Biological Synthesis and Characterization of Silver-Doped Nanocomposites: Antibacterial and Mechanistic Studies

1. Introduction

Diseases that spread from one person to another and are caused by bacteria, parasites, fungi, and viruses are termed as infectious diseases [1]. Noteworthy, infectious diseases are also transmitted through bug bites, contaminated food, water and soil, as well as poor sanitation [2]. Common infectious diseases include common cold, flu (influenza), stomach flu (gastroenteritis), hepatitis, respiratory syncytial virus (RSV) and COVID 19 [2]. Infectious diseases have a parade number of symptoms or manifestations that may lead to death, if the concerned illnesses are left untreated. Infectious diseases are one of the significant causes of morbidity and mortality across the world. Generally, antibiotics are used to diagnose, treat or prevent bacterial infections; however, mutations resulting from their use might cause bacterial drug resistance [3]. Remarkably, bacteria has seemingly won the battle against antibiotics since most of them have become resistant to these drugs [4]. It is important to mention that various antimicrobial agents interferes with (i) synthesis of cell wall, (ii) ribosomal function, (iii) folate synthesis, (iv) biofilm formation and (v) nucleic acid synthesis [5,6,7,8,9]. Resistance occurs when there are profound modifications to one of these functions. For instance, bacterial resistance to β-lactam antibiotics include modification of porins (cell wall proteins) and targets, production of the inactivating enzymes namely beta-lactamases and autolytic enzymes’ inactivation [10]. Inhibition of nucleic acid synthesis is observed with quinolones and fluoroquinolones that target DNA synthesis through inhibition of type 2 topoisomerases, such as DNA gyrase and topoisomerase IV [11]. Sulfonamides, such as trimethoprim, which is widely used to treat urinary tract infections and pneumonia binds on dihydrofolate reductase to inhibit the synthesis of folic acid [12]. In addition, the ribosomal function is affected by macrolide antibiotics [13]. Aminoglycosides and quinolones have been shown to inhibit biofilm formation by Pseudomonas aeruginosa [14,15]. These bacterial drug resistance events, combined with the problem of toxicity, high cost and unavailability of modern therapy justify the crucial need to search for new and safe antimicrobials. Infectious diseases cause over 4.8 % of mortality rate and an economic loss of more than USD 100 trillion per year worldwide worldwide [16]. Since their introduction into health care and clinical practice in the early 20^th century, with the discovery of penicillin in 1928, antibiotics have revolutionized medicine, many of them have been either isolated from natural products or chemically synthesized and numerous lives have been saved [17]. It is noted that, these drugs are increasingly threatened by bacteria that develop a wide variety of resistance mechanisms. Medicinal plants remain the most abundant natural source of active drugs and are invaluable in the traditional treatment of a number of infectious diseases since time antediluvian [18,19]. In fact, medicinal plants have been reported to contain numerous secondary metabolites, including terpenoids, flavonoids, phenolic compounds, alkaloids, tannins, saponins, among others. These metabolites have been reported to inhibit the growth of several microorganisms, including bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella spp., etc.).

However, the incorporation of these plant secondary metabolites into nanosized particles has been proven more valuable as this nanosize form can overcome biological barriers and augment the delivery of active principles to the target site thereby increasing the efficacy [20,21]. Undoubtedly, one of the latest approaches to combat resistant microorganisms include the use of nanotechnology-based antimicrobials [22,23]. Because of the non-specific mechanistic action of metal-based nanoparticles on bacteria, the development of resistance by these microorganisms is difficult. Thus, resistance to antibiotics is not relevant to nanoparticles (NPs) as there is a direct contact with the bacterial cell wall without the need of penetration into microbial cells [23]. Nanoparticles are spherical particles (size: 1-100 nm) that exhibit unique properties due to their high surface area to volume ratio. They can be classified into fullerenes and metal, ceramic, and polymeric NPs [24]. In addition, nanoparticles are bioactive products that are stable, dispersed, biocompatible and affordable [25]. Dakal et al. [26] revealed that silver nanoparticles are able to adhere onto the surface of cell wall or membrane, penetrate inside the cell and damage organelles, such as ribosomes, mitochondria and vacuoles by releasing free ions to produce reactive oxygen species (ROS) [26,27]. The metal used for the fabrication of nanoparticles undergo a reaction with chemical groups of components of the cell membrane (phosphorus and sulfur groups), such as proteins, lipids, and DNA bases to generate potential reactive oxygen species [28]. It has been reported that the accumulation of nanoparticles within bacterial cell walls and membranes can induce morphological changes in the bacteria. These include membrane detachment and disruption, shrinkage of cytoplasm, and formation of electron-dense holes [17]. The preparation of nanocomposites using activated carbons contributes to increasing the antibacterial action of these products. Because of their light weight, high specific surface areas and favorable electrical and mechanical properties, nanocarbons have recently gained considerable attention [29]. In fact, activated carbon is a porous material with amphoteric characteristics, which is used for the adsorption of organic and inorganic compounds [30]. The high content in oxygen onto the surface of activated carbons is crucial for an effective adsorption of bacteria, such as S. aureus and E. coli [31]. Parameters, such as pore size distribution, surface area, pH, and elemental analysis are used to characterize active carbon products [31,32]. Recent studies have demonstrated that activated supported metal nanoparticles prepared from plant (Cassia roxburghii, Aloe vera and Cinnamomum verum) extracts afford nanocomposites with superior antibacterial efficiency [33,34,35].

Based on the foregoing considerations, the present study aims to use Croton macrostachyus (a plant employed in the traditional treatment of a number of infectious diseases, including malaria and venereal diseases, among others) for the fabrication of carbon activated immobilized metal nanoparticles. Moreover, antibacterial screening of the fabricated nanocomposite and potential mechanistic basis of antimicrobial action are investigated.

2. Results and Discussion

2.1. Physical and UV-Vis Analysis of the Nanoparticles

A green synthesis of silver nanoparticles was successfully achieved using aqueous extract of C. macrostachyus stem bark along with a solution of silver nitrate (AgNO₃). The change in color observed from pale yellow to dark brown indicated the formation of the nanoparticles (Figure 1). The fabrication of the AgNPs was further confirmed by UV-vis visible spectroscopy in a wavelength range of 350-900 nm as described in Figure 2. On the spectrum of AgNPs, a spectrophotometric absorption peak was observed at a characteristic wavelength of 443 nm and was attributed to the surface plasmon resonance of the formed nanoparticles. Meanwhile, the spectra of the silver salt and aqueous extract solutions of C. macrostachyus showed no peaks. For silver nanoparticles, the λmax values were obtained in the wavelength range of the visible spectrum (400-500 nm) [36]. These results are consistent with a number of studies published on the synthesis of nanomaterials from other plants with wavelength values corresponding to the visible spectrum as for the aqueous extract of C. sparsiflorus (457 nm ; Kathiravan et al. [37]) and C. bonplandianum (415 nm ; Kapoor [38]).

2.2. FTIR Analysis C. Macrostachyus Extract and the Nanoparticles

To identify the plausible functional groups that are involved in the reduction of silver ion to AgNPs, FTIR spectroscopy was performed on the as-prepared extract (C. macrostachyus extract) and nanoparticles (AgNPs). The FTIR spectrum (Figure 3) of the aqueous extract of C. macrostachyus (blue color), shows a peak at 511 cm^-1, which corresponds to the band of alkyl halides. The distortion observed at 781 cm^-1 may be related to the CH=CH bonds of the benzylic aromatic ring of phenols, whereas the presence of an intense peak at 1021 cm^-1 is characteristic of an aliphatic amine (C-N). At 1304 cm^-1, a weak peak, which is characteristic of C-OH groups is observed, whilst another intense peak that corresponds to the C=O groups of protein amides is detected. Moreover, two distinct peaks, which were identified at 2912 cm^-1 and 3287 cm^-1 are potentially linked to the symmetrical elongation vibrations of the CH₂–CH₃ group of an alkane and the O-H bonds linked to alcohols and phenolic compounds, respectively. These chemical groups are well known to exist in plant extracts as evidenced by previously published research papers [39,40,41]. The FTIR spectrum (orange color) of AgNPs revealed major peaks at wavelength 3300 cm^-1 (OH bond stretching) and 1634 cm^-1 (carbonyl C=O group), which corresponded to the involvement of hydroxyl and carboxylic acid moieties (encountered in polyphenols, phenolic acids and proteins) of C. macrostachyus extract in the reduction of the silver ions (Ag⁺). Noteworthy, primary (proteins) and secondary (polyphenols) metabolites are responsible for the formation and stabilization of silver nanoparticles (AgNPs) [42,43].

As evidenced by the characteristic peak revealed at 1634 cm^-1, the presence of proteins prevented the agglomeration of the as-prepared nanoparticles. Indeed, it has been reported that capping proteins prevents agglomeration in the medium and are responsible for the fabrication of highly stable AgNPs [44]. As already reported by other authors [45,46], the absence of peak, which is characteristic to the Ag-O groups, indicates that silver is present in metallic form in the fabricated nanoparticles [45,46]. These results demonstrated that C. macrostachyus extract contains secondary metabolites, such as phenolics, flavonoids, alkaloids, and terpenoids, among others [47], which might have been incorporated into the prepared nanoparticles.

2.3. Characterization of the As-Prepared Activated Carbon and Nanocomposite

2.3.1. FTIR of the Activated Carbon

Figure 4 shows the IR spectra of the activated carbon (orange color) and C. macrostachyus dried powder (blue color). It appears that both spectra are different in number and type of peaks. The very broad band observed around 3200-2500 cm^-1 is attributed to the O-H elongation vibration association, and may be characteristic of carbonyl and phenolic hydroxyl groups. The peak observed at 1574 cm^-1 is characteristic of C=C alkene groups of benzene. The frail peak that appeared at 1156 cm^-1 might be attributed to the P-O elongation from the phosphoric acid used in the activation. Another weak signal peak, which appeared at 1071 cm^-1 corresponds to the in-plane deformation of the C-O grouping of the aromatic compounds and acetyl and carboxylic acid functions. The peaks that appeared at 988 and 876 cm^-1 indicate the presence of meta and di-substituted benzene groups. Moreover, the presence of a haloalkane was indicated by the signal observed at 488 cm^-1. Noteworthy, the peaks, which were observed with the plant dried powder at 3287 and 2921 cm^-1 disappeared after carbonization. In addition, the peak position was shifted to lower regions when C. macrostachyus dried powder was treated with phosphoric acid (H₃PO₄). The observed structural modifications have been attributed to the presence of organophosphorus compounds, which might overlay the outside plane deformation vibrations of C-H in the aromatic moieties [48].

2.3.2. FTIR of the Doped-Activated Carbon (Nanocomposite)

Figure 5 reveals the spectrum of the fabricated nanocomposite (orange color) compared to that obtained from the activated carbon (blue color). It can be seen that both spectra are having almost the same pattern, thus indicating that the obtained nanocomposite is not a hybrid material and is structurally identical to the nanoparticle. A number of research groups have obtained the same results while working on the green synthesis of nanofillers and their nanocomposites [49,50].

2.3.3. Iodine Number and Methylene Blue Index

The identification of the iodine number and methylene blue index tests allowed for the characterization of microporosity (iodine), mesoporosity and macroporosity (methylene blue) of the activated carbon and nanocomposite. Figure 6 shows the values of the iodine number and methylene blue index of activated carbon and nanocomposite. The results demonstrated that the iodine number of the activated carbon (1026.69 mg/g) is higher than that of the nanocomposite (975.29 mg/g). These values corroborate with those obtained by Islam et al. [51] (iodine number : 1000-1200 mg/g) and Raut et al. [52] (iodine number : 1140.69 mg/g) in their work on the fabrication and characterization of activated carbon from Jute Stick, and sugarcane bagasse and rice husk, respectively [51,52]. Furthermore, the values of the methylene blue indices were almost identical for the activated carbon (89.862 mg/g) and nanocomposites (89.854 mg/g).

As a partial conclusion, aqueous extract, silver nanoparticles, activated carbon and derived nanocomposites were successfully prepared from the stem bark of Croton macrostachyus. UV-visible spectra confirmed the synthesis of the nanoparticles. FTIR analysis revealed the presence of chemical functional groups that are characteristic to nanomaterials [53]. Noteworthy, the effect of the temperature and the activating agent (i.e. phosphoric acid) during the carbonization process (depolymerization and dehydratation) was clearly demonstrated by the difference in the spectra of the Croton macrostachyus extract and activated carbon [54]. In addition, there were no significant changes in the spectra obtained for CAC and CACAgNPs confirming the fabrication of the nanocomposite through van der waals interactions [55]. Furthermore, the percentage of iodine number was decreased by 5%, whereas the methylene blue index showed percent reduction of 0.008% inferring that, in lieu of macrospores, the activated carbon’s microspores constituted the main fixation site for the nanoparticles, leading to a decrease in their adsorption surface [45,56].

2.4. Antibacterial and Cytotoxic Activity

2.4.1. Minimum Inhibitory Concentration

Table 1 summarizes the MIC values (range : 62.5 µg/mL to >1000 µg/mL) of C. macrostachyus extract, and the as-prepared activated carbon, nanoparticles and nanocomposites. The median cytotoxic concentrations (CC₅₀s) of these entities on Vero cells are also presented in Table 1. The silver nanoparticles were the most active (MIC range : 62.5 to 125 µg/mL), followed by the nanocomposites (MIC range : 125 to 500 µg/mL). Although the nanocomposites were less active than the derived nanoparticles, their activity was superior when compared to that of the activated carbon (MIC > 1000 µg/mL). In fact, the antibacterial activity of activated carbon (MIC > 1000 µg/mL) was potentiated by that of the incorporated nanoparticles. To our knowledge, no report has previously described the involvement of silver nanoparticles in potentiating the antibacterial activity of activated carbon obtained from the aqueous extract of C. macrostachyus stem bark. On the other hand, metal nanoparticles are generally thought to be toxic because of the metal used during the fabrication as a result of their large specific surface area, high surface energy and magnetic interaction, as well as their easiness to agglomerate into micron- or millimeter-sized floccules [57]. Therefore, we attempted to overcome or minimize the toxicity caused by nanoparticles while maintaining the antibacterial activity by preparing the nanocomposites counterpart using an activated carbon.

2.5. Effect of the As-Prepared Nanomaterials on the Mortality Kinetics of S. aureus and E. coli

The growth of E. coli and S. aureus was monitored in the presence of nanoparticles (AgNPs) and nanocomposites (CAC/AgNPs) at a series of MIC value-based concentrations, including MIC/4, MIC/2, MIC, 2MIC and 4MIC for 24 h. As a result, both nanomaterials inhibited the growth of E. coli and S. aureus at MIC, 2MIC and 4MIC (Figure 7 and Figure 8). Referring to figure 7 A & B, treatment with AgNPs decreased the bacterial population after 2 to 4 h of incubation time, followed by a stationary state of bacterial growth from 4 to 10 h of incubation time. In figure 8 A & B, the same trend was observed with the treatment using various concentrations of the nanocomposites (CAC/AgNPs). In fact, after 4 h incubation time, there was a bacterial growth, which was followed by a 6 h period (from 4 to 10 h incubation time) of no growth. From 10 h onwards, both E. coli and S. aureus resumed an exponential growth up to 24 h of incubation time upon treatment with both nanomaterials. The ephemeral bacterial growth inhibition (after 4h incubation time) caused by AgNPs and CAC/AgNPs led us to the conclusion that both nanomaterials induced a bacteriostatic effect as the bacterial growth resumed after 10 h of incubation and beyond. Noteworthy, the growth inhibition was concentration dependent as the more the MIC value (2MIC and 4MIC), the higher was the inhibition as evidenced by the trend of curves presented in Figure 7 & 8. However, ciprofloxacin, which was used as positive control did not show the same trend as this compound inhibited the bacterial growth in a continuous manner, whereas the untreated control group displayed an exponential growth of bacteria from 0 to 24 h (Figure 7 and Figure 8).

2.6. Effect of Nanomaterials on the Membrane Integrity of Escherichia coli and Staphylococcus aureus

To evaluate the effect of the as-prepared nanomaterials on the bacterial membrane integrity, cells were treated with MIC value-based concentrations (MIC/2, 2MIC and MIC) and incubated at incubation times varying from 0 to 8 h (0, 0.5, 1, 2, 4, 6 and 8 h). Next, the absorbance was measured at 260 nm (wavelength at which nucleic acid absorbs ultraviolet light) after 8 h of incubation time. Irrespective of the concentration tested, there was no significant leakage of nucleic acids following bacterial treatment with AgNPs (Figure 9 A&C) and CAC/AgNPs (Figure 9 B & D). This observation suggests that the antibacterial mechanism of action of the as-prepared nanomaterials is far from being through cell membrane disruption. However, due to their nanoscale size, nanomaterials anchor onto and easily penetrate and pass through the cell wall and interact with the cell organelles [58,59]. Once in the bacteria, the nanomaterials interacts with proteins, lipids, DNA lysosomes, ribosomes, and enzymes to inhibit the normal cell function (oxidative stress, heterogeneous alterations, enzyme inhibition, changes in gene expression, among others) [28,60,61]. Although detailed antibacterial mechanisms of nanomaterials have not been fully unveiled, other reports point out the (i) induction of oxidative stress, (ii) release of the metal and metal oxide ions, (iii) and non-oxidative mechanisms [28,62].

2.7. Catalase Inhibition Assay of the Nanomaterials

To evaluate the activity of catalase in E. coli and S. aureus, cells were treated with AgNPs and CAC/AgNPs along with hydrogen peroxide and PBS (phosphate buffer saline) for 30 min [63]. After reading the optical densities of the supernatant (obtained via centrifugation of the incubated preparation), the percentage of remaining H₂O₂ was calculated. In E. coli, the percentages of H₂O₂ remaining in the bacterial cultures after treatment varied from 2.45 to 6.02%, whereas in S. aureus, these values ranged from 17.26 to 24.20% (Figure 10). Notably, the activity of the nanocomposites on catalase produced by both bacteria was superior as compared to that of the nanoparticles. Widely distributed in aerobic and some anaerobic bacteria, catalase is an enzyme that is crucial in bacterial defense against oxidative stress [64,65]. Thus, it is not unreasonable to speculate that the as-prepared nanomaterials might have exerted antibacterial action by inhibiting the activity of catalase in E. coli and S. aureus.

3. Material and Methods

3.1. Plant Collection and Identification

The plant material consisted of bark from the trunk of Croton macrostachyus that was collected at Baneghang (West Region, Cameroon) in March 2021. The identity of the plant was confirmed by Mr Nana Victor (Botanist) at the National Herbarium of Cameroon (Yaounde, Cameroon), where a voucher specimen was deposited under number 32264HNC.

3.2. Plant Extraction

The collected plant material was shade dried, then ground to fine powder using a grinder. Next, 10 g of bark powder was added to 500 mL of distilled water in an Erlenmeyer flask and boiled on a hot plate for 10 min. The resulting solution was allowed to cool to room temperature, then filtered through Wattman paper N°1 and the filtrate was dried under ventilation for 4 days and the extract was collected and kept at 4°C for future use.

3.3. Preparation of Activated Carbon from Croton Macrostachyus

The stem bark of Croton macrostachyus was washed with distilled water, cut into small pieces and dried at room temperature (25°C) in the dark. Next, the dried samples were ground and sieve to obtain finer particles. Ten grams (10 g) of the fine powder (CMP) was impregnated with H₃PO₄ solution (1:1). The reaction mixture was left at room temperature for 1 h to allow for a complete absorption into the C. macrostachyus stem bark. Afterward, the impregnated C. macrostachyus stem bark was dried at 110^oC for 24 h in an oven. The dried samples were kept temporarily in the desiccator, then carbonized at 432^oC for 40 min using a Carbolite Furnace to obtain the activated carbon (CAC).

3.4. Biological Synthesis and Characterization of Silver Nanoparticles Using Croton Macrostachyus

3.4.1. Preparation of Silver Nanoparticles

For the synthesis of nanoparticles (AgNPs), 10 g of fine bark powder was added to 500 mL of distilled water in an Erlenmeyer flask and boiled on a hot plate for 10 min. The resulting solution was allowed to cool to room temperature, and then filtered using Wattman paper number 1. One hundred milliliters (100 mL) of the obtained aqueous extract was added to 100 mL of 5 mM aqueous silver nitrate (AgNO₃) solution. Next, the mixture was stirred for 1 min using a magnetic stirrer (Horse shoe IKA magnetic agitator) in the absence of light to avoid any photochemical reactions of silver nitrate and stored in a dark chamber for 24 h. The generation of the nanoparticles was evidenced by the color change from pale yellow to dark brown. The absorbance of the resulting solution was read using a visible ultraviolet spectrophotometer.

3.4.2. Characterization of AgNPs

The as-prepared silver nanoparticles were characterized using analytical techniques, such as UV-visible (UV–Vis) spectrophotometry and Fourier transform infrared (FTIR).

3.4.2.1. UV-Vis Spectrophotometry Analysis

The UV-visible analysis of the extracts, and prepared nanoparticles (AgNPs) was carried out using shimadzu UV-visible spectrophotometer (UV-1800, Japan) in the wavelength range of 350–900 nm to obtain the UV-visible spectra of the sample.

3.4.2.2. Fourier Transform Infrared Spectroscopy Analysis of AgNPs

FTIR analysis of the aqueous extract and AgNPs was performed in the absorbance range of 400 to 4000 cm-1 using a Universal ATR, Crystal: Platinum, Diamond, Bounces: 1, Solvent: ethanol. In brief, the prepared extract or nanoparticles (AgNPs) were mixed with potassium bromide (KBr) and subsequently pressed into pellets. The pellets thus formed by compression were placed in the sample holder of the apparatus for analysis. The results of the transmittance as a function of the wavelengths were given by the screen. Afterwards, the obtained spectrograms were analyzed to identify the functional groups located on the surface of extracts and nanoparticles.

3.5. Preparation and Characterization of Nanocomposite from Croton Macrostachyus

3.5.1. Preparation of the Nanocomposite

To obtain the nanocomposites (CAC/AgNPs), the silver nanoparticles (AgNPs) were loaded on the activated carbon (CAC) by means of simple agitation. Briefly, 5.0 g of CAC was added to AgNPs solution, then the resulting solution was vigorously mixed by continuous stirring for 1 h at 150 rpm using a Horse shoe IKA agitator. The nanocomposite was obtained by drying the AgNPs-loaded activated carbon powder at 110^oC in an oven (Bluepard Instruments Co. Ltd, Shangai).

3.5.2. Characterization of the Adsorbents Activated Carbon and Nanocomposite

The as-prepared activated carbon (CAC) and nanocomposite (CAC/AgNP) were characterized by determination of methylene blue and iodine numbers. FTIR measurements were carried out to detect changes in the composition of biomolecules and their functional groups. FTIR analysis was performed in the absorbance range of 400 to 4000 cm-1 using Universal ATR, Crystal: Platinum, Diamond, Bounces:1, Solvent: ethanol.

3.5.3. Determination of the Methylene Blue Index and Iodine Number

Defined as the number of milligrams of iodine or methylene blue adsorbed by 1 g of activated carbon, iodine number and methylene blue index, respectively provide information on the microporosity (up to 2 nm) and mesoporosity of the activated carbon. The porosity of the activated carbon is critical to conclude about the degree of adsorption of metal loaded-activated carbon of molecular size.

3.5.3.1. Determination of the Iodine Number Using Batch Mode Adsorption

To determine the iodine number, 100 mg of CAC and CAC/AgNPs were separately introduced into 30 mL of iodine solution in 100 mL Erlenmeyer flasks. Then, the mixtures were sealed and stirred for 3 h at room temperature. After stirring, each mixture was filtered through a Wattman filter paper and the filtrate obtained was collected in a dry flask.

Next, 10 mL of each solution was titrated with sodium thiosulfate using starch as an indicator until the solution became transparent. The equation for of this the reaction is as follows:

The amount of iodine adsorbed is given by the reaction following formula (Nasehir et al., 2010) [66]:

$$\mathrm{Q}=\frac{\left(\mathit{C}\mathit{o}-\mathit{C}\mathit{e}\right)\times \mathit{V}}{{\mathit{m}}_{\mathit{C}\mathit{A}\mathit{P}}}$$

Where Co is the initial concentration of methylene blue ; Ce is the equilibrium concentration of the methylene blue ; m represents the mass (g) of sample (CAC or CAC/AgNPs) ; and V is the volume of methylene blue solution.

3.5.3.2. Determination of the Methylene Blue Index Using Batch Mode Adsorption

To determine the methylene blue index, 100 mg of CAC and CAC/AgNPs were separately introduced into 30 mL of methylene blue solution in 100 mL Erlenmeyer flasks. Then, the mixtures were sealed and stirred for 3 h at room temperature. After stirring, each mixture was filtered using a Wattman filter paper number 1 and the filtrate obtained was collected in a dry flask. Next, the absorbance was read at 660 nm against the blank using a spectrophotometer (Spectrumlab S23A). The amount of methylene blue adsorbed was calculated using the following formula [66]:

$$\mathrm{Q}=\frac{\left(\mathit{C}\mathit{o}-\mathit{C}\mathit{e}\right)\times \mathit{V}}{{\mathit{m}}_{\mathit{C}\mathit{A}\mathit{P}}}$$

Where Co is the initial concentration of methylene blue ; Ce is the equilibrium concentration of the methylene blue ; m represents the mass (g) of sample (CAC or CAC/AgNPs) ; and V is the volume of methylene blue solution.

3.6. Antibacterial Activity

The antibacterial activity of the C. macrostachyus extract and synthesized nanomaterials was assessed on five bacterial strains according to the guidelines set by the Clinical Laboratory Standards Institute [67] using 96-well microtitre plates. The bacterial strains used in this study are summarized in Table 2. Shigella flexneri and Shigella sonnei were generously obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Rockville, MD 20,852), whereas Escherichia coli and Staphylococcus aureus was obtained commercially from the American Type Culture Collection (ATCC, Manassas, VA, USA). Salmonella enteridis was generously obtained from the Centre Pasteur of Cameroon (CPC).

Briefly, 96 µL of Mueller Hinton Broth (MHB) culture medium was introduced into each well of a 96 well plate followed by a subsequent addition of 4 µL of stock solution of each prepared sample (extract of AgNPs) at 100 mg/mL. Next, 100 µL of 1 x 10⁶ mL of inoculum was added to each well except those for the sterility control. The positive control consisted of the inoculum and ciprofloxacin (10 µg/mL). The negative control comprised culture medium and inoculum, whereas the sterility control consisted of culture medium only. The plates were covered and incubated at 37°C for 24 h. At the end of the incubation period, 20 µL of freshly prepared resazurin solution (0.15 mg/mL) was added to each well and the plates were once again incubated under the same conditions for 30 min. The wells in which there was no visible bacterial growth corresponded to those containing active substances. Extracts and nanoparticles that inhibited the growth of bacteria at 1000 µg/mL concentration were selected for the determination of the minimum inhibitory concentrations (MICs).

3.7. Determination of the Minimum Inhibitory Concentrations

The minimum inhibitory concentrations (MICs) of the doped activated carbons and nanoparticles from C. macrostachyus were determined by the broth microdilution method, using the microdilution assay as described above. The assays were performed in duplicate in sterile 96-well microplates. In brief, 196 µL of MHB culture medium was introduced into the wells of column A, while 100 µL were added into the remaining wells of the microplate. Subsequently, 4 µL of a sterile solution of extract or nanoparticle (AgNPs), or nanocomposite (CAC/AgNPs) (100 mg/mL) was added into the wells of column A. This addition was followed by a series of 8 dilutions in geometric reasoning of order 2 and from column A to H. Next, 100 µL of bacterial load viz. 1x10⁶ UFC/mL was distributed to the remaining wells, including negative control wells. The concentrations of extract or nanoparticles, and ciprofloxacin in the wells of a row ranged from 1000 µg/mL to 7.8125 µg/mL, and from 5 µg/mL to 0.15625 µg/mL, respectively. The sterility control contained the culture medium only, whilst the positive group was made up of culture medium, inoculum and ciprofloxacin (stock solution at 1 mg/ml). The microplates were coated and then incubated at 37°C for 24 h. Next, 20 µL of freshly prepared resazurin solution (0.15 mg/mL) was added to all wells and the plates were incubated again at 37°C for 30 min. The lowest concentrations at which no color change (from blue to pink) were observed corresponded to absence of bacterial growth and were considered as MIC values.

3.8. Cytotoxicity Assays

The cytotoxicity of the most active nanomaterials was assessed on human mammalian cells (Vero cell line ATCC CRL 1586) using the resazurin colorimetric method as described by Bowling et al. [68]. The Vero cell line (ATCC CRL 1586) were cultured in complete medium containing 13.5 g/L DMEM (Gibco, Waltham, MA USA), 0.5% MEM (Gibco, Waltham, MA USA), 10% fetal bovine serum (Gibco, Waltham, MA USA), 0.21% bicarbonate (Sigma-Aldrich, New Delhi, India), and 10 mL (1%) of penicillin/streptomycin antibiotics. Briefly, 100 µL of cell suspension was introduced into 96-well (Costar, USA) microplates (10⁴ cells per well) and incubated overnight (18 h) to allow cell adhesion. Subsequently, the culture medium was replenished with 90 µL of new fresh complete culture medium, and then 10 µL of serially diluted extract or nanoparticles were added in triplicate to the corresponding wells. Podophyllotoxin at 50 µM was used as a positive control, and wells involving untreated cells were included as a 100% growth control. Next, the plates were incubated in a humidified atmosphere with 5 % CO₂ at 37°C for 48 h. Afterwards, 10 µL of resazurin stock solution (0.15 mg/mL in phosphate buffer saline, PBS) was added to each well and incubated for an additional 4 h. Fluorescence was then read using a multi-well plate reader (TECAN-Infinite M200) at excitation and emission wavelengths of 530 et 590 nm, respectively. From the optical densities (OD) obtained, the percentages of cell viability were calculated according to the following formula :

$$\mathbf{P}\mathbf{e}\mathbf{r}\mathbf{c}\mathbf{e}\mathbf{n}\mathbf{t}\mathbf{a}\mathbf{g}\mathbf{e}\mathbf{s}\mathbf{o}\mathbf{f}\mathbf{c}\mathbf{e}\mathbf{l}\mathbf{l}\mathbf{v}\mathbf{i}\mathbf{a}\mathbf{b}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{y}\left(\mathbf{\%}\right)=\frac{\mathbf{O}\mathbf{D}\mathbf{t}\mathbf{e}\mathbf{s}\mathbf{t}}{\mathbf{O}\mathbf{D}\mathbf{n}\mathbf{e}\mathbf{g}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{v}\mathbf{e}\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}}\times 100$$

Percentages of cell viability were used to determine the median cytotoxic concentrations (CC₅₀) using nonlinear regression curves using GraphPad Prism 5.0 (San Diego, Californie) software. ‘

3.9. Potential Mechanistic Studies of the Most Potent Nanomaterials

To understand the mechanism of antibacterial action, the growth kinetics, release of nucleic acids by bacteria and inhibition of catalase upon treatment with the most promising nanoparticules (AgNPs) and nanocomposite (CAC/AgNPs) were investigated.

3.9.1. Bacterial Growth Kinetics on Various Concentrations of the Nanomaterials

To study the bacterial growth curve, cultures containing E. coli and S. aureus strains were incubated with the most active antibacterial nanoparticles (AgNPs) and nanocomposites (CAC/AgNPs) as per the protocol reported by Nguimatsia et al. [69] with minor modifications. In short, 20 µL of AgNPs and 8 µL of CAC/AgNPs were added respectively in 180 µL and 192 µL of Mueller Hinton Broth in the first twelve wells of column A (A). In the remaining wells of the microplate, 100 µL of Mueller Hinton Broth was introduced. Next, a series of geometric dilutions of order 2 was operated from column A to H to achieve concentrations ranging from 250 to 15.625 µg/mL and from 2000 to 125 µg/mL for AgNPs and CAC/AgNPs, respectively. Afterwards, 100 µL of 1x10⁶ UFC/mL inoculum was introduced into each well except for control groups (sterility, AgNPs and CAC/AgNPs). Ciprofloxacin was used as a positive control. The microplate was coated and incubated at 37°C at diverse incubation time periods (0, 1, 2, 4, 6, 8, 10, 12 and 24 h) and the optical densities were read at 630 nm using a microplate reader (Infinite M200 microplate reader TECAN, Männedorf, Switzerland) after each incubation time was elapsed. The values obtained were used to plot OD = f(t) graphs.

3.9.2. Nucleic Acid Leakage Assays

This test was performed according to the protocol described by Carson et al. [70]. The assays were performed in triplicate in 2 mL eppendorf tubes. Colonies obtained from 24 h cultures were washed twice with NaCl solution (0.9%), then resuspended in sterile peptone water (0.1%) and calibrated to 0.5 MacFarland standard. Subsequently, 250 µL of inoculum (1.5 x 10⁸ UFC/mL) was introduced into 250 µL of peptone water containing the prepared extract at different concentrations including 2CMI, CMI et CMI/2, and the the tubes were incubated at 37°C at different times (0, 0.5, 1, 2, 4, 6 and 8 h). After each incubation period, the solution was centrifuged at 5000 rpm for 20 min. The supernatant was collected and transferred into wells of a microplate and the absorbance was measured at 260 nm using a TECAN infinite M200 plate reader. The obtained optical densities (OD) were plotted versus time using Excel software.

3.9.3. Catalase Inhibition Assay

The catalase inhibition assay of the most active nanoparticles (AgNPs) or nanocomposites (CAC/AgNPs) was determined on E. coli ATCC 25922 and S. aureus ATCC 43300, according to a previously described protocol [63]. The assays were performed in triplicate in 2 mL eppendorf tubes. Various concentrations of samples (AgNPs and CAC/AgNPs) were introduced in test tubes containing 200 µL of hydrogen peroxide (40 mM) and 200 µL of phosphate buffer saline (PBS 10X; pH= 7.3 – 7.7) (Sigma-Aldrich, Germany). Next, In the mixture, 100 µL of bacterial suspension at 1.5 x 10⁸ CFU/mL were added and the samples were incubated at 37°C for 30 min. Then, the test tubes were centrifuged at 1200 rpm for 10 min and the supernatants were further collected and transferred into wells of a microplate. At last, the optical density of the microplate was read against the blank (phosphate buffer saline, PBS 10X, pH : 7.3-7.7) and the negative control (bacteria in PBS without inhibitor) at 232 nm using an Infinite M200 microplate reader (TECAN, Männedorf, Switzerland). Ciprofloxacin (at 0.078 and 0.039 µg/mL for E. coli ATCC 25922 and S. aureus ATCC 43300, respectively) was used as a positive control. The percentage of remaining hydrogen peroxide was determined according to the following formula :

$$\mathbf{\%} \mathbf{de} {\mathbf{H}}_{\mathbf{2}}{\mathbf{O}}_{\mathbf{2}} \mathbf{restant}\mathbf{=}\frac{\left(\mathbf{A}\mathit{e}-\mathbf{A}\mathbf{c}\right)}{\mathbf{A}\mathbf{c}}\mathbf{x}\mathbf{100}$$

Ac is the negative control, whereas Ae is the absorbance of H₂O₂ in the presence of the sample.

3.9.4. Statistical Analysis

Results were expressed as mean ± standard deviation. The statistical analysis and plots were performed using Graphpad Prism 8 and Excel software. The means were analyse using one-way ANOVA (Analysis of variance) test, followed by a post hoc multiple comparison (Tukey test) and were considered significant for *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

4. Conclusion

To sum up, an eco-friendly method was developed to synthesize silver nanocomposite from the corresponding nanoparticle and C. macrostachyus-based activated carbon. The nanomaterials were further characterized using UV-visible spectroscopy, and FTIR analyses. The nanomaterials exhibited antibacterial activity against an array of bacterial strains, the most susceptible being Escherichia coli and Staphylococcus aureus. Although the activated carbon did not exhibit antibacterial action, their role in minimizing the cytotoxicity of the nanocomposite was paramount. Antibacterial mechanistic studies revealed that the as-prepared nanomaterials induced (i) bacteriostatic activity vis-à-vis E. coli and S. aureus and (ii) inhibition of catalase in these bacteria. This novel contribution on the antibacterial mechanisms of action of silver nanocomposite from C. macrostachyus-based activated carbon might help in better understanding the bacterial inhibition by this biomaterial. The results suggest that the prepared nanocomposite could be a promising antibacterial potential candidate with low toxicity to Vero cells.