHOR CONTRIBUTIONS
Alya Khaizura Azman was responsible for conducting the experiments, drafting the manuscript, and analysing experimental data.Mohammad Aidiel was involved in analysing experimental data and drafting the final version of the manuscript.Deborah Anna Van Oosterhout prepared for drafting the manuscript.Maisarah Abdul Mutalib was responsible for the conception and design of the research, drafting the final version of the manuscript, supervising students, research management, and funding acquisition.

FUNDING
This research was funded by Management and Science University Seed Research Grant, SG-011-022020-IMS.

Figure 3 UV
Figure 3UV-vis spectrum on biogenic fabrication of KP-AE AgNMs

Figure 4 SEM
Figure 4SEM of biogenic fabrication of KP-AgNMs

Table 2
Phytochemical analysis on Kaempferia parviflora extractNote: Major phytochemical groups such as alkaloids, saponins, tannins, flavonoids, terpenoids and glycosides are present in the crude extract.

Table 3
Yield production on biogenic fabrication of KP-AgNMs