Green synthesis of silver nanoparticles using Kenaf leaves extract and their antibacterial potential in acne management.  

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Wei Ting Jess Ong, Swee Pin Yeap, Jahurul Haque, Kar Lin Nyam This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4614655/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Acne vulgaris affects approximately 9.4% of the world population and was ranked 8th most prevalent disease. Concurrently, silver nanoparticles (AgNPs) are widely explored for their profound antibacterial properties which are ideal for acne management. With the current search for natural alternatives in both cosmetics and nanotechnology, plant extracts have garnered tremendous attention in the synthesis of AgNPs. Therefore, this study utilized Kenaf leaves extract (KLE) as a novel, natural reducing agent for the synthesis of AgNPs. The effects of different synthesis parameters were studied and KLE-synthesised AgNPs (KLE-AgNPs) were further analysed for their physicochemical properties and antimicrobial efficiency. Results showed that small-sized (60.32±2.41nm), stable (-43.03±2.55 mV) and monodispersed (0.28±0.01) KLE-AgNPs were successfully formed with 3mM silver nitrate, and 3mg/mL KLE along with the optimal conditions at pH 11, 48 hours incubation time, reaction temperature of 37°C, and centrifugation at 10000 g for purification. FTIR analysis confirmed the presence of functional groups that aid in the formation of AgNPs. Additionally, XRD result demonstrated that KLE-AgNPs recorded crystalline size of 58.59nm. The FESEM and EDX analyses displayed that the particles were spherical and silver was the main element respectively. The antimicrobial analysis proved that a lower dose of KLE-AgNPs demonstrated better antimicrobial effect on the three acne-causing bacteria compared to commercial AgNPs and chemically synthesized-AgNPs. The outcome of this research amplifies the role of KLE as a natural reducing agent in the synthesis of AgNPs for the development of hybrid nanocosmetics with increased efficacy due to the synergistic effect of KLE and AgNPs. Silver nanoparticles green synthesis kenaf leaves process optimization antibacterial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1.0 INTRODUCTION Acne vulgaris is a common skin complication that affects many adolescents and young adults globally. Roughly 9.4% of the general population suffer with acne vulgaris [ 1 ]. LeungOne of the most addressed factors that contributes to acne vulgaris is bacterial colonization of the skin microbiota which induces in vitro inflammatory effect and thus leads to formation of lesions (acne). Most of the acne remedies currently available in the market such as topical antibiotics and retinoids cause skin irritation such as dryness and flaking, erythema, thinning of the stratum corneum and burning sensation upon application as these are extremely potent anti-acne agents [ 1 ]. This raises interest in the use of silver nanoparticles (AgNPs) for acne management. This is contributed by the fact that AgNPs are well known for their profound therapeutic qualities such as antimicrobial, anticancer, anti-inflammatory and wound healing [ 2 , 3 ]. Among the diverse curative traits, the antibacterial capability of AgNPs is the most sought after as multiple studies had proved that AgNPs are destructive against a wide range of bacteria and viruses [ 4 ]. Universally, AgNPs are synthesized via conventional methods such as physical and chemical. However, these conventional techniques require high heat and energy treatment, hence they are costly. Additionally, the chemicals used are toxic to the environment. Subsequently, the surging of sustainability trends prompted the rapid progression of green or bio-synthesis that is simple yet efficient, eco-friendly, and cost-effective [ 5 ]. Green synthesis involves the use of natural and accessible raw materials such as microorganisms, algae and plant extracts as reductants. Plant extracts are often preferred in green synthesis as studies had proven that the rate of synthesis and completion can be achieved within minutes of reaction time between the metal salt and the plant extracts [ 6 ]. The procedure is also simple which enable for a straightforward scale-up practice [ 7 ]. In addition to that, plant mediated synthesis does not require special conditions for culture prepation and maintenance as compared to other biological entities such as microbes. Furthermore, biocompatibility of plant mediated AgNPs is much higher compared to those synthesised with other resources [ 8 ]. As a result, they are more widely accepted in the pharmaceutical and medical sectors. Kenaf ( Hibiscus cannabinus L .) is an annual, herbaceous crop that belongs to the plant family called Malvaceae. In Malaysia, Kenaf is a valuable crop as its fibre is extensively industrialised for paper and pulp. The fiber is also harvested as raw materials for ropes, sacks, animal beddings and biocomposites. Due to the exploitation of their fibrous stems, Kenaf leaves are often discarded as agricultural waste. However, there has been a shift of interest towards the exploitation of Kenaf leaves due to the advancement of circular economy where biowastes should be minimized via reusing and volarization. According to [ 9 ], ethanol-extracted kenaf leaves contained many phenolics and flavonoids such as tannic acid, catechin hydrate and caffeic acid. Similarly, the UPLC analysis reported that the major compounds present in the leaves extract were kaemperitrin, caffeic acid, myricetin glycoside, and p-hydroxybenzoic acid [ 10 ]. Previous studies had demonstrated that these bioactive compounds were capable of reducing silver ions (Ag + ) to Ag 0 . Furthermore, the compounds also serve as stabilizing and capping agents that prevent the agglomeration of the NPs [ 11 ]. To our knowledge, there is a lack of up-to-date and comprehensive research on the utilization of ethanol extracted Kenaf leaves extract (KLE) in the green synthesis of AgNPs and its application as a component in cosmeceuticals for acne management. Therefore, the present work focuses on the green synthesis of AgNPs using Kenaf leaves extract (KLE) in which the effect of concentration of silver nitrate and KLE, incubation time, medium pH, temperature and centrifugal force on the characteristics of KLE-synthesised AgNPs (KLE-AgNPs) were evaluated to dictate the optimal synthesis conditions. This approach warrants the production of tightly controlled AgNPs as the physicochemical properties of AgNPs are also strongly influenced by the operational variables during synthesis [ 12 ]. Subsequently, the physicochemical properties of KLE-AgNPs were characterized by UV-vis spectroscopy, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and Field Emission Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy (FESEM-EDX). Additionally, the antibacterial activity of KLE-AgNPs was studied against several bacteria that are responsible for the pathogenesis of acne. 2.0 MATERIALS AND METHODS 2.1 Materials and chemicals Fresh Kenaf ( Hibiscus cannabinus L .) leaves were obtained from Lembaga Kenaf & Tembakau Negara (LKTN) (Malaysia). Ethanol, silver nitrate, resazurin sodium salt and commercial silver nanoparticles were all purchased from Sigma-Aldrich (Missouri, United States). Bacteria strains, Cutibacterium acnes ( C.acnes ), Staphylococcus aureus ( S.aureus ) and Staphylococcus epidermidis ( S.epidermidis ), were acquired from ATCC (Manassas, Virginia). Nutrient broth was purchased from Merck (Germany) and tryptone soy broth as well as tryptone soy agar with sheep blood were purchased from Oxoid (England, United Kingdom). All chemicals used were of analytical reagent grade. The ultra-pure water (Millipore, USA) was used throughout the analysis. 2.1.1 Pulsed ultrasonic assisted extraction of powdered kenaf leaves The extraction of kenaf leaves extract (KLE) was carried out according to [ 9 ] with slight modifications. Firstly, 10g of dried, and grounded kenaf leaves was added to 100mL of pure ethanol as the extraction solvent. Then, the mixture was ultrasonicated using the labsonic ultrasonic homogenizer (Sartorius, Germany). The ultrasonic assisted extraction of kenaf leaves was performed at the sonication amplitude of 50%. One sonication cycle consisted of one minute pulse duration period and one minute pulse interval period. This cycle was repeated three times. An ice bath was used during the sonication process to maintain the temperature of KLE within the initial range of 18–22 ± 3℃. This was done to prevent the degradation of the bioactive compounds because of the heat generated during ultrasonication. The extracted mixture was then transferred to Falcon tubes with equal total weight and centrifuged at 5000rpm for 10 minutes. After centrifugation, the supernatant of the KLE was filtered with Whatman filter paper while the pellet was discarded. Next, about 95% of the extraction solvent, ethanol, in the filtered KLE was removed using a rotary evaporator (Buchi, Switzerland) at 45ºC under reduced pressure. After evaporation, the concentrated crude KLE stored in the freezer at − 20°C under dark conditions for future analysis. 2.1.2 Green synthesis of silver nanoparticles (KLE-AgNPs) Green synthesis of silver nanoparticles were generally conducted according to [ 13 ] with slight modifications. First, concentrated KLE solution was prepared by dissolving the desired amount in ethanol. Then, in a ratio of 1:9, 5 mL of KLE solution was added to 45 mL of aqueous silver nitrate (AgNO 3 ) drop by drop while the reaction mixture was stirred at 700 rpm to initiate the reduction process. The stirring was stopped once all the KLE solution was added. The reaction mixture was then quickly adjusted to desired pH with 1M sodium hydroxide (NaOH). The formation of AgNPs was observed by the immediate color change of the reaction mixture from greenish-yellow to reddish brown. The reaction mixtures were incubated at different temperatures under dark conditions and the UV-vis spectra of the reaction mixtures were measured at different time intervals up to 48 hours. 2.1.3 Chemical synthesis of silver nanoparticles (C-AgNPs) Chemical synthesis of silver nanoparticles (C-AgNPs) was operated by using sodium borohydride (NaBH 4 ) as described by [ 14 ] with slight changes. Firstly, aqueous solutions of 10mL of 0.01M of AgNO 3 and 30mL of 0.02M NaBH 4 were freshly prepared. The NaBH 4 solution was placed on an ice bath and allowed to cool for 30 minutes. After cooling, AgNO 3 solution was added drop by drop while the stirring was initiated at a speed of 700 rpm. Once all the AgNO 3 solution was added, the stirring was stopped. 2.1.4 Purification of KLE-AgNPs The purification of KLE-AgNPs was performed according to[ 15 ] with minor modifications. This procedure was required to remove any excess reactants before further analysis of the KLE-AgNPs. The reaction mixture was centrifuged at a speed of 10000 g at a temperature of 4°C for 150 minutes using 5415R microcentrifuge (Eppendorf, Germany). Then, the supernatant was discarded and the pellet was resuspended with ultra-pure water (washing). The resuspended pellet was further centrifuged again under the same conditions and the supernatant was removed (1 washing cycle). The washing cycle was repeated 3 times. Finally, the purified KLE-AgNPs were resuspended with ultra-pure water and the solution was stored at 4°C ± 2°C for further analysis. 2.1.5 Single factor optimization of the green synthesis (a) Effect of concentration of silver nitrate and KLE The concentration of silver nitrate used was 1mM, 3mM and 5mM while the concentration of KLE used was 1mg/mL, 3mg/mL and 5mg/mL. The medium pH, reaction temperature and incubation time were maintained at pH 11, 37°C and 48 hours respectively. The centrifugal force for the purification procedure described in Section 2.1.4 was maintained at 10000 g . The conditions for the parameters were shown in Table 1 . Table 1 Concentration of AgNO 3 and KLE in different samples Sample Concentration of AgNO 3 (mM) Concentration of KLE (mg/mL) S1 1 1 S2 1 3 S3 1 5 S4 3 1 S5 3 3 S6 3 5 S7 5 1 S8 5 3 S9 5 5 (b) Effect of incubation time The effect of incubation time on the formation of KLE-AgNPs was monitored using UV-vis spectroscopy of the reaction mixture at different time intervals, 0 hour, 1st hour, 2nd hour, 3rd hour, 4th hour, 5th hour, 6th hour, 24th hour and 48th hour. The concentration of AgNO 3 , concentration of KLE, medium pH and reaction temperature were maintained at 3mM, 3mg/mL, pH 11 and 37°C respectively. (c) Effect of medium pH The reaction mixtures were adjusted to pH 3, 5, 7, 9 and 11 after addition of KLE solution prior to incubation for 48 hours at 37°C. The concentration of AgNO 3 and KLE were maintained at 3mM and 3mg/mL respectively. After incubation, the reaction mixtures were purified via centrifugation at 10000 g with the conditions described in Section 2.1.4 . (d) Effect of reaction temperature The reaction mixtures were incubated at 15°C, 30°C, 37°C, 45°C, 60°C for 48 hours. The concentration of AgNO 3 , concentration of KLE and medium pH were maintained at 3mM, 3mg/mL and pH 11 respectively. After incubation, the reaction mixtures were purified via centrifugation at 10000 g with the conditions described in Section 2.1.4 . (e) Effect of centrifugal force Green synthesis was performed according to the described general procedure in Section 2.1.2 . The concentration of AgNO 3 and concentration of KLE used were 3mM and 3mg/mL respectively. The medium pH was adjusted to 11 and incubated at 37°C for 48 hours. After incubation, the reaction solution was purified according to the procedure described in Section 2.1.4 but with different centrifugal force, 4000 g , 8000 g , 10000 g and 16000 g . 2.1.6 Characterization of KLE-synthesised silver nanoparticles (KLE-AgNPs) Formation of KLE-AgNPs was observed with the FLUOStar Omega microplate reader (BMG Labtech, Germany) based on the method described by [ 16 ] with slight modification. The gradual reduction of Ag + ions in solution was monitored at a range of 300nm to 800nm. The mean particle size, zeta potential and polydispersity index (PdI) were measured by dynamic light scattering (DLS) analysis using Malvern Zetasizer Nano ZS (Malvern Panalytical, United Kingdom) whereby samples were diluted 10x with ultra-pure water and added into disposable folded capillary cells at 25°C. The detection of peak values and active functional groups of KLE-AgNPs were characterized using Fourier Transform Infrared (FTIR) spectrophotometer (Shimadzu, Japan) as stated by [ 17 ] with some modifications. The scan range was done through the wavenumber from 4000 cm − 1 to 400 cm − 1 with a resolution of 1 cm − 1 to acquire the spectra. The analysis was carried out at room temperature and single spectra were corrected against the background spectrum of air. The X-ray diffraction analysis of the AgNPs was carried out following the method as reported earlier by [ 18 ] with slight modifications. The purified KLE-AgNPs solution was freeze-dried and the KLE-AgNPs powder was collected for this analysis. The sample was observed using the Empyrean X-ray diffractometer (Malvern Panalytical, United Kingdom) with CuKα radiation at room temperature with 2θ angle in the range of 20–90° under continuous scanning at 40Ma and 45Kv. The scanning rate was 2° per min. The morphology, size and elemental mapping of KLE-AgNPs were observed using JSM-7600F Field Emission Scanning Electron Microscope (FESEM) equipped with Energy Dispersive X-Ray (EDX) (Jeol, Japan). Freeze-dried KLE-AgNPs were prepared and smeared on sample grid which was then coated with platinum prior to analysis. 2.1.7 Antimicrobial analysis of AgNPs The antimicrobial action of optimized KLE-AgNPs was determined via minimum inhibitory concentration (MIC) using broth micro-dilution technique in 96 well microtiter plate (Nest, China) with reference to [ 19 ] but with some alterations. The sample was prepared by diluting purified KLE-AgNPs with ultra-pure water to obtain the final concentration of 500µg/mL. Sixty microliters of respective broth were added into the wells containing 100 µL of two-fold serial diluted KLE-AgNPs in similar broth. Then, 20µL of resazurin indicator (0.6mg/mL) and 20µL of the standardized bacterial suspension were added to make up a total volume of 200µL in each well. The final concentration of the KLE-AgNPs sample ranged from 0.98 µg/mL to 250µg/mL. The positive control was tetracycline while the negative control was the medium broth without sample. The 96-well plates were incubated at 37ºC in aerobic condition for S. aureus and S.epidermidis (24 hours) and anaerobic condition for C.acnes (48 hours). The MIC value was recorded as the lowest concentration of KLE-AgNPs at which no visible colour change from blue to pink after incubation was observed. The optimized KLE-AgNPs were also tested on their bacteriostatic and bactericidal activity by inoculating the suspension from the wells that showed no colour change (blue coloured wells) onto NA for S. aureus and S. epidermidis and TSA sheep blood agar for C. acnes via the streak-plate method. Then, the agar plates were incubated at 37ºC for 24 and 48 hours respectively. The MBC value was recorded as the lowest concentration of KLE-AgNPs at which there were no bacteria growth on the plate observed [ 19 ]. 2.1.8 Statistical analysis All results were recorded in triplicates and analysed using One-way Analysis of variance (ANOVA), followed by Tukey’s test. The analysis was performed using MINITAB 16 (Minitab Inc, Pennsylvania, USA). All experimental data were expressed as mean ± standard deviation and the differences were considered significant at p < 0.05. 3.0 RESULTS AND DISCUSSION 3.1 Colour observation The visual colour change of the reacting solution preliminarily indicated the formation of KLE-AgNPs. As observed in Fig. 1 , the reacting solution turned from greenish yellow to reddish brown, indicating the rapid reduction of Ag + ions to Ag 0 by the KLE. Similar colour observations were reported by the authors in the green synthesis of AgNPs using Phyllanthus emblica fruit extract and Areca catechu nut aqueous extract [ 20 , 21 ]. The brown colour of AgNPs correlates strongly with their surface plasmon resonance (SPR) effect and any change in colour provides information on their physical properties. To illustrate, Badi’ah et al. (2019) [ 22 ] stated that further change of colour of the reaction mixture from yellow or brown to grey signified the aggregation of AgNPs to form larger-sized AgNPs. 3.2 UV-vis spectroscopy The green synthesis of AgNPs with KLE was further confirmed by UV-vis spectroscopy. The UV-vis spectra supplemented the SPR effect that was responsible for the colour change as perceived in Section 3.1 . Based on Fig. 2 (a), an absorption peak was observed at a wavelength of 400nm between 300nm to 800nm. This is in accordance with the study on the green synthesis of AgNPs using Aloe vera leaf extract which also reported maximum absorption at 400 nm [ 23 ]. It has been established that the spectroscopic nature of AgNPs exhibit absorbance peaks ranging from 400-500nm [ 24 ]. This phenomenon could be attributed to the free electrons present which vibrate in resonance with the light wave [ 25 ]. According to Rautela et al., (2019) [ 26 ], the reduction process was aided by the donation of electrons to Ag + ions by the phenolic contents in KLE to form Ag 0 nanoparticles. Moreover, the sharp and narrow distribution of the absorption peak indicated that the AgNPs formed were predominantly smaller in size and monodispersed whereas the broader peaks signified the production of larger and polydisperse AgNPs. Absorption spectra of silver nitrate and KLE were performed and served as control. 3.3 Single factor optimization of the green synthesis (a) Effect of concentration of AgNO 3 and KLE The KLE-mediated synthesis of AgNPs were subjected to combinations of various concentrations of AgNO 3 and KLE. The SPR peaks of different samples were detected via UV-vis spectroscopy. Referring to Fig. 2 (b), the increase in concentration of both AgNO 3 and KLE simultaneously led to an increase in the absorption peak. The highest absorption intensity was recorded with Sample 9 which consisted of 5mM AgNO 3 and 5mg/mL of KLE. This outcome was apparent as more silver precursors and KLE were available for the reduction to take place, thus a greater amount of KLE-AgNPs were formed. However, despite the increase in concentration of AgNO 3 , there was a significant decrease in absorption peaks for Sample 4 and 7 whereby the concentrations of AgNO 3 + KLE were 3mM + 1mg/mL and 5mM + 1mg/mL respectively. This pattern showed that the increase in AgNO 3 without the increase of KLE led to low conversion of Ag + to Ag 0 and thus lesser number of AgNPs were formed. It could be deduced that KLE was the limiting reactant in this reaction as lesser bioactive compounds were available to fully reduce the Ag + ions [ 27 ]. The broadness of the peaks reflected the possible aggregation of AgNPs in the reaction mixture of Sample 4 and Sample 7. This outcome was comparable with a past study which concluded that the increase Tabernaemontana heyneana leaves extract led to increase in absorption intensity while increase in AgNO 3 could lead to a broad spectrum especially when the Ag + ions is in excess [ 28 ]. Similar effects of higher concentration of AgNO 3 and lower concentration of KLE as in Sample 4 and Sample 7 were reflected in their dynamic light scattering (DLS) analysis. From Table 2 , particle sizes of S4 and S7 were significantly larger than the rest of the samples except Sample 8. This finding aligned with the broadness of their SPR peaks as discussed earlier. Nonetheless, Sample 8, with the concentration of 5mM AgNO 3 and 3mg/mL KLE respectively, recorded the largest particle size (94.73 ± 2.73nm). The significantly larger size of S4, S7 and S8 could be explained by the lack of bioactive compounds from KLE. These compounds are also responsible for the capping and stabilizing action of AgNPs that prevent the formation of larger aggregates. Besides the factor of KLE, Zayed et al. (2015) [ 29 ] stated that excess Ag + ions could promote the growth of NPs. Consequently, the KLE-AgNPs produced from the three samples were significantly larger. Meanwhile, the smallest particle size produced was from Sample 3 with 1mM AgNO 3 and 5mg/mL KLE. High concentration of KLE enabled the full conversion Ag + ions to KLE-AgNPs while preserving the size of the NPs formed. Table 2 Effect of varying concentration of AgNO 3 and KLE on the particle size, zeta potential and polydispersity index Sample Particle size (nm) Zeta potential (mV) Polydispersity index (PdI) S1 64.10 ± 0.23 d -49.50 ± 0.79 b 0.29 ± 0.00 c S2 37.95 ± 0.89 f -50.13 ± 2.85 b 0.62 ± 0.02 a S3 26.28 ± 0.72 g -47.90 ± 0.56 b 0.40 ± 0.02 b S4 72.03 ± 1.06 c -49.33 ± 1.25 b 0.25 ± 0.01 d S5 59.73 ± 1.63 e -44.17 ± 1.63 a 0.28 ± 0.01 cd S6 58.42 ± 0.42 e -49.27 ± 0.40 b 0.39 ± 0.00 b S7 78.89 ± 0.68 b -48.43 ± 0.38 b 0.28 ± 0.01 cd S8 94.73 ± 2.73 a -48.70 ± 0.92 b 0.39 ± 0.02 b S9 58.84 ± 1.18 e -47.77 ± 0.62 ab 0.41 ± 0.00 b Values were presented as mean ± standard deviation (n = 3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p < 0.05. The optimal concentration of AgNO 3 and KLE in the green synthesis of AgNPs were determined based on the DLS analysis of the KLE-AgNPs complementary to the UV-Vis spectra. Besides particle size, the polydispersity index (PdI) and zeta potential of the KLE-AgNPs were taken into account as the PdI values estimate the uniformity in terms of size distribution while zeta potential values represent the stability of the NPs. Nanoparticles are known to be stable when their zeta potentials are either greater than + 30 mV or less than − 30 mV [ 30 , 31 ]. Based on Table 2 , the KLE-AgNPs produced from all samples had particle sizes of less than 100nm and were relatively stable according to their zeta potential readings. As it is known that the particle sizes of NPs significantly affect the pharmacokinetics properties, acquiring samples with uniform particle size distribution is vital. Generally, NPs are regarded as highly monodisperse if the PdI value is less 0.1 and moderately disperse if the PdI value is within 0.1 to 0.4 [ 32 ]. Therefore, samples with PdI values closest to 0.1 were considered to minimize any sort of biological fluctuations of the AgNPs to ensure consistency of future analysis. This narrowed down the selection to S1, S4, S5 and S7. As S1, S4 and S7 had low reduction efficiency and larger particle sizes, S5 (3mM AgNO 3 and 3mg/mL KLE) were chosen as the optimal condition for future analysis. (b) Effect of incubation time The reaction mixture was continuously incubated at 37°C for a total of 48 hours to determine the complete formation of KLE-AgNPs. As revealed in the UV-vis spectra (Fig. 2 (c)), the absorption spectra gradually increased every hour for the first 6 hours and by the 24th hour, there was an exponential rise. The SPR peaks were less intense and broader in the first 6 hours and therefore, an extended duration of 24 and 48 hours were added to further confirm the completion of the biosynthesis [ 33 ]. Consequently, the SPR peaks observed at the later stage were more intense and sharper. The increase in absorption reading during incubation signified the increase in the yield of KLE-AgNPs [ 34 ]. This could be deduced by the continuous reaction that occurred when the silver precursor (silver nitrate) and the reducing agent (KLE) remain in contact for a longer period. Additionally, the intensity no longer increased at the 48th hour and thus implied the complete reduction of Ag + ions to Ag 0 . This finding is in correspondence with the synthesis of AgNPs using Kawista ( Limonia Acidissima Groff .) leaves ethanol extract [ 35 ]. (c) Effect of pH on the synthesis of KLE-AgNPs In this study, pH of the reaction mixtures was adjusted to pH 3, pH 5, pH 7, pH 9 and pH 11. The effect of pH on the formation of KLE-AgNPs were observed in Fig. 2 (d). The UV-Vis spectra portrayed lower and broader peaks at low to neutral pH (pH 5 and 7). At pH 3, no absorbance peak was observed due to the nucleation of AgNPs, thus pH 3 was unfavorable for the synthesis. Meanwhile at pH 9 and 11, higher maximum absorption peaks at 410nm with narrower bands were observed. This observation evidently proved that pH could impact the formation of AgNPs by altering their surface charges. In one recent study, the authors deciphered that the decrease in absorbance values at low pH was due to the occurrence of severe agglomeration stimulated by denser surface charges [ 36 ]. This clarification is supported by Fernando and Zhou (2019) [ 37 ] who claimed that at low pH, the energy barrier between the nanoparticles is neutralized and suppressed by the kinetic energy of the Brownian motion, resulting in particle agglomeration. Besides UV-vis analysis, the samples were also subjected to DLS measurements to determine the size, zeta potential and PdI of KLE-AgNPs formed under different pH. As presented in Table 3 , the mean size of KLE-AgNPs at pH 3 was 1427.30 ± 490.60nm. This further confirmed that nano-sized particles were not formed when the synthesis was conducted in a highly acidic medium. Despite that, AgNPs with sizes less than 100nm were formed at pH 5, 7 and 9, which were 92.30 ± 26.70nm, 74.10 ± 11.50nm and 79.70 ± 24.60nm respectively. The smallest-sized KLE-AgNPs (60.32 ± 2.4nm) were formed in the most alkaline medium (pH 11). Succinctly, the size of KLE-AgNPs was inversely proportional to the pH values. Previous study had published comparable finding using Bacillus brevis culture for AgNPs synthesis [ 38 ]. According to these authors, more -OH ions were available at high pH which further elevated the nucleation process, leading to the formation of smaller-sized AgNPs. Additionally, the dispersion of the KLE-AgNPs were determined through their PdI values and it could be deduced that KLE-AgNPs synthesised at pH 11 exhibited the lowest polydispersity. With reference to the zeta potential readings, it could be deduced from Table 3 that the increase in pH led to the increase in the degree of stability of the KLE-AgNPs. The high zeta potential values implied that the AgNPs produced in this study have developed a high net surface charge that can promote electrostatic repulsion. To be brief, at low or high pH, the H + ions and OH − ions can alter the positive and negative charges on the surface of NPs which will lead to electrostatic repulsion between particles and thus prevent aggregation and formation of larger-sized NPs [ 39 ]. This also means that nanoparticles are least stable in pH 7, the isoelectric point, as the surface charges are neutralized (zero charge) [ 40 ]. However, in plant-mediated synthesis, alkaline medium appeared to be the ideal condition as it promoted the ionization of the functional groups such as COOH groups to -COO- which simultaneously increased the rate of reduction of Ag + ions and the negative surface charges [ 41 , 42 ]. This explained the formation of smaller particle size with higher stability (lower zeta potential values) in pH 11. As a result, the optimal pH for the formation of relatively stable and small-sized KLE-AgNPs would be pH 11. Table 3 Effect of pH on the average size, zeta potential and polydispersity index pH Particle size (nm) Zeta potential (mV) Polydispersity index (PdI) 3 1427.30 ± 490.60 a -11.66 ± 1.44 a 0.72 ± 0.15 a 5 92.30 ± 26.70 b -26.07 ± 6.00 b 0.44 ± 0.02 b 7 74.10 ± 11.50 b -25.52 ± 3.30 b 0.38 ± 0.12 b 9 79.70 ± 24.60 b -41.33 ± 5.61 c 0.45 ± 0.16 b 11 60.32 ± 2.41 b -43.03 ± 2.55 c 0.28 ± 0.01 b Values were presented as mean ± standard deviation (n = 3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p < 0.05. (d) Effect of temperature Temperature is one of the vital parameters that can significantly affect the properties of KLE-AgNPs formed. In this study, the reacting solution was incubated at different temperatures (15°C, 30°C, 37°C, 45°C and 60°C) and the effect was analyzed using UV-Vis spectroscopy and DLS. As portrayed in Fig. 2 (e), the increase in temperature led to the increase in maximum absorption wavelength. This meant that the rate of formation of KLE-AgNPs was elevated as the temperature increased. The absorption bands also appeared sharper with the increase in temperature. Equivalent observation was reported by Rousta and Ghasemi (2019). However, unlike pH, results from the DLS analysis (Table 4 ) for the different temperatures did not complement the UV-vis spectra results. The order, in terms of mean size, was 37°C < 30°C < 15°C < 60°C < 45°C while in terms of PdI, it showed a trend of 37°C < 30°C < 15°C < 45°C < 60°C. Although the UV-Vis spectra illustrated that the highest and sharpest absorbance peak belonged to KLE-AgNPs synthesized at 60°C, Table 4 documented that KLE-AgNPs synthesized at 37°C were the smallest in size with the lowest value of PdI (0.28 ± 0.01). This extraordinary phenomenon was vindicated by Liu et al. (2020) [ 44 ]. The authors reported that sufficient Ag + precursors led to the increase in particle size at 70°C to 80°C due to the linear increase of growth rate constant with the increased of synthesis temperature. In other words, growth process superseded the nucleation process, causing the increase in size. Hence, it was confirmed that physical properties of AgNPs differ accordingly at varying temperatures. Additionally, KLE-AgNPs synthesised at all temperatures exhibited high degrees of stability (zeta potential values of less than − 30mV). Based on the above elucidation, the optimal reaction and incubation temperature was 37°C. Table 4 Effect of temperature on the average size, zeta potential and polydispersity index Temperature (°C) Particle size (nm) Zeta potential (mV) Polydispersity index (PdI) 15 76.44 ± 22.51 ab -40.92 ± 2.03 a 0.31 ± 0.03 bc 30 68.82 ± 11.21 ab -41.70 ± 1.73 a 0.33 ± 0.05 b 37 60.32 ± 2.41 b -43.03 ± 2.55 ab 0.28 ± 0.01 bc 45 83.27 ± 6.15 a -43.23 ± 1.49 ab 0.27 ± 0.04 c 60 77.69 ± 7.71 ab -45.22 ± 1.30 b 0.40 ± 0.01 a Values were presented as mean ± standard deviation (n = 3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p < 0.05. (e) Effect of centrifugal force From Table 5 , the average sizes of KLE-AgNPs were directly proportional with the increase in centrifugal force and they were statistically significant. On the contrary, the increase in centrifugal force led to a decrease in PdI, suggesting an increase in monodispersity of the KLE-AgNPs formed. In particular, the particles purified at 10000 g recorded the lowest PdI, suggesting their moderately dispersed nature with a mean size of 60.32 ± 2.41nm. The magnitude of zeta potentials of all samples was lower than − 30mV, indicating that the KLE-AgNPs were highly stable regardless of the change in the speed of centrifugation. In general, centrifugation allows the separation of AgNPs from excess ligands or impurities, with the aim to convert polydisperse silver suspensions into isolated, monodisperse samples [ 45 ]. A higher centrifugal force is required to separate smaller-sized AgNPs. However, in this study, higher centrifugal force showed a contrasting effect. This phenomenon could be explained by the correlation between surface charges and particle size. Asnaashari Kahnouji et al. (2019) [ 46 ] conferred that smaller-sized particles have higher surface charges which leads to stronger tendency to bind together and agglomerate. Besides, the pressure induced by the high centrifugal force could potentially flatten the KLE-AgNPs, resulting in shape deformation and size increment. Nonetheless, comprehensive analysis on the optimal centrifugal force remained scarce as specific parameters and centrifugal conditions used by researchers were not disclosed in many experimental and research papers [ 47 ]. Thus, the data obtained in this study may serve as a reference for future researchers who intend to review or compare centrifugation techniques for their work. In addition to that, Table 3.6 comparatively highlights the optimized synthesis conditions of AgNPs using plant extracts as reported in previous studies and the present. Table 5 Effect of centrifugal force on the average size, zeta potential and polydispersity index Centrifugal force ( g ) Particle size (nm) Zeta potential (mV) Polydispersity index (PdI) 4000 33.81 ± 1.85 d -48.62 ± 1.91 b 0.51 ± 0.03 a 8000 46.78 ± 14.58 c -46.83 ± 2.32 b 0.48 ± 0.03 a 10000 60.32 ± 2.41 b -43.03 ± 2.55 a 0.28 ± 0.01 b 16000 101.80 ± 2.76 a -45.83 ± 1.31 ab 0.24 ± 0.03 c Values were presented as mean ± standard deviation (n = 3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p < 0.05. 3.4 Comparison between optimized KLE-AgNPs, commercial and chemically synthesised AgNPs Based on the evaluation done on section 3.3 (a) to (e), it could be deduced that the optimal concentrations for both silver precursor and reducing agent were 3mM AgNO 3 and 3mg/mL KLE while the optimal conditions were 48 hours incubation time at 37°C with pH 11 medium. After synthesis, 10000 g of centrifugal force was employed for the purification of KLE-AgNPs. The particle size, zeta potential and PdI of the optimized KLE-AgNPs were compared with the particle size, zeta potential and PdI of commercial AgNPs (X-AgNPs) and chemically synthesized AgNPs (C-AgNPs). With reference to Table 6 the optimized KLE-AgNPs were smaller, more stable and monodispersed in comparison to the other two AgNPs. This proved that the green synthesis of AgNPs using KLE in which the synthesis process was optimized would produce particles with tightly controlled properties. Table 6 The particle size, zeta potential and polydispersity index of KLE-AgNPs, X-AgNPs and C-AgNPs Sample Particle size (nm) Zeta potential (mV) Polydispersity index (PdI) KLE-AgNPs 60.32 ± 2.41 b -43.03 ± 2.55 b 0.28 ± 0.01 b X-AgNPs 146.10 ± 6.96 a -36.03 ± 1.31 a 0.47 ± 0.11 a C-AgNPs 149.83 ± 6.99 a -41.37 ± 1.07 b 0.45 ± 0.04 a Values were presented as mean ± standard deviation (n = 3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p < 0.05. 3.5 Characterization of KLE-AgNPs (a) FTIR spectroscopy The optimized KLE-AgNPs were subjected to FTIR analysis to determine the functional groups of the bioactive compounds present in KLE that were responsible for reducing Ag + ions to Ag 0 and stabilizing the KLE-AgNPs formed by acting as capping agents [ 48 ]. It could be observed that the IR spectra of AgNPs contained peaks ranging from 3329.14cm − 1 to 599.86 cm − 1 . The broad peak at 3329.14cm − 1 represents the H-bonded, O-H stretching, revealing the presence of alcohols and phenols. Meanwhile, a sharper peak at 1633.71cm − 1 , which symbolizes the C = O band, served as evidence that there were carbonyl groups. In addition to that, the less intense peak at 667.37 cm − 1 corresponded to the presence of N-H wag amines. The data presented in this research were similar to the findings of several other studies involving the biosynthesis of AgNPs, confirming the feasibility of using KLE for AgNPs synthesis [ 49 – 51 ]. The FTIR spectra of KLE-AgNPs was also compared with the FTIR spectra of commercially bought AgNPs (X-AgNPs) and chemically synthesized-AgNPs (C-AgNPs). The similarities of peaks between the three AgNPs further confirmed the formation of AgNPs mediated by KLE. (b) X-Ray Diffraction (XRD) analysis Further characterization of the optimized KLE-AgNPs was conducted using the X-Ray diffraction (XRD) analysis. This technique serves to identify the crystalline structure and the phase purity of the NPs formed. In accordance with Fig. 4 , there were four major diffraction peaks observed at 2θ values of 38.26°, 44.41°, 64.65° and 77.49° with the maximum peak at position 38.26°. These peaks correspond to the crystallographic lattice planes of AgNPs which are (111), (200), (220) and (311) under the standard set by the Joint Committee on Powder Diffraction Standards, JCPDS (File no. 04-0783) [ 52 ]. A similar XRD pattern was observed in a study conducted by Baran et al. (2023) [ 53 ] on the green synthesis of AgNPs using Allium cepa peel extract. These planes represent the face centered cubic structure of silver. The appearance of additional peaks as seen on the XRD spectrum can be attributed to the crystallization of bioactive compounds from the KLE on the surface of AgNPs. The Debye-Scherrer equation, d = \(\frac{K\lambda }{{\beta }\text{c}\text{o}\text{s} {\theta }}\) , was used to determine the average size of the AgNPs [ 54 ]. With reference to that, the average crystalline size of KLE-AgNPs was estimated to be 58.59nm. (c) Field Emission Scanning Electron Microsopy – Energy Dispersive X-Ray (FESEM-EDX) The shape and size of the KLE-AgNPs were identified using FESEM coupled with EDX for elemental mapping. Based on Fig. 5 , the optimized KLE-AgNPs appeared to be in spherical shape and monodisperse with the average diameters ranging from 20nm to 55nm. In comparison with the size detected via DLS analysis, the KLE-AgNPs captured by FESEM were smaller. According to Pryshchepa et al. (2020) [ 55 ], DLS often overestimates particle sizes as it measures the hydrodynamic diameter of the NPs. Moreover, the FESEM image also displayed several larger particles due to the agglomeration of smaller KLE-AgNPs. The occurrence of aggregation could be caused by the freeze-drying process that was done prior to the analysis and presence of high surface activity [ 56 ]. Thin layers were noted on the outer parts of the KLE-AgNPs as shown in Fig. 5 which demonstrated the capping layers of organic molecules present during the synthesis. With reference to Fig. 6 , the EDX spectrum exhibited that the strongest peak representing the major element was silver thereby confirming the formation of KLE-AgNPs. The biomolecules from KLE acting as the capping agents, were represented by other weak peaks such as carbon, oxygen, silicon, sulphur, sodium and potassium [ 57 , 58 ]. According to Jagtap and Bapat (2013) [ 59 ], the absorption peak of metallic silver nanoparticles usually appears at 3keV as evident in Fig. 6 due to their SPR. The FESEM image suggested that with the optimum conditions, green synthesis of AgNPs via KLE was successfully achieved. 3.6 Antimicrobial activity The antimicrobial activity of optimized KLE-AgNPs on Cutibacterium acnes , Staphylococcus aureus and Staphylococcus epidermidis were evaluated using the minimum inhibitory concentration (MIC) technique. The MIC values of KLE-AgNPs on the 3 bacteria were recorded in Table 7 . Based on Table 7 , KLE-AgNPs showed the highest inhibitory effect on C.acnes followed by both S. aureus and S.epidermidis . The MIC values recorded for C.acnes and S.aureus and S.epidermidis were 3.91µg/mL, 62.5 µg/mL and 62.5µg/mL respectively. The bactericidal activity of KLE-AgNPs on the 3 bacteria was also determined and recorded as minimum bactericidal concentration (MBC) values. Similar to MIC analysis, KLE-AgNPs induced bactericidal effect most effectively on C.acnes among all tested pathogens (MIC = 3.91µg/mL). The antibacterial activity of KLE-AgNPs were again compared with commercially bought AgNPs (X-AgNPs) and chemically synthesized-AgNPs (C-AgNPs). From the comparison, it can be observed that KLE-AgNPs required much lower concentration to inhibit the proliferation of all 3 bacteria compared to X-AgNPs and C-AgNPs. This could be attributed to the difference in physical properties as displayed in Table 6 . Out of the three types of AgNPs, KLE-AgNPs had the smallest size compared to X-AgNPs and C-AgNPs. The antibacterial efficacy of AgNPs correlates with their structural properties, primarily their size and shape [ 60 ]. As reported by [ 61 ], the increase in particle size led to a decrease in antibacterial activity. Similarly, the bactericidal activity of KLE-AgNPs was stronger among all three types of AgNPs. This suggested that AgNPs synthesized by KLE were better antibacterial agents than the commercially available AgNPs and AgNPs that were synthesized from a conventional method. The antibacterial effect of AgNPs on the 3 bacteria can be elucidated by several proposed mechanisms. For instance, AgNPs attack and damage lipoteichoic acid which is a major component of the cell wall of Gram positive bacteria like C.acnes , S.aureus and S.epidermidis . Consequently, growth and metabolism of the bacteria are disrupted and eventually lead to cell apoptosis [ 62 ]. As claimed by Jemal et al. (2017) [ 63 ], AgNPs also release positively charged Ag + ions that interact with the negative electrostatic charges located on the surface of cell membrane of bacteria. This explains the ability of NPs to retard the structure of pathogens and formation of biofilm as demonstrated by [ 64 ]. Table 7 Antimicrobial activity of KLE-AgNPs, X-AgNPs and C-AgNPs Bacteria MIC/MBC (µg/mL) KLE-AgNPs X-AgNPs C-AgNPs P.acnes 3.91/ 7.81 625/ 1250 62.5/250 S.aureus 62.5/250 625/ > 625 > 250/ >250 S.epidermidis 62.5/125 1250/ >1250 125/ >250 4.0 CONCLUSION Conclusively, plant-mediated synthesis of AgNPs using ethanolic KLE was successfully executed due to the presence of phytochemical compounds in the extract that efficiently converted Ag + ions to AgNPs. This study had shown that under optimal parameters with 3mM AgNO 3 , 3mg/mL KLE, 48 hours incubation time at 37°C, pH 11 medium and centrifugal force of 10000 g for purification, KLE-AgNPs that possessed an average size of 60.32 ± 2.41nm, zeta potential of -43.03 ± 2.55mV with a low PdI value of 0.28 ± 0.01 could be obtained. The formation of KLE-AgNPs was further confirmed by FTIR in which the spectrum of KLE-AgNPs were similar to commercial AgNPs and chemically synthesised-AgNPs. Additionally, FESEM-EDX analysis revealed spherical shaped KLE-AgNPs with diameter size ranging between 20nm to 55nm and XRD estimated the size to be 58.59nm. The optimized KLE-AgNPs showed potent inhibitory effect on the colonization of the three bacteria, C.acnes , S.aureus and S.epidermidis which are associated with the pathogenesis of acne vulgaris. The outcome of this research will not only provide a deeper understanding on the role of Kenaf leaves in the manufacturing of nanoparticles and the potential of KLE-AgNPs to be incorporated into cosmetic products for acne management. Stability of KLE-AgNPs can be further evaluated based on their storage conditions to ensure that physicochemical changes of KLE-AgNPs are minimized when kept long-term. Declarations Author contributions Conceptualization: Wei Ting Jess Ong, Kar Lin Nyam, Swee Pin Yeap; Methodology: Wei Ting Jess Ong, Kar Lin Nyam, Swee Pin Yeap; Formal analysis and investigation: Wei Ting Jess Ong; Writing – original draft preparation: Wei Ting Jess Ong; Writing – review and editing: Kar Lin Nyam, Swee Pin Yeap, Md Jahurul Haque Akanda; Supervision: Kar Lin Nyam, Swee Pin Yeap. All authors read and approved the final manuscript. Conflict of interest The authors declare no conflict of interest. Funding declaration This work was supported by UCSI University Kuala Lumpur through Research Excellence & Innovation Grant, Project number REIG-FAS-2020/029. Data Availability declaration Data is available upon request. References Leung AKC, Barankin B, Lam JM, et al (2021) Dermatology: how to manage acne vulgaris. Drugs Context 10:1–18. https://doi.org/10.7573/DIC.2021-8-6 Ong WTJ, Nyam KL (2022) Evaluation of silver nanoparticles in cosmeceutical and potential biosafety complications. Saudi J Biol Sci 29:2085–2094. https://doi.org/10.1016/J.SJBS.2022.01.035 Lara HH, Ixtepan-Turrent L, Jose Yacaman M, Lopez-Ribot J (2020) Inhibition of Candida auris Biofilm Formation on Medical and Environmental Surfaces by Silver Nanoparticles. ACS Appl Mater Interfaces 12:21183–21191. https://doi.org/10.1021/ACSAMI.9B20708 Neupane NP, Kushwaha AK, Karn AK, et al (2022) Anti-bacterial efficacy of bio-fabricated silver nanoparticles of aerial part of Moringa oleifera lam: Rapid green synthesis, In-Vitro and In-Silico screening. Biocatal Agric Biotechnol 39:102229. https://doi.org/10.1016/J.BCAB.2021.102229 Pal G, Rai P, Pandey A (2019) Green synthesis of nanoparticles: A greener approach for a cleaner future. In: Shukla AK, Iravani S (eds) Green Synthesis, Characterization and Applications of Nanoparticles. Elsevier, pp 1–26 Rauwel P, Kuunal S, Ferdov S, Rauwel E (2015) A Review on the Green Synthesis of Silver Nanoparticles and Their Morphologies Studied via TEM. Advances in Materials Science and Engineering 2015:1–9 Mittal AK, Chisti Y, Banerjee UC (2013) Synthesis of metallic nanoparticles using plant extracts. Biotechnol Adv 31:346–356. https://doi.org/10.1016/J.BIOTECHADV.2013.01.003 Shah M, Fawcett D, Sharma S, et al (2015) Green Synthesis of Metallic Nanoparticles via Biological Entities. Materials (Basel) 8:7308. https://doi.org/10.3390/MA8115377 Sim YY, Jess Ong WT, Nyam KL (2019) Effect of various solvents on the pulsed ultrasonic assisted extraction of phenolic compounds from Hibiscus cannabinus L. leaves. Ind Crops Prod 140:111708. https://doi.org/10.1016/j.indcrop.2019.111708 Ryu J, Kwon S-J, Ahn J-W, et al (2017) Phytochemicals and antioxidant activity in the kenaf plant ( Hibiscus cannabinus L.). J Plant Biotechnol 44:191–202. https://doi.org/10.5010/JPB.2017.44.2.191 Roy A, Bulut O, Some S, et al (2019) Green synthesis of silver nanoparticles: Biomolecule-nanoparticle organizations targeting antimicrobial activity. RSC Adv 9:2673–2702. https://doi.org/10.1039/C8RA08982E Jameel MS, Aziz AA, Dheyab MA (2020) Green synthesis: Proposed mechanism and factors influencing the synthesis of platinum nanoparticles. Green Processing and Synthesis 9:386–398. https://doi.org/10.1515/GPS-2020-0041/XML Khan MZH, Tareq FK, Hossen MA, Roki MNAM (2018) Green Synthesis And Characterization Of Silver Nanoparticles Using Coriandrum Sativum Leaf Extract. Journal of Engineering Science and Technology 13:158–166 Mavani K, Shah M (2013) Synthesis of Silver Nanoparticles by using Sodium Borohydride as a Reducing Agent. International Journal of Engineering Research & Technology (IJERT) 2:1–5 Hemlata, Meena PR, Singh AP, Tejavath KK (2020) Biosynthesis of Silver Nanoparticles Using Cucumis prophetarum Aqueous Leaf Extract and Their Antibacterial and Antiproliferative Activity against Cancer Cell Lines. ACS Omega 5:5520–5528. https://doi.org/10.1021/ACSOMEGA.0C00155/ASSET/IMAGES/MEDIUM/AO0C00155_M003.GIF Sougandhi PR, Ramanaiah S (2020) Green synthesis and spectral characterization of silver nanoparticles from Psidium guajava leaf extract. Inorganic and Nano-Metal Chemistry 50:1290–1294. https://doi.org/10.1080/24701556.2020.1745839 Erdogan O, Abbak M, Demirbolat GM, et al (2019) Green synthesis of silver nanoparticles via Cynara scolymus leaf extracts: The characterization, anticancer potential with photodynamic therapy in MCF7 cells. PLoS One 14:e0216496. https://doi.org/10.1371/journal.pone.0216496 Asadi S, Charati FR, Akbari R, Razavi SA (2018) Green synthesis of silver nanoparticles using Taxus baccata Leaves extract and identify its specifications. J Mater Environ Sci 9:2798–2803 Loo YY, Rukayadi Y, Nor-Khaizura MAR, et al (2018) In Vitro antimicrobial activity of green synthesized silver nanoparticles against selected Gram-negative foodborne pathogens. Front Microbiol 9:1–9. https://doi.org/10.3389/fmicb.2018.01555 Hu X, Wu L, Du M, Wang L (2022) Eco-friendly synthesis of size-controlled silver nanoparticles by using Areca catechu nut aqueous extract and investigation of their potent antioxidant and anti-bacterial activities. Arabian Journal of Chemistry 15:103763. https://doi.org/10.1016/J.ARABJC.2022.103763 Masum MI, Siddiqa MM, Ali KA, et al (2019) Biogenic synthesis of silver nanoparticles using phyllanthus emblica fruit extract and its inhibitory action against the pathogen acidovorax oryzaestrain RS-2 of rice bacterial brown stripe. Front Microbiol 10:820. https://doi.org/10.3389/FMICB.2019.00820/BIBTEX Badi’ah HI, Seedeh F, Supriyanto G, Zaidan AH (2019) Synthesis of Silver Nanoparticles and the Development in Analysis Method. IOP Conf Ser Earth Environ Sci 217:012005. https://doi.org/10.1088/1755-1315/217/1/012005 Medda S, Hajra A, Dey U, et al (2015) Biosynthesis of silver nanoparticles from Aloe vera leaf extract and antifungal activity against Rhizopus sp. and Aspergillus sp. Applied Nanoscience (Switzerland) 5:875–880. https://doi.org/10.1007/S13204-014-0387-1/FIGURES/7 Ashraf JM, Ansari MA, Khan HM, et al (2016) Green synthesis of silver nanoparticles and characterization of their inhibitory effects on AGEs formation using biophysical techniques. Sci Rep 6:20414. https://doi.org/10.1038/SREP20414 Anandalakshmi K, Venugobal J, Ramasamy V (2016) Characterization of silver nanoparticles by green synthesis method using Pedalium murex leaf extract and their antibacterial activity. Applied Nanoscience (Switzerland) 6:399–408. https://doi.org/10.1007/S13204-015-0449-Z/TABLES/2 Rautela A, Rani J, Debnath (Das) M (2019) Green synthesis of silver nanoparticles from Tectona grandis seeds extract: characterization and mechanism of antimicrobial action on different microorganisms. J Anal Sci Technol 10:1–10. https://doi.org/10.1186/S40543-018-0163-Z/FIGURES/14 Bamsaoud SF, Basuliman MM, Bin-Hameed EA, et al (2021) The effect of volume and concentration of AgNO 3 aqueous solutions on silver nanoparticles synthesized using Ziziphus Spina–Christi leaf extract and their antibacterial activity. J Phys Conf Ser 1900:012005. https://doi.org/10.1088/1742-6596/1900/1/012005 Karekalammanavar G, David M (2018) Optimization of Green Synthesis of Silver Nanoparticles from Leaf Extracts of Tabernaemontana heyneana and evaluation of their catalytic activity on reduction of methylene blue. Haya: Saudi J Life Sci 3:248–254. https://doi.org/10.21276/HAYA.2018.3.3.5 Zayed MF, Eisa WH, Abdel-Moneam YK, et al (2015) Ziziphus spina-christi based bio-synthesis of Ag nanoparticles. J Ind Eng Chem 23:50–56. https://doi.org/10.1016/J.JIEC.2014.07.041 Clayton KN, Salameh JW, Wereley ST, Kinzer-Ursem TL (2016) Physical characterization of nanoparticle size and surface modification using particle scattering diffusometry. Biomicrofluidics 10:054107. https://doi.org/10.1063/1.4962992 Htwe YZN, Chow WS, Suda Y, Mariatti M (2019) Effect of silver nitrate concentration on the production of silver nanoparticles by green method. Mater Today Proc 17:568–573. https://doi.org/10.1016/J.MATPR.2019.06.336 Ardani HK, Imawan C, Handayani W, et al (2017) Enhancement of the stability of silver nanoparticles synthesized using aqueous extract of Diospyros discolor Willd . leaves using polyvinyl alcohol. IOP Conf Ser Mater Sci Eng 188:012056. https://doi.org/10.1088/1757-899X/188/1/012056 Ramalingam B, Parandhaman T, Das SK (2016) Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of Gram-Negative Bacteria viz. Escherichia coli and Pseudomonas aeruginosa . ACS Appl Mater Interfaces 8:4963–4976. https://doi.org/10.1021/ACSAMI.6B00161 Singh S, Bharti A, Meena VK (2015) Green synthesis of multi-shaped silver nanoparticles: optical, morphological and antibacterial properties. J Mater Sci: Mater Electron 26:3638–3648. https://doi.org/10.1007/S10854-015-2881-Y Wisnuwardhani HA, Lukmayani Y, Hazar S, Hoeruniswah H (2019) Optimization of Silver Nanoparticles Synthesis using Kawista ( Limonia Acidissima Groff .) leaves ethanol extract. J Phys Conf Ser 1375:12077. https://doi.org/10.1088/1742-6596/1375/1/012077 Fu LM, Hsu JH, Shih MK, et al (2021) Process Optimization of Silver Nanoparticle Synthesis and Its Application in Mercury Detection. Micromachines 12:1123. https://doi.org/10.3390/MI12091123 Fernando I, Zhou Y (2019) Impact of pH on the stability, dissolution and aggregation kinetics of silver nanoparticles. Chemosphere 216:297–305. https://doi.org/10.1016/J.CHEMOSPHERE.2018.10.122 Chitra K, Annadurai G (2014) Antibacterial activity of pH-dependent biosynthesized silver nanoparticles against clinical pathogen. Biomed Res Int 2014:1–6. https://doi.org/10.1155/2014/725165 Xiong Y, Liu X, Xiong H (2021) Aggregation modeling of the influence of pH on the aggregation of variably charged nanoparticles. Sci Rep 11:1–10. https://doi.org/10.1038/s41598-021-96798-3 Titma T (2018) The effect of surface charge and pH on the physiological behaviour of cobalt, copper, manganese, antimony, zinc and titanium oxide nanoparticles in vitro. Toxicol In Vitro 50:11–21. https://doi.org/10.1016/J.TIV.2018.02.010 Handayani W, Ningrum AS, Imawan C (2020) The Role of pH in Synthesis Silver Nanoparticles Using Pometia pinnata (Matoa) Leaves Extract as Bioreductor. J Phys Conf Ser 1428:012021. https://doi.org/10.1088/1742-6596/1428/1/012021 Miranda A, Akpobolokemi T, Chung E, et al (2022) pH Alteration in Plant-Mediated Green Synthesis and Its Resultant Impact on Antimicrobial Properties of Silver Nanoparticles (AgNPs). Antibiotics (Basel) 11:1592. https://doi.org/10.3390/ANTIBIOTICS11111592 Rousta MH, Ghasemi N (2019) Green synthesis of silver nanoparticles using a mountain plant extract. Rev Roum Chim 64:143–152. https://doi.org/10.33224/RRCH/2019.64.2.04 Liu H, Zhang H, Wang J, Wei J (2020) Effect of temperature on the size of biosynthesized silver nanoparticle: Deep insight into microscopic kinetics analysis. Arab J Chem 13:1011–1019. https://doi.org/10.1016/J.ARABJC.2017.09.004 Robertson JD, Rizzello L, Avila-Olias M, et al (2016) Purification of Nanoparticles by Size and Shape. Sci Rep 2016 6:1 6:1–9. https://doi.org/10.1038/srep27494 Asnaashari Kahnouji Y, Mosaddegh E, Bolorizadeh MA (2019) Detailed analysis of size-separation of silver nanoparticles by density gradient centrifugation method. Mater Sci Eng: C 103:109817. https://doi.org/10.1016/J.MSEC.2019.109817 Martins CSM, Sousa HBA, Prior JAV (2021) From Impure to Purified Silver Nanoparticles: Advances and Timeline in Separation Methods. Nanomaterials (Basel) 11:3407. https://doi.org/10.3390/NANO11123407 Banerjee P, Satapathy M, Mukhopahayay A, Das P (2014) Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: Synthesis, characterization, antimicrobial property and toxicity analysis. Bioresour Bioprocess 1:1–10. https://doi.org/10.1186/S40643-014-0003-Y/FIGURES/6 Venil CK, Malathi M, Velmurugan P, Renuka Devi P (2021) Green synthesis of silver nanoparticles using canthaxanthin from Dietzia maris AURCCBT01 and their cytotoxic properties against human keratinocyte cell line. J Appl Microbiol 130:1730–1744. https://doi.org/10.1111/JAM.14889 Karthik C, Suresh S, Sneha Mirulalini G, Kavitha S (2020) A FTIR approach of green synthesized silver nanoparticles by Ocimum sanctum and Ocimum gratissimum on mung bean seeds. Inorg Nano-Met Chem 50:606–612. https://doi.org/10.1080/24701556.2020.1723025 Francis S, Joseph S, Koshy EP, Mathew B (2018) Microwave assisted green synthesis of silver nanoparticles using leaf extract of elephantopus scaber and its environmental and biological applications. Artif Cells Nanomed Biotechnol 46:795–804. https://doi.org/10.1080/21691401.2017.1345921 Ali MH, Azad MAK, Khan KA, et al (2023) Analysis of Crystallographic Structures and Properties of Silver Nanoparticles Synthesized Using PKL Extract and Nanoscale Characterization Techniques. ACS Omega 8:28133–28142. https://doi.org/10.1021/ACSOMEGA.3C01261/ASSET/IMAGES/LARGE/AO3C01261_0007.JPEG Baran MF, Keskin C, Baran A, et al (2023) Green Synthesis of Silver Nanoparticles from Allium cepa L. Peel Extract, Their Antioxidant, Antipathogenic, and Anticholinesterase Activity. Molecules 28:2310. https://doi.org/10.3390/MOLECULES28052310 Sagadevan S, Vennila S, Singh P, et al (2019) Facile synthesis of silver nanoparticles using Averrhoa bilimbi L and Plum extracts and investigation on the synergistic bioactivity using in vitro models. Green Processing and Synthesis 8:873–884. https://doi.org/10.1515/GPS-2019-0058 Pryshchepa O, Pomastowski P, Buszewski B (2020) Silver nanoparticles: Synthesis, investigation techniques, and properties. Adv Colloid Interface Sci 284:102246. https://doi.org/10.1016/J.CIS.2020.102246 Ibraheim MH, Ibrahiem AA, Dalloul TR (2016) Biosynthesis of silver nanoparticles using Pomegranate juice extract and its antibacterial activity. Int J Appl Sci Biotechnol 4:254–258. https://doi.org/10.3126/IJASBT.V4I3.15417 Padman AJ, Henderson J, Hodgson S, Rahman PKSM (2014) Biomediated synthesis of silver nanoparticles using Exiguobacterium mexicanum . Biotechnol Lett 36:2079–2084. https://doi.org/10.1007/S10529-014-1579-1/FIGURES/5 Banerjee P, Satapathy M, Mukhopahayay A, Das P (2014) Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: Synthesis, characterization, antimicrobial property and toxicity analysis. Bioresour Bioprocess 1:1–10. https://doi.org/10.1186/S40643-014-0003-Y/FIGURES/6 Jagtap UB, Bapat VA (2013) Green synthesis of silver nanoparticles using Artocarpus heterophyllus Lam. seed extract and its antibacterial activity. Ind Crops Prod 46:132–137. https://doi.org/10.1016/J.INDCROP.2013.01.019 Helmlinger J, Sengstock C, Groß-Heitfeld C, et al (2016) Silver nanoparticles with different size and shape: equal cytotoxicity, but different antibacterial effects. RSC Adv 6:18490–18501. https://doi.org/10.1039/C5RA27836H Dong Y, Zhu H, Shen Y, et al (2019) Antibacterial activity of silver nanoparticles of different particle size against Vibrio Natriegens . PLoS One 14:e0222322. https://doi.org/10.1371/JOURNAL.PONE.0222322 Swolana D, Wojtyczka RD (2022) Activity of Silver Nanoparticles against Staphylococcus spp. Int J Mol Sci 23:4298. https://doi.org/10.3390/IJMS23084298 Jemal K, Sandeep B V., Pola S (2017) Synthesis, Characterization, and Evaluation of the Antibacterial Activity of Allophylus serratus Leaf and Leaf Derived Callus Extracts Mediated Silver Nanoparticles. J Nanomater 2017:1–11. https://doi.org/10.1155/2017/4213275 Paosen S, Bilhman S, Wunnoo S, et al (2023) Control of biomaterial-associated infections through biofabrication of gold nanoparticles using Musa sapientum extract. Biotechnol J 2300008. https://doi.org/10.1002/BIOT.202300008 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4614655","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319007848,"identity":"043d2b6f-f683-4a95-a4ca-788df78c5405","order_by":0,"name":"Wei Ting Jess Ong","email":"","orcid":"","institution":"UCSI University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"Ting Jess","lastName":"Ong","suffix":""},{"id":319007849,"identity":"2913dbb6-4a21-4861-99a7-8bcbd62eefc3","order_by":1,"name":"Swee Pin Yeap","email":"","orcid":"","institution":"UCSI University","correspondingAuthor":false,"prefix":"","firstName":"Swee","middleName":"Pin","lastName":"Yeap","suffix":""},{"id":319007850,"identity":"12aa1300-3036-4ba3-8bbb-bc4ff98f2996","order_by":2,"name":"Jahurul Haque","email":"","orcid":"","institution":"University of Central Arkansas","correspondingAuthor":false,"prefix":"","firstName":"Jahurul","middleName":"","lastName":"Haque","suffix":""},{"id":319007851,"identity":"a4c745e7-521d-4d6e-8f03-7224e01558e6","order_by":3,"name":"Kar Lin Nyam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHUlEQVRIiWNgGAWjYJACxoYCGzDFwGDAAGXASJxaDNJQtDQ2EKHlMJoAPi3mEslPN84wOJ/Y33+4Tbqg4LCcef/h9sc8DDayGw5wp0lg0WI5I83s5gaD24kzbiS2Sc8wOGwscyOxsZmHIc14wwHebdi0GNxIMLv5AKhlgwRjmzSPQVriDAlGkJbDibi1pH8DajmXuIH/IFhL/Qz+gyAt//FoyQE57EDiBoZEkBabBAkGsMMO4NZy5k3ZzRkGycZAvzRbA7UYzpBIbJw5Bygy8zDvZgtsWo6nb7vZU2En299//OFtnj8S8hL8xx98eAMU6Tveu/EGtlAWSMAiyMQDilNmBhZsDmPgP4BFkPEHhGb+gE3LKBgFo2AUjDQAAAoUbeBOyS7tAAAAAElFTkSuQmCC","orcid":"","institution":"UCSI University","correspondingAuthor":true,"prefix":"","firstName":"Kar","middleName":"Lin","lastName":"Nyam","suffix":""}],"badges":[],"createdAt":"2024-06-21 03:40:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4614655/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4614655/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60511177,"identity":"445460ff-a018-49d2-96d2-7f5ddaf696cd","added_by":"auto","created_at":"2024-07-17 14:26:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReaction mixture (a) before formation and (b) after formation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/7246e12a52c50cb89fd3c6a5.jpg"},{"id":60509984,"identity":"1aa37f4f-31d1-43e6-88c2-738a0e3a6def","added_by":"auto","created_at":"2024-07-17 14:18:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV-vis spectroscopy of KLE-AgNPs (a) UV-vis spectra of KLE-AgNPs, KLE and silver nitrate (b) effect of concentration of AgNO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and KLE (c) effect of incubation time (d) effect of pH (e) and effect of temperature\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/590fe853f44c4cafc3c611be.jpg"},{"id":60509986,"identity":"147c3d69-e98a-4864-8b20-5d9190edcb5b","added_by":"auto","created_at":"2024-07-17 14:18:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectrum of X-AgNPs, KLE-AgNPs and C-AgNPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/f930f0f797dbbbe477cd7317.jpg"},{"id":60511669,"identity":"295f7129-96f8-4e37-bdaa-bd3bf25b7042","added_by":"auto","created_at":"2024-07-17 14:34:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD spectrum of KLE-AgNPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/ad052671299aaf98045e5b3d.jpg"},{"id":60509981,"identity":"321c369d-eaa0-4ab2-af1d-8f1571200e38","added_by":"auto","created_at":"2024-07-17 14:18:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":52125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFESEM image of freeze-dried KLE-AgNPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/c374e08fc333e56166cf6113.jpg"},{"id":60509983,"identity":"c13f2015-a3fc-4614-ad42-ce4d955323fb","added_by":"auto","created_at":"2024-07-17 14:18:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEDX spectrum of freeze-dried KLE-AgNPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/d38ba69b99192ca2923bb9ee.jpg"},{"id":60512245,"identity":"1507ec92-ab07-4995-88c1-f867ba1bf655","added_by":"auto","created_at":"2024-07-17 14:42:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1315741,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4614655/v1/e4f375ed-9393-4c0f-94e3-073dd12c4075.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green synthesis of silver nanoparticles using Kenaf leaves extract and their antibacterial potential in acne management. ","fulltext":[{"header":"1.0 INTRODUCTION","content":"\u003cp\u003eAcne vulgaris is a common skin complication that affects many adolescents and young adults globally. Roughly 9.4% of the general population suffer with acne vulgaris [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. LeungOne of the most addressed factors that contributes to acne vulgaris is bacterial colonization of the skin microbiota which induces \u003cem\u003ein vitro\u003c/em\u003e inflammatory effect and thus leads to formation of lesions (acne). Most of the acne remedies currently available in the market such as topical antibiotics and retinoids cause skin irritation such as dryness and flaking, erythema, thinning of the stratum corneum and burning sensation upon application as these are extremely potent anti-acne agents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This raises interest in the use of silver nanoparticles (AgNPs) for acne management. This is contributed by the fact that AgNPs are well known for their profound therapeutic qualities such as antimicrobial, anticancer, anti-inflammatory and wound healing [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among the diverse curative traits, the antibacterial capability of AgNPs is the most sought after as multiple studies had proved that AgNPs are destructive against a wide range of bacteria and viruses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUniversally, AgNPs are synthesized via conventional methods such as physical and chemical. However, these conventional techniques require high heat and energy treatment, hence they are costly. Additionally, the chemicals used are toxic to the environment. Subsequently, the surging of sustainability trends prompted the rapid progression of green or bio-synthesis that is simple yet efficient, eco-friendly, and cost-effective [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Green synthesis involves the use of natural and accessible raw materials such as microorganisms, algae and plant extracts as reductants. Plant extracts are often preferred in green synthesis as studies had proven that the rate of synthesis and completion can be achieved within minutes of reaction time between the metal salt and the plant extracts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The procedure is also simple which enable for a straightforward scale-up practice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition to that, plant mediated synthesis does not require special conditions for culture prepation and maintenance as compared to other biological entities such as microbes. Furthermore, biocompatibility of plant mediated AgNPs is much higher compared to those synthesised with other resources [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As a result, they are more widely accepted in the pharmaceutical and medical sectors.\u003c/p\u003e \u003cp\u003eKenaf (\u003cem\u003eHibiscus cannabinus L\u003c/em\u003e.) is an annual, herbaceous crop that belongs to the plant family called Malvaceae. In Malaysia, Kenaf is a valuable crop as its fibre is extensively industrialised for paper and pulp. The fiber is also harvested as raw materials for ropes, sacks, animal beddings and biocomposites. Due to the exploitation of their fibrous stems, Kenaf leaves are often discarded as agricultural waste. However, there has been a shift of interest towards the exploitation of Kenaf leaves due to the advancement of circular economy where biowastes should be minimized via reusing and volarization. According to [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], ethanol-extracted kenaf leaves contained many phenolics and flavonoids such as tannic acid, catechin hydrate and caffeic acid. Similarly, the UPLC analysis reported that the major compounds present in the leaves extract were kaemperitrin, caffeic acid, myricetin glycoside, and p-hydroxybenzoic acid [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous studies had demonstrated that these bioactive compounds were capable of reducing silver ions (Ag\u003csup\u003e+\u003c/sup\u003e) to Ag\u003csup\u003e0\u003c/sup\u003e. Furthermore, the compounds also serve as stabilizing and capping agents that prevent the agglomeration of the NPs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, there is a lack of up-to-date and comprehensive research on the utilization of ethanol extracted Kenaf leaves extract (KLE) in the green synthesis of AgNPs and its application as a component in cosmeceuticals for acne management. Therefore, the present work focuses on the green synthesis of AgNPs using Kenaf leaves extract (KLE) in which the effect of concentration of silver nitrate and KLE, incubation time, medium pH, temperature and centrifugal force on the characteristics of KLE-synthesised AgNPs (KLE-AgNPs) were evaluated to dictate the optimal synthesis conditions. This approach warrants the production of tightly controlled AgNPs as the physicochemical properties of AgNPs are also strongly influenced by the operational variables during synthesis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Subsequently, the physicochemical properties of KLE-AgNPs were characterized by UV-vis spectroscopy, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and Field Emission Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy (FESEM-EDX). Additionally, the antibacterial activity of KLE-AgNPs was studied against several bacteria that are responsible for the pathogenesis of acne.\u003c/p\u003e"},{"header":"2.0 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and chemicals\u003c/h2\u003e \u003cp\u003eFresh Kenaf (\u003cem\u003eHibiscus cannabinus L\u003c/em\u003e.) leaves were obtained from Lembaga Kenaf \u0026amp; Tembakau Negara (LKTN) (Malaysia). Ethanol, silver nitrate, resazurin sodium salt and commercial silver nanoparticles were all purchased from Sigma-Aldrich (Missouri, United States). Bacteria strains, \u003cem\u003eCutibacterium acnes\u003c/em\u003e (\u003cem\u003eC.acnes\u003c/em\u003e), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS.aureus\u003c/em\u003e) and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (\u003cem\u003eS.epidermidis\u003c/em\u003e), were acquired from ATCC (Manassas, Virginia). Nutrient broth was purchased from Merck (Germany) and tryptone soy broth as well as tryptone soy agar with sheep blood were purchased from Oxoid (England, United Kingdom). All chemicals used were of analytical reagent grade. The ultra-pure water (Millipore, USA) was used throughout the analysis.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Pulsed ultrasonic assisted extraction of powdered kenaf leaves\u003c/h2\u003e \u003cp\u003eThe extraction of kenaf leaves extract (KLE) was carried out according to [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] with slight modifications. Firstly, 10g of dried, and grounded kenaf leaves was added to 100mL of pure ethanol as the extraction solvent. Then, the mixture was ultrasonicated using the labsonic ultrasonic homogenizer (Sartorius, Germany). The ultrasonic assisted extraction of kenaf leaves was performed at the sonication amplitude of 50%. One sonication cycle consisted of one minute pulse duration period and one minute pulse interval period. This cycle was repeated three times. An ice bath was used during the sonication process to maintain the temperature of KLE within the initial range of 18\u0026ndash;22\u0026thinsp;\u0026plusmn;\u0026thinsp;3℃. This was done to prevent the degradation of the bioactive compounds because of the heat generated during ultrasonication. The extracted mixture was then transferred to Falcon tubes with equal total weight and centrifuged at 5000rpm for 10 minutes. After centrifugation, the supernatant of the KLE was filtered with Whatman filter paper while the pellet was discarded. Next, about 95% of the extraction solvent, ethanol, in the filtered KLE was removed using a rotary evaporator (Buchi, Switzerland) at 45\u0026ordm;C under reduced pressure. After evaporation, the concentrated crude KLE stored in the freezer at \u0026minus;\u0026thinsp;20\u0026deg;C under dark conditions for future analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Green synthesis of silver nanoparticles (KLE-AgNPs)\u003c/h2\u003e \u003cp\u003eGreen synthesis of silver nanoparticles were generally conducted according to [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] with slight modifications. First, concentrated KLE solution was prepared by dissolving the desired amount in ethanol. Then, in a ratio of 1:9, 5 mL of KLE solution was added to 45 mL of aqueous silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e) drop by drop while the reaction mixture was stirred at 700 rpm to initiate the reduction process. The stirring was stopped once all the KLE solution was added. The reaction mixture was then quickly adjusted to desired pH with 1M sodium hydroxide (NaOH). The formation of AgNPs was observed by the immediate color change of the reaction mixture from greenish-yellow to reddish brown. The reaction mixtures were incubated at different temperatures under dark conditions and the UV-vis spectra of the reaction mixtures were measured at different time intervals up to 48 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 Chemical synthesis of silver nanoparticles (C-AgNPs)\u003c/h2\u003e \u003cp\u003eChemical synthesis of silver nanoparticles (C-AgNPs) was operated by using sodium borohydride (NaBH\u003csub\u003e4\u003c/sub\u003e) as described by [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] with slight changes. Firstly, aqueous solutions of 10mL of 0.01M of AgNO\u003csub\u003e3\u003c/sub\u003e and 30mL of 0.02M NaBH\u003csub\u003e4\u003c/sub\u003e were freshly prepared. The NaBH\u003csub\u003e4\u003c/sub\u003e solution was placed on an ice bath and allowed to cool for 30 minutes. After cooling, AgNO\u003csub\u003e3\u003c/sub\u003e solution was added drop by drop while the stirring was initiated at a speed of 700 rpm. Once all the AgNO\u003csub\u003e3\u003c/sub\u003e solution was added, the stirring was stopped.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4 Purification of KLE-AgNPs\u003c/h2\u003e \u003cp\u003eThe purification of KLE-AgNPs was performed according to[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] with minor modifications. This procedure was required to remove any excess reactants before further analysis of the KLE-AgNPs. The reaction mixture was centrifuged at a speed of 10000\u003cem\u003eg\u003c/em\u003e at a temperature of 4\u0026deg;C for 150 minutes using 5415R microcentrifuge (Eppendorf, Germany). Then, the supernatant was discarded and the pellet was resuspended with ultra-pure water (washing). The resuspended pellet was further centrifuged again under the same conditions and the supernatant was removed (1 washing cycle). The washing cycle was repeated 3 times. Finally, the purified KLE-AgNPs were resuspended with ultra-pure water and the solution was stored at 4\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.1.5 Single factor optimization of the green synthesis\u003c/h2\u003e \u003cp\u003e \u003cb\u003e(a) Effect of concentration of silver nitrate and KLE\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe concentration of silver nitrate used was 1mM, 3mM and 5mM while the concentration of KLE used was 1mg/mL, 3mg/mL and 5mg/mL. The medium pH, reaction temperature and incubation time were maintained at pH 11, 37\u0026deg;C and 48 hours respectively. The centrifugal force for the purification procedure described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.1.4\u003c/span\u003e was maintained at 10000\u003cem\u003eg\u003c/em\u003e. The conditions for the parameters were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eConcentration of AgNO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eand KLE in different samples\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration of AgNO\u003csub\u003e3\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration of KLE (mg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(b) Effect of incubation time\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effect of incubation time on the formation of KLE-AgNPs was monitored using UV-vis spectroscopy of the reaction mixture at different time intervals, 0 hour, 1st hour, 2nd hour, 3rd hour, 4th hour, 5th hour, 6th hour, 24th hour and 48th hour. The concentration of AgNO\u003csub\u003e3\u003c/sub\u003e, concentration of KLE, medium pH and reaction temperature were maintained at 3mM, 3mg/mL, pH 11 and 37\u0026deg;C respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(c) Effect of medium pH\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe reaction mixtures were adjusted to pH 3, 5, 7, 9 and 11 after addition of KLE solution prior to incubation for 48 hours at 37\u0026deg;C. The concentration of AgNO\u003csub\u003e3\u003c/sub\u003e and KLE were maintained at 3mM and 3mg/mL respectively. After incubation, the reaction mixtures were purified via centrifugation at 10000\u003cem\u003eg\u003c/em\u003e with the conditions described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.1.4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(d) Effect of reaction temperature\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe reaction mixtures were incubated at 15\u0026deg;C, 30\u0026deg;C, 37\u0026deg;C, 45\u0026deg;C, 60\u0026deg;C for 48 hours. The concentration of AgNO\u003csub\u003e3\u003c/sub\u003e, concentration of KLE and medium pH were maintained at 3mM, 3mg/mL and pH 11 respectively. After incubation, the reaction mixtures were purified via centrifugation at 10000\u003cem\u003eg\u003c/em\u003e with the conditions described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.1.4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(e) Effect of centrifugal force\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGreen synthesis was performed according to the described general procedure in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.1.2\u003c/span\u003e. The concentration of AgNO\u003csub\u003e3\u003c/sub\u003e and concentration of KLE used were 3mM and 3mg/mL respectively. The medium pH was adjusted to 11 and incubated at 37\u0026deg;C for 48 hours. After incubation, the reaction solution was purified according to the procedure described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.1.4\u003c/span\u003e but with different centrifugal force, 4000\u003cem\u003eg\u003c/em\u003e, 8000\u003cem\u003eg\u003c/em\u003e, 10000\u003cem\u003eg\u003c/em\u003e and 16000\u003cem\u003eg\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.1.6 Characterization of KLE-synthesised silver nanoparticles (KLE-AgNPs)\u003c/h2\u003e \u003cp\u003eFormation of KLE-AgNPs was observed with the FLUOStar Omega microplate reader (BMG Labtech, Germany) based on the method described by [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] with slight modification. The gradual reduction of Ag\u003csup\u003e+\u003c/sup\u003e ions in solution was monitored at a range of 300nm to 800nm. The mean particle size, zeta potential and polydispersity index (PdI) were measured by dynamic light scattering (DLS) analysis using Malvern Zetasizer Nano ZS (Malvern Panalytical, United Kingdom) whereby samples were diluted 10x with ultra-pure water and added into disposable folded capillary cells at 25\u0026deg;C. The detection of peak values and active functional groups of KLE-AgNPs were characterized using Fourier Transform Infrared (FTIR) spectrophotometer (Shimadzu, Japan) as stated by [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] with some modifications. The scan range was done through the wavenumber from 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a resolution of 1 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to acquire the spectra. The analysis was carried out at room temperature and single spectra were corrected against the background spectrum of air. The X-ray diffraction analysis of the AgNPs was carried out following the method as reported earlier by [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] with slight modifications. The purified KLE-AgNPs solution was freeze-dried and the KLE-AgNPs powder was collected for this analysis. The sample was observed using the Empyrean X-ray diffractometer (Malvern Panalytical, United Kingdom) with CuKα radiation at room temperature with 2θ angle in the range of 20\u0026ndash;90\u0026deg; under continuous scanning at 40Ma and 45Kv. The scanning rate was 2\u0026deg; per min. The morphology, size and elemental mapping of KLE-AgNPs were observed using JSM-7600F Field Emission Scanning Electron Microscope (FESEM) equipped with Energy Dispersive X-Ray (EDX) (Jeol, Japan). Freeze-dried KLE-AgNPs were prepared and smeared on sample grid which was then coated with platinum prior to analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.1.7 Antimicrobial analysis of AgNPs\u003c/h2\u003e \u003cp\u003eThe antimicrobial action of optimized KLE-AgNPs was determined via minimum inhibitory concentration (MIC) using broth micro-dilution technique in 96 well microtiter plate (Nest, China) with reference to [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] but with some alterations. The sample was prepared by diluting purified KLE-AgNPs with ultra-pure water to obtain the final concentration of 500\u0026micro;g/mL. Sixty microliters of respective broth were added into the wells containing 100 \u0026micro;L of two-fold serial diluted KLE-AgNPs in similar broth. Then, 20\u0026micro;L of resazurin indicator (0.6mg/mL) and 20\u0026micro;L of the standardized bacterial suspension were added to make up a total volume of 200\u0026micro;L in each well. The final concentration of the KLE-AgNPs sample ranged from 0.98 \u0026micro;g/mL to 250\u0026micro;g/mL. The positive control was tetracycline while the negative control was the medium broth without sample. The 96-well plates were incubated at 37\u0026ordm;C in aerobic condition for \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS.epidermidis\u003c/em\u003e (24 hours) and anaerobic condition for \u003cem\u003eC.acnes\u003c/em\u003e (48 hours). The MIC value was recorded as the lowest concentration of KLE-AgNPs at which no visible colour change from blue to pink after incubation was observed. The optimized KLE-AgNPs were also tested on their bacteriostatic and bactericidal activity by inoculating the suspension from the wells that showed no colour change (blue coloured wells) onto NA for \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e and TSA sheep blood agar for \u003cem\u003eC. acnes\u003c/em\u003e via the streak-plate method. Then, the agar plates were incubated at 37\u0026ordm;C for 24 and 48 hours respectively. The MBC value was recorded as the lowest concentration of KLE-AgNPs at which there were no bacteria growth on the plate observed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.1.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll results were recorded in triplicates and analysed using One-way Analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s test. The analysis was performed using MINITAB 16 (Minitab Inc, Pennsylvania, USA). All experimental data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and the differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3.0 RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Colour observation\u003c/h2\u003e \u003cp\u003eThe visual colour change of the reacting solution preliminarily indicated the formation of KLE-AgNPs. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the reacting solution turned from greenish yellow to reddish brown, indicating the rapid reduction of Ag\u003csup\u003e+\u003c/sup\u003e ions to Ag\u003csup\u003e0\u003c/sup\u003e by the KLE. Similar colour observations were reported by the authors in the green synthesis of AgNPs using \u003cem\u003ePhyllanthus emblica\u003c/em\u003e fruit extract and \u003cem\u003eAreca catechu\u003c/em\u003e nut aqueous extract [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The brown colour of AgNPs correlates strongly with their surface plasmon resonance (SPR) effect and any change in colour provides information on their physical properties. To illustrate, Badi\u0026rsquo;ah et al. (2019) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] stated that further change of colour of the reaction mixture from yellow or brown to grey signified the aggregation of AgNPs to form larger-sized AgNPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 UV-vis spectroscopy\u003c/h2\u003e \u003cp\u003eThe green synthesis of AgNPs with KLE was further confirmed by UV-vis spectroscopy. The UV-vis spectra supplemented the SPR effect that was responsible for the colour change as perceived in Section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e. Based on Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a), an absorption peak was observed at a wavelength of 400nm between 300nm to 800nm. This is in accordance with the study on the green synthesis of AgNPs using \u003cem\u003eAloe vera\u003c/em\u003e leaf extract which also reported maximum absorption at 400 nm [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been established that the spectroscopic nature of AgNPs exhibit absorbance peaks ranging from 400-500nm [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This phenomenon could be attributed to the free electrons present which vibrate in resonance with the light wave [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. According to Rautela et al., (2019) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the reduction process was aided by the donation of electrons to Ag\u003csup\u003e+\u003c/sup\u003e ions by the phenolic contents in KLE to form Ag\u003csup\u003e0\u003c/sup\u003e nanoparticles. Moreover, the sharp and narrow distribution of the absorption peak indicated that the AgNPs formed were predominantly smaller in size and monodispersed whereas the broader peaks signified the production of larger and polydisperse AgNPs. Absorption spectra of silver nitrate and KLE were performed and served as control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Single factor optimization of the green synthesis\u003c/h2\u003e \u003cp\u003e \u003cb\u003e(a) Effect of concentration of AgNO\u003c/b\u003e \u003csub\u003e \u003cb\u003e3\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand KLE\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe KLE-mediated synthesis of AgNPs were subjected to combinations of various concentrations of AgNO\u003csub\u003e3\u003c/sub\u003e and KLE. The SPR peaks of different samples were detected via UV-vis spectroscopy. Referring to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b), the increase in concentration of both AgNO\u003csub\u003e3\u003c/sub\u003e and KLE simultaneously led to an increase in the absorption peak. The highest absorption intensity was recorded with Sample 9 which consisted of 5mM AgNO\u003csub\u003e3\u003c/sub\u003e and 5mg/mL of KLE. This outcome was apparent as more silver precursors and KLE were available for the reduction to take place, thus a greater amount of KLE-AgNPs were formed. However, despite the increase in concentration of AgNO\u003csub\u003e3\u003c/sub\u003e, there was a significant decrease in absorption peaks for Sample 4 and 7 whereby the concentrations of AgNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;KLE were 3mM\u0026thinsp;+\u0026thinsp;1mg/mL and 5mM\u0026thinsp;+\u0026thinsp;1mg/mL respectively. This pattern showed that the increase in AgNO\u003csub\u003e3\u003c/sub\u003e without the increase of KLE led to low conversion of Ag\u003csup\u003e+\u003c/sup\u003e to Ag\u003csup\u003e0\u003c/sup\u003e and thus lesser number of AgNPs were formed. It could be deduced that KLE was the limiting reactant in this reaction as lesser bioactive compounds were available to fully reduce the Ag\u003csup\u003e+\u003c/sup\u003e ions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The broadness of the peaks reflected the possible aggregation of AgNPs in the reaction mixture of Sample 4 and Sample 7. This outcome was comparable with a past study which concluded that the increase \u003cem\u003eTabernaemontana heyneana\u003c/em\u003e leaves extract led to increase in absorption intensity while increase in AgNO\u003csub\u003e3\u003c/sub\u003e could lead to a broad spectrum especially when the Ag\u003csup\u003e+\u003c/sup\u003e ions is in excess [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilar effects of higher concentration of AgNO\u003csub\u003e3\u003c/sub\u003e and lower concentration of KLE as in Sample 4 and Sample 7 were reflected in their dynamic light scattering (DLS) analysis. From Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, particle sizes of S4 and S7 were significantly larger than the rest of the samples except Sample 8. This finding aligned with the broadness of their SPR peaks as discussed earlier. Nonetheless, Sample 8, with the concentration of 5mM AgNO\u003csub\u003e3\u003c/sub\u003e and 3mg/mL KLE respectively, recorded the largest particle size (94.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73nm). The significantly larger size of S4, S7 and S8 could be explained by the lack of bioactive compounds from KLE. These compounds are also responsible for the capping and stabilizing action of AgNPs that prevent the formation of larger aggregates. Besides the factor of KLE, Zayed et al. (2015) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] stated that excess Ag\u003csup\u003e+\u003c/sup\u003e ions could promote the growth of NPs. Consequently, the KLE-AgNPs produced from the three samples were significantly larger. Meanwhile, the smallest particle size produced was from Sample 3 with 1mM AgNO\u003csub\u003e3\u003c/sub\u003e and 5mg/mL KLE. High concentration of KLE enabled the full conversion Ag\u003csup\u003e+\u003c/sup\u003e ions to KLE-AgNPs while preserving the size of the NPs formed.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of varying concentration of AgNO\u003csub\u003e3\u003c/sub\u003e and KLE on the particle size, zeta potential and polydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolydispersity index (PdI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-49.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-50.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-47.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-49.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-44.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-49.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-48.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-48.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-47.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eThe optimal concentration of AgNO\u003csub\u003e3\u003c/sub\u003e and KLE in the green synthesis of AgNPs were determined based on the DLS analysis of the KLE-AgNPs complementary to the UV-Vis spectra. Besides particle size, the polydispersity index (PdI) and zeta potential of the KLE-AgNPs were taken into account as the PdI values estimate the uniformity in terms of size distribution while zeta potential values represent the stability of the NPs. Nanoparticles are known to be stable when their zeta potentials are either greater than +\u0026thinsp;30 mV or less than \u0026minus;\u0026thinsp;30 mV [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Based on Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the KLE-AgNPs produced from all samples had particle sizes of less than 100nm and were relatively stable according to their zeta potential readings. As it is known that the particle sizes of NPs significantly affect the pharmacokinetics properties, acquiring samples with uniform particle size distribution is vital. Generally, NPs are regarded as highly monodisperse if the PdI value is less 0.1 and moderately disperse if the PdI value is within 0.1 to 0.4 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, samples with PdI values closest to 0.1 were considered to minimize any sort of biological fluctuations of the AgNPs to ensure consistency of future analysis. This narrowed down the selection to S1, S4, S5 and S7. As S1, S4 and S7 had low reduction efficiency and larger particle sizes, S5 (3mM AgNO\u003csub\u003e3\u003c/sub\u003e and 3mg/mL KLE) were chosen as the optimal condition for future analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(b) Effect of incubation time\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe reaction mixture was continuously incubated at 37\u0026deg;C for a total of 48 hours to determine the complete formation of KLE-AgNPs. As revealed in the UV-vis spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (c)), the absorption spectra gradually increased every hour for the first 6 hours and by the 24th hour, there was an exponential rise. The SPR peaks were less intense and broader in the first 6 hours and therefore, an extended duration of 24 and 48 hours were added to further confirm the completion of the biosynthesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consequently, the SPR peaks observed at the later stage were more intense and sharper. The increase in absorption reading during incubation signified the increase in the yield of KLE-AgNPs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This could be deduced by the continuous reaction that occurred when the silver precursor (silver nitrate) and the reducing agent (KLE) remain in contact for a longer period. Additionally, the intensity no longer increased at the 48th hour and thus implied the complete reduction of Ag\u003csup\u003e+\u003c/sup\u003e ions to Ag\u003csup\u003e0\u003c/sup\u003e. This finding is in correspondence with the synthesis of AgNPs using Kawista (\u003cem\u003eLimonia Acidissima Groff\u003c/em\u003e.) leaves ethanol extract [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e(c) Effect of pH on the synthesis of KLE-AgNPs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, pH of the reaction mixtures was adjusted to pH 3, pH 5, pH 7, pH 9 and pH 11. The effect of pH on the formation of KLE-AgNPs were observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (d). The UV-Vis spectra portrayed lower and broader peaks at low to neutral pH (pH 5 and 7). At pH 3, no absorbance peak was observed due to the nucleation of AgNPs, thus pH 3 was unfavorable for the synthesis. Meanwhile at pH 9 and 11, higher maximum absorption peaks at 410nm with narrower bands were observed. This observation evidently proved that pH could impact the formation of AgNPs by altering their surface charges. In one recent study, the authors deciphered that the decrease in absorbance values at low pH was due to the occurrence of severe agglomeration stimulated by denser surface charges [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This clarification is supported by Fernando and Zhou (2019) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] who claimed that at low pH, the energy barrier between the nanoparticles is neutralized and suppressed by the kinetic energy of the Brownian motion, resulting in particle agglomeration.\u003c/p\u003e \u003cp\u003eBesides UV-vis analysis, the samples were also subjected to DLS measurements to determine the size, zeta potential and PdI of KLE-AgNPs formed under different pH. As presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the mean size of KLE-AgNPs at pH 3 was 1427.30\u0026thinsp;\u0026plusmn;\u0026thinsp;490.60nm. This further confirmed that nano-sized particles were not formed when the synthesis was conducted in a highly acidic medium. Despite that, AgNPs with sizes less than 100nm were formed at pH 5, 7 and 9, which were 92.30\u0026thinsp;\u0026plusmn;\u0026thinsp;26.70nm, 74.10\u0026thinsp;\u0026plusmn;\u0026thinsp;11.50nm and 79.70\u0026thinsp;\u0026plusmn;\u0026thinsp;24.60nm respectively. The smallest-sized KLE-AgNPs (60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4nm) were formed in the most alkaline medium (pH 11). Succinctly, the size of KLE-AgNPs was inversely proportional to the pH values. Previous study had published comparable finding using \u003cem\u003eBacillus brevis\u003c/em\u003e culture for AgNPs synthesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. According to these authors, more -OH ions were available at high pH which further elevated the nucleation process, leading to the formation of smaller-sized AgNPs. Additionally, the dispersion of the KLE-AgNPs were determined through their PdI values and it could be deduced that KLE-AgNPs synthesised at pH 11 exhibited the lowest polydispersity. With reference to the zeta potential readings, it could be deduced from Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e that the increase in pH led to the increase in the degree of stability of the KLE-AgNPs. The high zeta potential values implied that the AgNPs produced in this study have developed a high net surface charge that can promote electrostatic repulsion.\u003c/p\u003e \u003cp\u003eTo be brief, at low or high pH, the H\u003csup\u003e+\u003c/sup\u003e ions and OH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions can alter the positive and negative charges on the surface of NPs which will lead to electrostatic repulsion between particles and thus prevent aggregation and formation of larger-sized NPs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This also means that nanoparticles are least stable in pH 7, the isoelectric point, as the surface charges are neutralized (zero charge) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, in plant-mediated synthesis, alkaline medium appeared to be the ideal condition as it promoted the ionization of the functional groups such as COOH groups to -COO- which simultaneously increased the rate of reduction of Ag\u003csup\u003e+\u003c/sup\u003e ions and the negative surface charges [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This explained the formation of smaller particle size with higher stability (lower zeta potential values) in pH 11. As a result, the optimal pH for the formation of relatively stable and small-sized KLE-AgNPs would be pH 11.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of pH on the average size, zeta potential and polydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolydispersity index (PdI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1427.30\u0026thinsp;\u0026plusmn;\u0026thinsp;490.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-11.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.30\u0026thinsp;\u0026plusmn;\u0026thinsp;26.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-26.07\u0026thinsp;\u0026plusmn;\u0026thinsp;6.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.10\u0026thinsp;\u0026plusmn;\u0026thinsp;11.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.30 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.70\u0026thinsp;\u0026plusmn;\u0026thinsp;24.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-41.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.61\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-43.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(d) Effect of temperature\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTemperature is one of the vital parameters that can significantly affect the properties of KLE-AgNPs formed. In this study, the reacting solution was incubated at different temperatures (15\u0026deg;C, 30\u0026deg;C, 37\u0026deg;C, 45\u0026deg;C and 60\u0026deg;C) and the effect was analyzed using UV-Vis spectroscopy and DLS. As portrayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (e), the increase in temperature led to the increase in maximum absorption wavelength. This meant that the rate of formation of KLE-AgNPs was elevated as the temperature increased. The absorption bands also appeared sharper with the increase in temperature. Equivalent observation was reported by Rousta and Ghasemi (2019). However, unlike pH, results from the DLS analysis (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) for the different temperatures did not complement the UV-vis spectra results. The order, in terms of mean size, was 37\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;30\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;15\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;60\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;45\u0026deg;C while in terms of PdI, it showed a trend of 37\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;30\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;15\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;45\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;60\u0026deg;C. Although the UV-Vis spectra illustrated that the highest and sharpest absorbance peak belonged to KLE-AgNPs synthesized at 60\u0026deg;C, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e documented that KLE-AgNPs synthesized at 37\u0026deg;C were the smallest in size with the lowest value of PdI (0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01). This extraordinary phenomenon was vindicated by Liu et al. (2020) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The authors reported that sufficient Ag\u003csup\u003e+\u003c/sup\u003e precursors led to the increase in particle size at 70\u0026deg;C to 80\u0026deg;C due to the linear increase of growth rate constant with the increased of synthesis temperature. In other words, growth process superseded the nucleation process, causing the increase in size. Hence, it was confirmed that physical properties of AgNPs differ accordingly at varying temperatures. Additionally, KLE-AgNPs synthesised at all temperatures exhibited high degrees of stability (zeta potential values of less than \u0026minus;\u0026thinsp;30mV). Based on the above elucidation, the optimal reaction and incubation temperature was 37\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of temperature on the average size, zeta potential and polydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolydispersity index (PdI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.44\u0026thinsp;\u0026plusmn;\u0026thinsp;22.51\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-40.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.82\u0026thinsp;\u0026plusmn;\u0026thinsp;11.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-41.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-43.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.27\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-43.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.69\u0026thinsp;\u0026plusmn;\u0026thinsp;7.71\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-45.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(e) Effect of centrifugal force\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFrom Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the average sizes of KLE-AgNPs were directly proportional with the increase in centrifugal force and they were statistically significant. On the contrary, the increase in centrifugal force led to a decrease in PdI, suggesting an increase in monodispersity of the KLE-AgNPs formed. In particular, the particles purified at 10000\u003cem\u003eg\u003c/em\u003e recorded the lowest PdI, suggesting their moderately dispersed nature with a mean size of 60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41nm. The magnitude of zeta potentials of all samples was lower than \u0026minus;\u0026thinsp;30mV, indicating that the KLE-AgNPs were highly stable regardless of the change in the speed of centrifugation. In general, centrifugation allows the separation of AgNPs from excess ligands or impurities, with the aim to convert polydisperse silver suspensions into isolated, monodisperse samples [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A higher centrifugal force is required to separate smaller-sized AgNPs. However, in this study, higher centrifugal force showed a contrasting effect. This phenomenon could be explained by the correlation between surface charges and particle size. Asnaashari Kahnouji et al. (2019) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] conferred that smaller-sized particles have higher surface charges which leads to stronger tendency to bind together and agglomerate. Besides, the pressure induced by the high centrifugal force could potentially flatten the KLE-AgNPs, resulting in shape deformation and size increment. Nonetheless, comprehensive analysis on the optimal centrifugal force remained scarce as specific parameters and centrifugal conditions used by researchers were not disclosed in many experimental and research papers [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Thus, the data obtained in this study may serve as a reference for future researchers who intend to review or compare centrifugation techniques for their work. In addition to that, Table\u0026nbsp;3.6 comparatively highlights the optimized synthesis conditions of AgNPs using plant extracts as reported in previous studies and the present.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of centrifugal force on the average size, zeta potential and polydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCentrifugal force (\u003cem\u003eg\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolydispersity index (PdI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-48.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.78\u0026thinsp;\u0026plusmn;\u0026thinsp;14.58\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-46.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-43.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-45.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Comparison between optimized KLE-AgNPs, commercial and chemically synthesised AgNPs\u003c/h2\u003e \u003cp\u003eBased on the evaluation done on section \u003cspan refid=\"Sec15\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e (a) to (e), it could be deduced that the optimal concentrations for both silver precursor and reducing agent were 3mM AgNO\u003csub\u003e3\u003c/sub\u003e and 3mg/mL KLE while the optimal conditions were 48 hours incubation time at 37\u0026deg;C with pH 11 medium. After synthesis, 10000\u003cem\u003eg\u003c/em\u003e of centrifugal force was employed for the purification of KLE-AgNPs. The particle size, zeta potential and PdI of the optimized KLE-AgNPs were compared with the particle size, zeta potential and PdI of commercial AgNPs (X-AgNPs) and chemically synthesized AgNPs (C-AgNPs). With reference to Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e the optimized KLE-AgNPs were smaller, more stable and monodispersed in comparison to the other two AgNPs. This proved that the green synthesis of AgNPs using KLE in which the synthesis process was optimized would produce particles with tightly controlled properties.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe particle size, zeta potential and polydispersity index of KLE-AgNPs, X-AgNPs and C-AgNPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolydispersity index (PdI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKLE-AgNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-43.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eX-AgNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e146.10\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-36.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-AgNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e149.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-41.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3); mean values within the same column indicated by different superscript uppercase letters are significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Characterization of KLE-AgNPs\u003c/h2\u003e \u003cp\u003e \u003cb\u003e(a) FTIR spectroscopy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe optimized KLE-AgNPs were subjected to FTIR analysis to determine the functional groups of the bioactive compounds present in KLE that were responsible for reducing Ag\u003csup\u003e+\u003c/sup\u003e ions to Ag\u003csup\u003e0\u003c/sup\u003e and stabilizing the KLE-AgNPs formed by acting as capping agents [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. It could be observed that the IR spectra of AgNPs contained peaks ranging from 3329.14cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 599.86 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The broad peak at 3329.14cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the H-bonded, O-H stretching, revealing the presence of alcohols and phenols. Meanwhile, a sharper peak at 1633.71cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which symbolizes the C\u0026thinsp;=\u0026thinsp;O band, served as evidence that there were carbonyl groups. In addition to that, the less intense peak at 667.37 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to the presence of N-H wag amines. The data presented in this research were similar to the findings of several other studies involving the biosynthesis of AgNPs, confirming the feasibility of using KLE for AgNPs synthesis [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The FTIR spectra of KLE-AgNPs was also compared with the FTIR spectra of commercially bought AgNPs (X-AgNPs) and chemically synthesized-AgNPs (C-AgNPs). The similarities of peaks between the three AgNPs further confirmed the formation of AgNPs mediated by KLE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(b) X-Ray Diffraction (XRD) analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFurther characterization of the optimized KLE-AgNPs was conducted using the X-Ray diffraction (XRD) analysis. This technique serves to identify the crystalline structure and the phase purity of the NPs formed. In accordance with Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, there were four major diffraction peaks observed at 2θ values of 38.26\u0026deg;, 44.41\u0026deg;, 64.65\u0026deg; and 77.49\u0026deg; with the maximum peak at position 38.26\u0026deg;. These peaks correspond to the crystallographic lattice planes of AgNPs which are (111), (200), (220) and (311) under the standard set by the Joint Committee on Powder Diffraction Standards, JCPDS (File no. 04-0783) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. A similar XRD pattern was observed in a study conducted by Baran et al. (2023) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] on the green synthesis of AgNPs using \u003cem\u003eAllium cepa\u003c/em\u003e peel extract. These planes represent the face centered cubic structure of silver. The appearance of additional peaks as seen on the XRD spectrum can be attributed to the crystallization of bioactive compounds from the KLE on the surface of AgNPs.\u003c/p\u003e \u003cp\u003eThe Debye-Scherrer equation, d =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{K\\lambda }{{\\beta }\\text{c}\\text{o}\\text{s} {\\theta }}\\)\u003c/span\u003e\u003c/span\u003e, was used to determine the average size of the AgNPs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. With reference to that, the average crystalline size of KLE-AgNPs was estimated to be 58.59nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(c) Field Emission Scanning Electron Microsopy \u0026ndash; Energy Dispersive X-Ray (FESEM-EDX)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe shape and size of the KLE-AgNPs were identified using FESEM coupled with EDX for elemental mapping. Based on Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the optimized KLE-AgNPs appeared to be in spherical shape and monodisperse with the average diameters ranging from 20nm to 55nm. In comparison with the size detected via DLS analysis, the KLE-AgNPs captured by FESEM were smaller. According to Pryshchepa et al. (2020) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], DLS often overestimates particle sizes as it measures the hydrodynamic diameter of the NPs. Moreover, the FESEM image also displayed several larger particles due to the agglomeration of smaller KLE-AgNPs. The occurrence of aggregation could be caused by the freeze-drying process that was done prior to the analysis and presence of high surface activity [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Thin layers were noted on the outer parts of the KLE-AgNPs as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e which demonstrated the capping layers of organic molecules present during the synthesis. With reference to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the EDX spectrum exhibited that the strongest peak representing the major element was silver thereby confirming the formation of KLE-AgNPs. The biomolecules from KLE acting as the capping agents, were represented by other weak peaks such as carbon, oxygen, silicon, sulphur, sodium and potassium [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. According to Jagtap and Bapat (2013) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], the absorption peak of metallic silver nanoparticles usually appears at 3keV as evident in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e due to their SPR. The FESEM image suggested that with the optimum conditions, green synthesis of AgNPs via KLE was successfully achieved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Antimicrobial activity\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of optimized KLE-AgNPs on \u003cem\u003eCutibacterium acnes\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e were evaluated using the minimum inhibitory concentration (MIC) technique. The MIC values of KLE-AgNPs on the 3 bacteria were recorded in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Based on Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, KLE-AgNPs showed the highest inhibitory effect on \u003cem\u003eC.acnes\u003c/em\u003e followed by both \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eS.epidermidis\u003c/em\u003e. The MIC values recorded for \u003cem\u003eC.acnes\u003c/em\u003e and \u003cem\u003eS.aureus\u003c/em\u003e and \u003cem\u003eS.epidermidis\u003c/em\u003e were 3.91\u0026micro;g/mL, 62.5 \u0026micro;g/mL and 62.5\u0026micro;g/mL respectively. The bactericidal activity of KLE-AgNPs on the 3 bacteria was also determined and recorded as minimum bactericidal concentration (MBC) values. Similar to MIC analysis, KLE-AgNPs induced bactericidal effect most effectively on \u003cem\u003eC.acnes\u003c/em\u003e among all tested pathogens (MIC\u0026thinsp;=\u0026thinsp;3.91\u0026micro;g/mL). The antibacterial activity of KLE-AgNPs were again compared with commercially bought AgNPs (X-AgNPs) and chemically synthesized-AgNPs (C-AgNPs). From the comparison, it can be observed that KLE-AgNPs required much lower concentration to inhibit the proliferation of all 3 bacteria compared to X-AgNPs and C-AgNPs. This could be attributed to the difference in physical properties as displayed in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Out of the three types of AgNPs, KLE-AgNPs had the smallest size compared to X-AgNPs and C-AgNPs. The antibacterial efficacy of AgNPs correlates with their structural properties, primarily their size and shape [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. As reported by [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], the increase in particle size led to a decrease in antibacterial activity. Similarly, the bactericidal activity of KLE-AgNPs was stronger among all three types of AgNPs. This suggested that AgNPs synthesized by KLE were better antibacterial agents than the commercially available AgNPs and AgNPs that were synthesized from a conventional method.\u003c/p\u003e \u003cp\u003eThe antibacterial effect of AgNPs on the 3 bacteria can be elucidated by several proposed mechanisms. For instance, AgNPs attack and damage lipoteichoic acid which is a major component of the cell wall of Gram positive bacteria like \u003cem\u003eC.acnes\u003c/em\u003e, \u003cem\u003eS.aureus\u003c/em\u003e and \u003cem\u003eS.epidermidis\u003c/em\u003e. Consequently, growth and metabolism of the bacteria are disrupted and eventually lead to cell apoptosis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. As claimed by Jemal et al. (2017) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], AgNPs also release positively charged Ag\u003csup\u003e+\u003c/sup\u003e ions that interact with the negative electrostatic charges located on the surface of cell membrane of bacteria. This explains the ability of NPs to retard the structure of pathogens and formation of biofilm as demonstrated by [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAntimicrobial activity of KLE-AgNPs, X-AgNPs and C-AgNPs\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMIC/MBC (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKLE-AgNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX-AgNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC-AgNPs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP.acnes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.91/ 7.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e625/ 1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.5/250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS.aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62.5/250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e625/ \u0026gt; 625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;250/ \u0026gt;250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS.epidermidis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62.5/125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1250/ \u0026gt;1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125/ \u0026gt;250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4.0 CONCLUSION","content":"\u003cp\u003eConclusively, plant-mediated synthesis of AgNPs using ethanolic KLE was successfully executed due to the presence of phytochemical compounds in the extract that efficiently converted Ag\u003csup\u003e+\u003c/sup\u003e ions to AgNPs. This study had shown that under optimal parameters with 3mM AgNO\u003csub\u003e3\u003c/sub\u003e, 3mg/mL KLE, 48 hours incubation time at 37\u0026deg;C, pH 11 medium and centrifugal force of 10000\u003cem\u003eg\u003c/em\u003e for purification, KLE-AgNPs that possessed an average size of 60.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41nm, zeta potential of -43.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55mV with a low PdI value of 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 could be obtained. The formation of KLE-AgNPs was further confirmed by FTIR in which the spectrum of KLE-AgNPs were similar to commercial AgNPs and chemically synthesised-AgNPs. Additionally, FESEM-EDX analysis revealed spherical shaped KLE-AgNPs with diameter size ranging between 20nm to 55nm and XRD estimated the size to be 58.59nm. The optimized KLE-AgNPs showed potent inhibitory effect on the colonization of the three bacteria, \u003cem\u003eC.acnes\u003c/em\u003e, \u003cem\u003eS.aureus\u003c/em\u003e and \u003cem\u003eS.epidermidis\u003c/em\u003e which are associated with the pathogenesis of acne vulgaris. The outcome of this research will not only provide a deeper understanding on the role of Kenaf leaves in the manufacturing of nanoparticles and the potential of KLE-AgNPs to be incorporated into cosmetic products for acne management. Stability of KLE-AgNPs can be further evaluated based on their storage conditions to ensure that physicochemical changes of KLE-AgNPs are minimized when kept long-term.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization:\u0026nbsp;Wei Ting Jess Ong, Kar Lin Nyam, Swee Pin Yeap; Methodology: Wei Ting Jess Ong, Kar Lin Nyam, Swee Pin Yeap; Formal analysis and investigation: Wei Ting Jess Ong; Writing – original draft preparation: Wei Ting Jess Ong; Writing – review and editing: Kar Lin Nyam, Swee Pin Yeap, Md Jahurul Haque Akanda; Supervision: Kar Lin Nyam, Swee Pin Yeap.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by UCSI University Kuala Lumpur through Research Excellence \u0026amp; Innovation Grant, Project number REIG-FAS-2020/029. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeung AKC, Barankin B, Lam JM, et al (2021) Dermatology: how to manage acne vulgaris. Drugs Context 10:1\u0026ndash;18. https://doi.org/10.7573/DIC.2021-8-6\u003c/li\u003e\n\u003cli\u003eOng WTJ, Nyam KL (2022) Evaluation of silver nanoparticles in cosmeceutical and potential biosafety complications. Saudi J Biol Sci 29:2085\u0026ndash;2094. https://doi.org/10.1016/J.SJBS.2022.01.035\u003c/li\u003e\n\u003cli\u003eLara HH, Ixtepan-Turrent L, Jose Yacaman M, Lopez-Ribot J (2020) Inhibition of \u003cem\u003eCandida auris\u003c/em\u003e Biofilm Formation on Medical and Environmental Surfaces by Silver Nanoparticles. ACS Appl Mater Interfaces 12:21183\u0026ndash;21191. https://doi.org/10.1021/ACSAMI.9B20708\u003c/li\u003e\n\u003cli\u003eNeupane NP, Kushwaha AK, Karn AK, et al (2022) Anti-bacterial efficacy of bio-fabricated silver nanoparticles of aerial part of \u003cem\u003eMoringa oleifera\u003c/em\u003e lam: Rapid green synthesis, \u003cem\u003eIn-Vitro\u003c/em\u003e and \u003cem\u003eIn-Silico\u003c/em\u003e screening. Biocatal Agric Biotechnol 39:102229. https://doi.org/10.1016/J.BCAB.2021.102229\u003c/li\u003e\n\u003cli\u003ePal G, Rai P, Pandey A (2019) Green synthesis of nanoparticles: A greener approach for a cleaner future. In: Shukla AK, Iravani S (eds) Green Synthesis, Characterization and Applications of Nanoparticles. Elsevier, pp 1\u0026ndash;26\u003c/li\u003e\n\u003cli\u003eRauwel P, Kuunal S, Ferdov S, Rauwel E (2015) A Review on the Green Synthesis of Silver Nanoparticles and Their Morphologies Studied via TEM. Advances in Materials Science and Engineering 2015:1\u0026ndash;9\u003c/li\u003e\n\u003cli\u003eMittal AK, Chisti Y, Banerjee UC (2013) Synthesis of metallic nanoparticles using plant extracts. Biotechnol Adv 31:346\u0026ndash;356. https://doi.org/10.1016/J.BIOTECHADV.2013.01.003\u003c/li\u003e\n\u003cli\u003eShah M, Fawcett D, Sharma S, et al (2015) Green Synthesis of Metallic Nanoparticles via Biological Entities. Materials (Basel) 8:7308. https://doi.org/10.3390/MA8115377\u003c/li\u003e\n\u003cli\u003eSim YY, Jess Ong WT, Nyam KL (2019) Effect of various solvents on the pulsed ultrasonic assisted extraction of phenolic compounds from \u003cem\u003eHibiscus cannabinus\u003c/em\u003e L. leaves. Ind Crops Prod 140:111708. https://doi.org/10.1016/j.indcrop.2019.111708 \u003c/li\u003e\n\u003cli\u003eRyu J, Kwon S-J, Ahn J-W, et al (2017) Phytochemicals and antioxidant activity in the kenaf plant (\u003cem\u003eHibiscus cannabinus\u003c/em\u003e L.). J Plant Biotechnol 44:191\u0026ndash;202. https://doi.org/10.5010/JPB.2017.44.2.191\u003c/li\u003e\n\u003cli\u003eRoy A, Bulut O, Some S, et al (2019) Green synthesis of silver nanoparticles: Biomolecule-nanoparticle organizations targeting antimicrobial activity. RSC Adv 9:2673\u0026ndash;2702. https://doi.org/10.1039/C8RA08982E\u003c/li\u003e\n\u003cli\u003eJameel MS, Aziz AA, Dheyab MA (2020) Green synthesis: Proposed mechanism and factors influencing the synthesis of platinum nanoparticles. Green Processing and Synthesis 9:386\u0026ndash;398. https://doi.org/10.1515/GPS-2020-0041/XML\u003c/li\u003e\n\u003cli\u003eKhan MZH, Tareq FK, Hossen MA, Roki MNAM (2018) Green Synthesis And Characterization Of Silver Nanoparticles Using \u003cem\u003eCoriandrum Sativum\u003c/em\u003e Leaf Extract. Journal of Engineering Science and Technology 13:158\u0026ndash;166\u003c/li\u003e\n\u003cli\u003eMavani K, Shah M (2013) Synthesis of Silver Nanoparticles by using Sodium Borohydride as a Reducing Agent. International Journal of Engineering Research \u0026amp; Technology (IJERT) 2:1\u0026ndash;5\u003c/li\u003e\n\u003cli\u003eHemlata, Meena PR, Singh AP, Tejavath KK (2020) Biosynthesis of Silver Nanoparticles Using \u003cem\u003eCucumis prophetarum\u003c/em\u003e Aqueous Leaf Extract and Their Antibacterial and Antiproliferative Activity against Cancer Cell Lines. ACS Omega 5:5520\u0026ndash;5528. https://doi.org/10.1021/ACSOMEGA.0C00155/ASSET/IMAGES/MEDIUM/AO0C00155_M003.GIF\u003c/li\u003e\n\u003cli\u003eSougandhi PR, Ramanaiah S (2020) Green synthesis and spectral characterization of silver nanoparticles from \u003cem\u003ePsidium guajava\u003c/em\u003e leaf extract. Inorganic and Nano-Metal Chemistry 50:1290\u0026ndash;1294. https://doi.org/10.1080/24701556.2020.1745839\u003c/li\u003e\n\u003cli\u003eErdogan O, Abbak M, Demirbolat GM, et al (2019) Green synthesis of silver nanoparticles via \u003cem\u003eCynara scolymus\u003c/em\u003e leaf extracts: The characterization, anticancer potential with photodynamic therapy in MCF7 cells. PLoS One 14:e0216496. https://doi.org/10.1371/journal.pone.0216496\u003c/li\u003e\n\u003cli\u003eAsadi S, Charati FR, Akbari R, Razavi SA (2018) Green synthesis of silver nanoparticles using \u003cem\u003eTaxus baccata\u003c/em\u003e Leaves extract and identify its specifications. J Mater Environ Sci 9:2798\u0026ndash;2803\u003c/li\u003e\n\u003cli\u003eLoo YY, Rukayadi Y, Nor-Khaizura MAR, et al (2018) In Vitro antimicrobial activity of green synthesized silver nanoparticles against selected Gram-negative foodborne pathogens. Front Microbiol 9:1\u0026ndash;9. https://doi.org/10.3389/fmicb.2018.01555\u003c/li\u003e\n\u003cli\u003eHu X, Wu L, Du M, Wang L (2022) Eco-friendly synthesis of size-controlled silver nanoparticles by using \u003cem\u003eAreca catechu\u003c/em\u003e nut aqueous extract and investigation of their potent antioxidant and anti-bacterial activities. Arabian Journal of Chemistry 15:103763. https://doi.org/10.1016/J.ARABJC.2022.103763\u003c/li\u003e\n\u003cli\u003eMasum MI, Siddiqa MM, Ali KA, et al (2019) Biogenic synthesis of silver nanoparticles using \u003cem\u003ephyllanthus emblica\u003c/em\u003e fruit extract and its inhibitory action against the pathogen acidovorax oryzaestrain RS-2 of rice bacterial brown stripe. Front Microbiol 10:820. https://doi.org/10.3389/FMICB.2019.00820/BIBTEX\u003c/li\u003e\n\u003cli\u003eBadi\u0026rsquo;ah HI, Seedeh F, Supriyanto G, Zaidan AH (2019) Synthesis of Silver Nanoparticles and the Development in Analysis Method. IOP Conf Ser Earth Environ Sci 217:012005. https://doi.org/10.1088/1755-1315/217/1/012005\u003c/li\u003e\n\u003cli\u003eMedda S, Hajra A, Dey U, et al (2015) Biosynthesis of silver nanoparticles from Aloe vera leaf extract and antifungal activity against Rhizopus sp. and Aspergillus sp. Applied Nanoscience (Switzerland) 5:875\u0026ndash;880. https://doi.org/10.1007/S13204-014-0387-1/FIGURES/7\u003c/li\u003e\n\u003cli\u003eAshraf JM, Ansari MA, Khan HM, et al (2016) Green synthesis of silver nanoparticles and characterization of their inhibitory effects on AGEs formation using biophysical techniques. Sci Rep 6:20414. https://doi.org/10.1038/SREP20414\u003c/li\u003e\n\u003cli\u003eAnandalakshmi K, Venugobal J, Ramasamy V (2016) Characterization of silver nanoparticles by green synthesis method using \u003cem\u003ePedalium murex\u003c/em\u003e leaf extract and their antibacterial activity. Applied Nanoscience (Switzerland) 6:399\u0026ndash;408. https://doi.org/10.1007/S13204-015-0449-Z/TABLES/2\u003c/li\u003e\n\u003cli\u003eRautela A, Rani J, Debnath (Das) M (2019) Green synthesis of silver nanoparticles from \u003cem\u003eTectona grandis\u003c/em\u003e seeds extract: characterization and mechanism of antimicrobial action on different microorganisms. J Anal Sci Technol 10:1\u0026ndash;10. https://doi.org/10.1186/S40543-018-0163-Z/FIGURES/14\u003c/li\u003e\n\u003cli\u003eBamsaoud SF, Basuliman MM, Bin-Hameed EA, et al (2021) The effect of volume and concentration of AgNO\u003csub\u003e3\u003c/sub\u003e aqueous solutions on silver nanoparticles synthesized using \u003cem\u003eZiziphus Spina\u0026ndash;Christi\u003c/em\u003e leaf extract and their antibacterial activity. J Phys Conf Ser 1900:012005. https://doi.org/10.1088/1742-6596/1900/1/012005\u003c/li\u003e\n\u003cli\u003eKarekalammanavar G, David M (2018) Optimization of Green Synthesis of Silver Nanoparticles from Leaf Extracts of \u003cem\u003eTabernaemontana heyneana\u003c/em\u003e and evaluation of their catalytic activity on reduction of methylene blue. Haya: Saudi J Life Sci 3:248\u0026ndash;254. https://doi.org/10.21276/HAYA.2018.3.3.5\u003c/li\u003e\n\u003cli\u003eZayed MF, Eisa WH, Abdel-Moneam YK, et al (2015) \u003cem\u003eZiziphus spina-christi\u003c/em\u003e based bio-synthesis of Ag nanoparticles. J Ind Eng Chem 23:50\u0026ndash;56. https://doi.org/10.1016/J.JIEC.2014.07.041\u003c/li\u003e\n\u003cli\u003eClayton KN, Salameh JW, Wereley ST, Kinzer-Ursem TL (2016) Physical characterization of nanoparticle size and surface modification using particle scattering diffusometry. Biomicrofluidics 10:054107. https://doi.org/10.1063/1.4962992\u003c/li\u003e\n\u003cli\u003eHtwe YZN, Chow WS, Suda Y, Mariatti M (2019) Effect of silver nitrate concentration on the production of silver nanoparticles by green method. Mater Today Proc 17:568\u0026ndash;573. https://doi.org/10.1016/J.MATPR.2019.06.336\u003c/li\u003e\n\u003cli\u003eArdani HK, Imawan C, Handayani W, et al (2017) Enhancement of the stability of silver nanoparticles synthesized using aqueous extract of \u003cem\u003eDiospyros discolor Willd\u003c/em\u003e. leaves using polyvinyl alcohol. IOP Conf Ser Mater Sci Eng 188:012056. https://doi.org/10.1088/1757-899X/188/1/012056\u003c/li\u003e\n\u003cli\u003eRamalingam B, Parandhaman T, Das SK (2016) Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of Gram-Negative Bacteria viz. \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. ACS Appl Mater Interfaces 8:4963\u0026ndash;4976. https://doi.org/10.1021/ACSAMI.6B00161\u003c/li\u003e\n\u003cli\u003eSingh S, Bharti A, Meena VK (2015) Green synthesis of multi-shaped silver nanoparticles: optical, morphological and antibacterial properties. J Mater Sci: Mater Electron 26:3638\u0026ndash;3648. https://doi.org/10.1007/S10854-015-2881-Y\u003c/li\u003e\n\u003cli\u003eWisnuwardhani HA, Lukmayani Y, Hazar S, Hoeruniswah H (2019) Optimization of Silver Nanoparticles Synthesis using Kawista (\u003cem\u003eLimonia Acidissima Groff\u003c/em\u003e.) leaves ethanol extract. J Phys Conf Ser 1375:12077. https://doi.org/10.1088/1742-6596/1375/1/012077\u003c/li\u003e\n\u003cli\u003eFu LM, Hsu JH, Shih MK, et al (2021) Process Optimization of Silver Nanoparticle Synthesis and Its Application in Mercury Detection. Micromachines 12:1123. https://doi.org/10.3390/MI12091123\u003c/li\u003e\n\u003cli\u003eFernando I, Zhou Y (2019) Impact of pH on the stability, dissolution and aggregation kinetics of silver nanoparticles. Chemosphere 216:297\u0026ndash;305. https://doi.org/10.1016/J.CHEMOSPHERE.2018.10.122\u003c/li\u003e\n\u003cli\u003eChitra K, Annadurai G (2014) Antibacterial activity of pH-dependent biosynthesized silver nanoparticles against clinical pathogen. Biomed Res Int 2014:1\u0026ndash;6. https://doi.org/10.1155/2014/725165\u003c/li\u003e\n\u003cli\u003eXiong Y, Liu X, Xiong H (2021) Aggregation modeling of the influence of pH on the aggregation of variably charged nanoparticles. Sci Rep 11:1\u0026ndash;10. https://doi.org/10.1038/s41598-021-96798-3\u003c/li\u003e\n\u003cli\u003eTitma T (2018) The effect of surface charge and pH on the physiological behaviour of cobalt, copper, manganese, antimony, zinc and titanium oxide nanoparticles in vitro. Toxicol In Vitro 50:11\u0026ndash;21. https://doi.org/10.1016/J.TIV.2018.02.010\u003c/li\u003e\n\u003cli\u003eHandayani W, Ningrum AS, Imawan C (2020) The Role of pH in Synthesis Silver Nanoparticles Using \u003cem\u003ePometia pinnata\u003c/em\u003e (Matoa) Leaves Extract as Bioreductor. J Phys Conf Ser 1428:012021. https://doi.org/10.1088/1742-6596/1428/1/012021\u003c/li\u003e\n\u003cli\u003eMiranda A, Akpobolokemi T, Chung E, et al (2022) pH Alteration in Plant-Mediated Green Synthesis and Its Resultant Impact on Antimicrobial Properties of Silver Nanoparticles (AgNPs). Antibiotics (Basel) 11:1592. https://doi.org/10.3390/ANTIBIOTICS11111592\u003c/li\u003e\n\u003cli\u003eRousta MH, Ghasemi N (2019) Green synthesis of silver nanoparticles using a mountain plant extract. Rev Roum Chim 64:143\u0026ndash;152. https://doi.org/10.33224/RRCH/2019.64.2.04\u003c/li\u003e\n\u003cli\u003eLiu H, Zhang H, Wang J, Wei J (2020) Effect of temperature on the size of biosynthesized silver nanoparticle: Deep insight into microscopic kinetics analysis. Arab J Chem 13:1011\u0026ndash;1019. https://doi.org/10.1016/J.ARABJC.2017.09.004\u003c/li\u003e\n\u003cli\u003eRobertson JD, Rizzello L, Avila-Olias M, et al (2016) Purification of Nanoparticles by Size and Shape. Sci Rep 2016 6:1 6:1\u0026ndash;9. https://doi.org/10.1038/srep27494\u003c/li\u003e\n\u003cli\u003eAsnaashari Kahnouji Y, Mosaddegh E, Bolorizadeh MA (2019) Detailed analysis of size-separation of silver nanoparticles by density gradient centrifugation method. Mater Sci Eng: C 103:109817. https://doi.org/10.1016/J.MSEC.2019.109817\u003c/li\u003e\n\u003cli\u003eMartins CSM, Sousa HBA, Prior JAV (2021) From Impure to Purified Silver Nanoparticles: Advances and Timeline in Separation Methods. Nanomaterials (Basel) 11:3407. https://doi.org/10.3390/NANO11123407\u003c/li\u003e\n\u003cli\u003eBanerjee P, Satapathy M, Mukhopahayay A, Das P (2014) Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: Synthesis, characterization, antimicrobial property and toxicity analysis. Bioresour Bioprocess 1:1\u0026ndash;10. https://doi.org/10.1186/S40643-014-0003-Y/FIGURES/6\u003c/li\u003e\n\u003cli\u003eVenil CK, Malathi M, Velmurugan P, Renuka Devi P (2021) Green synthesis of silver nanoparticles using canthaxanthin from \u003cem\u003eDietzia maris\u003c/em\u003e AURCCBT01 and their cytotoxic properties against human keratinocyte cell line. J Appl Microbiol 130:1730\u0026ndash;1744. https://doi.org/10.1111/JAM.14889\u003c/li\u003e\n\u003cli\u003eKarthik C, Suresh S, Sneha Mirulalini G, Kavitha S (2020) A FTIR approach of green synthesized silver nanoparticles by \u003cem\u003eOcimum sanctum\u003c/em\u003e and \u003cem\u003eOcimum gratissimum\u003c/em\u003e on mung bean seeds. Inorg Nano-Met Chem 50:606\u0026ndash;612. https://doi.org/10.1080/24701556.2020.1723025\u003c/li\u003e\n\u003cli\u003eFrancis S, Joseph S, Koshy EP, Mathew B (2018) Microwave assisted green synthesis of silver nanoparticles using leaf extract of \u003cem\u003eelephantopus scaber\u003c/em\u003e and its environmental and biological applications. Artif Cells Nanomed Biotechnol 46:795\u0026ndash;804. https://doi.org/10.1080/21691401.2017.1345921\u003c/li\u003e\n\u003cli\u003eAli MH, Azad MAK, Khan KA, et al (2023) Analysis of Crystallographic Structures and Properties of Silver Nanoparticles Synthesized Using PKL Extract and Nanoscale Characterization Techniques. ACS Omega 8:28133\u0026ndash;28142. https://doi.org/10.1021/ACSOMEGA.3C01261/ASSET/IMAGES/LARGE/AO3C01261_0007.JPEG\u003c/li\u003e\n\u003cli\u003eBaran MF, Keskin C, Baran A, et al (2023) Green Synthesis of Silver Nanoparticles from Allium cepa L. Peel Extract, Their Antioxidant, Antipathogenic, and Anticholinesterase Activity. Molecules 28:2310. https://doi.org/10.3390/MOLECULES28052310\u003c/li\u003e\n\u003cli\u003eSagadevan S, Vennila S, Singh P, et al (2019) Facile synthesis of silver nanoparticles using \u003cem\u003eAverrhoa bilimbi\u003c/em\u003e L and Plum extracts and investigation on the synergistic bioactivity using in vitro models. Green Processing and Synthesis 8:873\u0026ndash;884. https://doi.org/10.1515/GPS-2019-0058\u003c/li\u003e\n\u003cli\u003ePryshchepa O, Pomastowski P, Buszewski B (2020) Silver nanoparticles: Synthesis, investigation techniques, and properties. Adv Colloid Interface Sci 284:102246. https://doi.org/10.1016/J.CIS.2020.102246\u003c/li\u003e\n\u003cli\u003eIbraheim MH, Ibrahiem AA, Dalloul TR (2016) Biosynthesis of silver nanoparticles using Pomegranate juice extract and its antibacterial activity. Int J Appl Sci Biotechnol 4:254\u0026ndash;258. https://doi.org/10.3126/IJASBT.V4I3.15417\u003c/li\u003e\n\u003cli\u003ePadman AJ, Henderson J, Hodgson S, Rahman PKSM (2014) Biomediated synthesis of silver nanoparticles using \u003cem\u003eExiguobacterium mexicanum\u003c/em\u003e. Biotechnol Lett 36:2079\u0026ndash;2084. https://doi.org/10.1007/S10529-014-1579-1/FIGURES/5\u003c/li\u003e\n\u003cli\u003eBanerjee P, Satapathy M, Mukhopahayay A, Das P (2014) Leaf extract mediated green synthesis of silver nanoparticles from widely available Indian plants: Synthesis, characterization, antimicrobial property and toxicity analysis. Bioresour Bioprocess 1:1\u0026ndash;10. https://doi.org/10.1186/S40643-014-0003-Y/FIGURES/6\u003c/li\u003e\n\u003cli\u003eJagtap UB, Bapat VA (2013) Green synthesis of silver nanoparticles using \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e Lam. seed extract and its antibacterial activity. Ind Crops Prod 46:132\u0026ndash;137. https://doi.org/10.1016/J.INDCROP.2013.01.019\u003c/li\u003e\n\u003cli\u003eHelmlinger J, Sengstock C, Gro\u0026szlig;-Heitfeld C, et al (2016) Silver nanoparticles with different size and shape: equal cytotoxicity, but different antibacterial effects. RSC Adv 6:18490\u0026ndash;18501. https://doi.org/10.1039/C5RA27836H\u003c/li\u003e\n\u003cli\u003eDong Y, Zhu H, Shen Y, et al (2019) Antibacterial activity of silver nanoparticles of different particle size against \u003cem\u003eVibrio Natriegens\u003c/em\u003e. PLoS One 14:e0222322. https://doi.org/10.1371/JOURNAL.PONE.0222322\u003c/li\u003e\n\u003cli\u003eSwolana D, Wojtyczka RD (2022) Activity of Silver Nanoparticles against Staphylococcus spp. Int J Mol Sci 23:4298. https://doi.org/10.3390/IJMS23084298\u003c/li\u003e\n\u003cli\u003eJemal K, Sandeep B V., Pola S (2017) Synthesis, Characterization, and Evaluation of the Antibacterial Activity of \u003cem\u003eAllophylus serratus\u003c/em\u003e Leaf and Leaf Derived Callus Extracts Mediated Silver Nanoparticles. J Nanomater 2017:1\u0026ndash;11. https://doi.org/10.1155/2017/4213275\u003c/li\u003e\n\u003cli\u003ePaosen S, Bilhman S, Wunnoo S, et al (2023) Control of biomaterial-associated infections through biofabrication of gold nanoparticles using \u003cem\u003eMusa sapientum\u003c/em\u003e extract. Biotechnol J 2300008. https://doi.org/10.1002/BIOT.202300008\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Silver nanoparticles, green synthesis, kenaf leaves, process optimization, antibacterial","lastPublishedDoi":"10.21203/rs.3.rs-4614655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4614655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcne vulgaris affects approximately 9.4% of the world population and was ranked 8th most prevalent disease. Concurrently, silver nanoparticles (AgNPs) are widely explored for their profound antibacterial properties which are ideal for acne management. With the current search for natural alternatives in both cosmetics and nanotechnology, plant extracts have garnered tremendous attention in the synthesis of AgNPs. Therefore, this study utilized Kenaf leaves extract (KLE) as a novel, natural reducing agent for the synthesis of AgNPs. \u0026nbsp;The effects of different synthesis parameters were studied and KLE-synthesised AgNPs (KLE-AgNPs) were further analysed for their physicochemical properties and antimicrobial efficiency. Results showed that small-sized (60.32±2.41nm), stable (-43.03±2.55 mV) and monodispersed (0.28±0.01) KLE-AgNPs were successfully formed with 3mM silver nitrate, and 3mg/mL KLE along with the optimal conditions at pH 11, 48 hours incubation time, reaction temperature of 37°C, and centrifugation at 10000\u003cem\u003eg\u003c/em\u003e for purification. FTIR analysis confirmed the presence of functional groups that aid in the formation of AgNPs. Additionally, XRD result demonstrated that KLE-AgNPs recorded crystalline size of 58.59nm. The FESEM and EDX analyses displayed that the particles were spherical and silver was the main element respectively. The antimicrobial analysis proved that a lower dose of KLE-AgNPs demonstrated better antimicrobial effect on the three acne-causing bacteria compared to commercial AgNPs and chemically synthesized-AgNPs. The outcome of this research amplifies the role of KLE as a natural reducing agent in the synthesis of AgNPs for the development of hybrid nanocosmetics with increased efficacy due to the synergistic effect of KLE and AgNPs.\u003c/p\u003e","manuscriptTitle":"Green synthesis of silver nanoparticles using Kenaf leaves extract and their antibacterial potential in acne management. 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