Green synthesis of silver nanoparticles and evaluation of their anti-bacterial activities: use of Aloe barbadensis miller and Ocimum tenuiflorum leaf extracts


 The presence of various phytochemicals makes the leaf extract-based green synthesis advantageous to other conventional methods, as it facilitates the production of non-toxic by-product. In the present study, leaf extracts from two different plants: Aloe barbadensis miller and Ocimum tenuiflorum, were used to synthesise Ag nanoparticles. The absorbance at 419-432 nm from UV-visible spectroscopy indicates the formation of Ag in the synthesised samples. The effect of precursors’ concentration on the stability, size and shape of the synthesised samples has also been investigated at constant heating temperature, stirring time, and the pH of the solution. The TEM results showed that all the synthesised samples of nanoparticles demonstrated stability with a size range of 7-70 and 9-48 nm with Aloe barbadensis miller and Ocimum tenuiflorum leaf extracts, respectively. The formation of smaller Ag nanoparticles due to utilisation of different precursor concentration and leaf extracts was also explained. The synthesised samples’ anti-bacterial activity was examined against the pathogens, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. In general, the green synthesis approach established a prospective for developing highly stable Ag nanoparticles with rigid particle shape/size distribution from different leaf extracts for the development of better anti-bacterial agents.


Introduction
Plant-based green synthesis of metal oxide nanoparticles (NPs) are gaining prominence due to its simplicity, commercial scalability, and versatility in using imperishable and lower-risk value-added components. At present, nanoscience and nanotechnology is one of the fastestgrowing and promising fields, which requires considerable attention to develop environmentally benevolent methods of synthesis of NPs that avoid the use of deadly chemicals [1][2][3][4][5]. In this context, the deployment of green chemistry reduces the use or generation of toxic chemicals during the synthesis processes [6]. Consequently, the research interest in fabricating metal NPs through biological approaches, especially leaf extracts, is developing rapidly. This is because of simple experimentation, environmental friendliness, reproducibility, and cost-effectiveness of biological routes compared to physical and chemical-based methods [7]. Thus, there is a boost in demand for green nanotechnology [8,9]. Generally, a variety of bioactive phytochemicals present in micro and macro organisms like algae, bacteria, fungi, and plants can be used for the green synthesis of NPs [10][11][12]. However, in green nanotechnology, the plant leaf extracts have been extensively used compared to microorganism approaches because plants are safe to handle, widely available, and possess a variety of phytochemicals that act as reducing/stabilising agents during the synthesis of NPs [13][14][15].
Undoubtedly, various novel NPs like ZnO, CuO, Au, Ag, Fe, Pd [16,17] and so on, have been synthesised by using green technology. However, the silver and gold NPs attracted tremendous interest due to extensive applicability in various areas such as drug delivery systems, electronics, energy-efficient systems, and in the field biomedical science as promising antimicrobial, wound healing, bone repairing, and anticancer agents [18][19][20][21]. Recently, NPs are explored as nanomedicines for early diagnosis and treatment of many deadly diseases like cancer [22,23]. Nanomedicines are very effective in the treatment of the cancer cells like Osteosarcoma (a type of bone cancer) [24], Retinoblastoma (an ophthalmological cancer) [25], etc. In addition, NPs are also playing their role as a safer drug carrier in the effective drug delivery systems for the drugs like crosin [26] and deferasirox [27].
Despite this all, the confined availability and high cost of the majority of the medicinal plants compel the researchers to think about the use of commonly available and cost effective plants. Toward this, the use of medicinal plants that are locally or easily available like Aloe barbadensis miller (aloe-vera) and Ocimum tenuiflorum (tulsi) is significant. Many studies have been reported on the biosynthesis of silver NPs [41,[55][56][57][58][59][60][61][62][63][64][65]. However, maximum of the reported work has been performed at a minimal precursor concentration. In addition, the effect of the change in the precursor concentration on the shape and size of synthesised Ag NPs is also not much explored. Nowadays, the frightening phenomenon of occurrence of drug-resistant bacteria is a major concern of the universal healthcare system. Hence, silver NPs represent the novel biocompatible nanostructured materials for unconventional antimicrobial applications [66]. This is due to their inherent broad bactericidal effects demonstrated against both Gram-positive and Gram-negative bacteria. Silver nano-structures are one of the most used metallic NPs in contemporary anti-bacterial applications [67]. In their anti-bacterial action, Ag NPs penetrate the cell and produce a drastic disturbance regarding structural damage, proper cell function, and ultimately results into cell death [68].
Therefore, in this present work, plant leaf extracts from Aloe barbadensis miller and Ocimum tenuiflorum were used as feedstock for the synthesis of Ag NPs. The leaves of Aloe barbadensis miller typically consist of active constituents: flavonoids, vitamins, sugars, saponins, amino acids, lignins, alkaloids, steroidal lactones, and tannin [69,70]. On the other hand, Ocimum tenuiflorum or tulsi (commonly known in India) is native to the old world tropics and contains several phytochemical compounds such as phenols, amino acids, flavonoids, etc. in large quantities [71]. The choice of these two plants was motivated by the presence of high flavonoid contents, as flavonoids are reported to be essential for bio-synthesis of required NPs [71,72]. Moreover, extensive characterisations of the synthesised samples using ultraviolet-visible (UV-visible) spectroscopy, X-ray diffraction (XRD) spectroscopy, energy dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were also carried out to investigate the impact of synthesis conditions on the yield, the morphology, and stability of the NPs. Further, the possible application of the synthesised Ag NPs in anti-bacterial field has also been explored.

Materials
Silver nitrate (AgNO 3 ) and potassium hydroxide (KOH) were obtained from the Merck Pvt. Ltd. and were used without any further purification. The distilled water (DW) with a pH of 6.8-7.0 and a conductivity of 2-3 × 10 −6 s·cm −1 (at 298.15 K) was used as a medium in all the experiments. The human pathogens, Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 737), and Escherichia coli (MTCC 739) were procured from the CSIR-IMTECH, Chandigarh in the freeze-dried ampoule. Aloe barbadensis miller and Ocimum tenuiflorum plant leaves were collected from the nearby region of Career Point University Hamirpur (HP) India.

Preparation of the Aloe barbadensis miller or Ocimum tenuiflorum plant leaf extract
Fresh leaves were collected and thoroughly washed under the tap water to remove any dust or lose material. The washed leaves were further rinsed with distilled water forremoval of impurities if any. The leaves were then dried to remove excess water and exactly 25 gm of dry leaves of Aloe barbadensis miller or 10 gm leaves of Ocimum tenuiflorum were chopped and crushed well by using mortar and pestle. Further, a well-minced leaves were boiled for about 15 minutes in 100 ml of distilled water in each case. After boiling, the hot extract solution was cooled and filtered by using the Whatman No.1 filter paper. The so-obtained plant leaf extract was stored in the refrigerator for further use in the biosynthesis of Ag NPs.

Synthesis of the Silver nanoparticles
The stock aqueous solutions (5, 10, and 50 mmol·kg −1 ) of the precursor silver nitrate (AgNO 3 ) were prepared in the distilled water. Then the prepared known concentration solution was taken in a flat bottom flask placed in the oil bath. The temperature of the solution was maintained at 70±1 ∘ C. Then 20 ml Aloe barbadensis miller or Ocimum tenuiflorum plant leaf extract as prepared above was added dropwise at a rate of approximately 5 ml/min with constant magnetic stirring. After about 15 minutes of adding Aloe barbadensis miller leaf extract, the pH of the reaction mixture was maintained at 11.5 with the addition of freshly prepared 0.2 mol·kg −1 KOH solution. Initially, the change in color of the solution from colourless to redish brown and appearance of the dark black color precipitates confirm the formation of Ag NPs. The reaction mixture was allowed to reflux at 70±1 ∘ C under constant stirring condition for 4 hrs. After refluxing, black color precipitates were allowed to settle down at room temperature. The so-obtained nanosamples were washed 3-4 times with distilled water followed by the washing with ethanol. Finally, the material was dried at 55-60 ∘ C in a hot air oven. Similarly, the Ag NPs were also synthesised at 10 and 50 mmol·kg −1 of precursor AgNO 3 .

Characterisation of the silver nanoparticles
The synthesised materials have been confirmed with the UV-Visible, XRD, SEM, TEM, and EDS spectral analysis. The Cary 100 Bio UV-Visible instrument, which is a doublebeam spectrophotometer capable of acquiring data in the spectral range from 190 to 900 nm has been employed to carry out the spectral studies. The Panalytical Empyrean XRS-45 kV, XRD instrument has been used to examine the crystallinity and phase identification of the samples. Further, the morphology and shape/size of the synthesised NPs were examined by using the Carl-Zeiss Ultra 55 field emission-scanning electron microscope and JEOL JEM 2100F-200 kV transmission electron microscope. However, the elemental detection analysis of the synthesised NPs has been carried out by using the EDS attached with the SEM micrograph.

Anti-bacterial activity of silver nanoparticles
The anti-bacterial potential of so synthesised silver oxide NPs has been tested against the pathogens, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli by using the agar well diffusion method [73].

UV-Visible Spectroscopy
A confirmatory and optical analysis of the prepared samples has been executed by using the UV-Visible spectrophotometer. The UV-Visible spectra of Ag NPs synthesised by using Aloe barbadensis miller and Ocimum tenuiflorum leaf extracts at 5, 10, and 50 mmol·kg −1 of AgNO 3 as precursor exhibit the characteristic absorbance peaks in the range of 419-432 nm ( Figure 1). It has been reported in the literature that the Ag NPs generally show absorption band in the range 380-470 nm depending upon the size and morphology of material [75,76]. Further, the band gap (Eg) values of so-obtained Ag NPs, are calculated by using equation 1, and are tabulated in Table 1.  The change in the λmax and Eg values with precursors' concentration suggests the formation of Ag NPs with variable shape/size. It is also observed that such type of change in shape/size with λmax/Eg values, ultimately, affecting the antibacterial activity of the synthesised samples, as discussed in sub-section anti-bacterial activity.

XRD Analysis
The XRD spectral analysis of each of the prepared samples represents the number of patterns as delineated in Figure 2A   tion of monoclinic unit cell volume and lattice parameters, respectively. The calculated values are tabulated in Table 2.
Here, λ = 1.5406 Å is the wavelength used during XRD analysis, β is full width at half maximum (FWHM) for the most intense peak, θ is Bragg's angle, (h, k, l) are Miller indices, d hkl is inter-planar spacing, (a = b = c) are unit cell lattice parameters, and V is the unit cell volume of the fcc crystal system of Ag NPs.

SEM/EDS Analysis
The surface morphology and topography of synthesised Ag NPs was determined by employing the SEM technique. From SEM images displayed in Figure 3A-3C, it is clear that Ag NPs are showing the fluffy arrangement of small spheres, when synthesised at 5, 10, and 50 mmol·kg −1 of the precursor AgNO 3 by using the Aloe barbadensis miller leaf extract. In addition, the SEM images of Ag NPs synthesised by using the Ocimum tenuiflorum leaf extract are representing the formation of NPs with spherical/spheroidal shape ( Figure 3D-3F) The representative EDS spectra (Figure 4) enumerate the elemental analysis of the Ag NPs synthesised by using Aloe barbadensis miller leaf extract. All such spectra show the presence of both silver and oxygen elements in the synthesised nano-material. The low-intensity peaks can be attributed to the occurrence of Au in coating the material used for EDS analysis experiments. However, the existence of elemental carbon peak may be due to the phytochemicals involved in the stabilisation of Ag NPs synthesised from the leaf extract. Similar spectra were obtained for the Ag NPs synthesised by sing Ocimum tenuiflorum leaf extract.

TEM Analysis
Both the size and shape of the biosynthesised Ag NPs were further characterised by transmission electron microscopy (TEM) analysis. From the analysis of the TEM micrograph images ( Figure 5), it is found that the all samples of NPs synthesised at 5, 10, and 50 mmol·kg −1 of AgNO 3 were obviously exhibiting nearly similar spherical and spheroidal shapes for both the investigated plants. In contrast, the size of NPs is reflecting the effect of the type of leaf extract used and the precursors' concentration as well. At 5 mmol·kg −1 of AgNO 3 , the Aloe barbadensis miller and Ocimum tenuiflorum synthesised Ag NPs show average size around 31.9 and 13.5 nm, respectively. Further, the particle size generally increases with an increase in the concentration of AgNO 3 , as reported in Table 3. The obtained results are found in good   Table 3, it is interesting to note that the particles synthesized by using the Ocimum tenuiflorum leaf extract are comparatively smaller in size as compare to those synthesised by using Aloe barbadensis miller leaf extract. However, at 50 mmol·kg −1 of AgNO 3 a remarkable wide range smaller size (7-65 nm) Ag NPs were formed when syn-thesised by using the Aloe barbadensis miller leaf extract ( Figure 5 and Table 3). Such type of large range in size can be attributed to the formation of mixed silver and silver oxide NPs, as also indicated in XRD patterns. Moreover, the exceptional results of Aloe barbadensis miller at 50 mmol·kg −1 of AgNO 3 may be due to active stabilisation or capping of the synthesised nano-crystals by aloin or other phytochemicals present in the leaf extract.
In the light of the above discussion, the mechanism of bio-synthesis of Ag NPs can be demonstrated as shown in Figure 6. Both, the Aloe barbadensis miller and Ocimum tenuiflorum leaf extracts are the rich source of various phyto-chemicals. Jain and Mehata [57], Logaranjan et al. [81], and Brahmachari et al. [82] have reported the role of Quercetin (present in Ocimum tenuiflorum), Aloin (found in Aloe barbadensis miller), and the Eugenol phytochemicals, respectively, in the mechanism of the bio-synthesis of Ag NPs. The presence of metabolites like anthraquinones, polyphenols, polysaccharides, lignins, saponins, alcohols, amino acids, fatty acids, sterols, etc. [83,84] in Aloe barbadensis miller and biologically active compounds like eugenol, urosolic acid, linalool, carvacrol, limatrol, luteolin, vitexin, orientin, chlorogenic acid, caffeic acid, rosmarinic acid, ascorbic acid, etc. in Ocimum tenuiflorum are the phytochemicals which are held responsible for their involvement in the synthesis of NPs [85]. Figure 7 illustrates the anti-bacterial activity of biosynthesised Ag NPs in terms of zones of inhibition, as investigated under the Agar well diffusion method against pathogens, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The so-calculated values of zones of inhibition (Figure 7) are summarised in Table 4 for both Aloe barbadensis miller and Ocimum tenuiflorum mediated nanosamples. The maximum zones of inhibition for the Aloe barbadensis miller (15.67±1.28 mm) and Ocimum tenuiflorum (12.45±1.18 mm) synthesised Ag NPs have been observed against Escherichia coli, whereas against Bacillus subtilis, almost all the synthesised samples show minimum zone of inhibition. Further, all the synthesised samples, except few, found to be inactive against Staphylococcus aureus (Ta-   [86]. Similar results have been demonstrated by Chauhan et al. [87] for the anti-bacterial activity of Ag NPs synthesised by using pathogen Pichia fermentans JA2. Exceptionally, the samples obtained at 50 mmol·kg −1 of AgNO 3 in the presence of both Aloe barbadensis miller and Ocimum tenuiflorum plant extracts, are very effective to inhibit the growth of all the studied human pathogens (Figure 7 and Table 4). This exceptional behaviour may be attributed to the presence of AgO nano-structure in addition to Ag in the samples synthesised at the 50 mmol·kg −1 of AgNO 3 , as already concluded from the XRD studies. A very small information has been found in the literature on the anti-bacterial activity of the AgO NPs. Vithiya et al. [88] have carried out the anti-bacterial activity study of Ag 2 O Figure 8: Mechanism for the anti-bacterial action of Ag NPs NPs at 1.69 mg/ml and Dharmaraj et al. [89] have examined the antibiofilm activity and cytotoxic potentiality of Ag 2 O at a very low amount. Moreover, the high activity of the AgO NPs as compared to the Ag NPs may be correlated with the ease of the generation of the active Ag ion during the anti-bacterial action. The Ag ions are stronger anti-bacterial agents than the Ag metal [90]. In the possible mechanism for the anti-bacterial action (Figure 8), the Ag ions penetrate the cell wall and disrupt the cellular function by interacting with the sulphur component present in the plasma membrane. The Van der Walls forces, electrostatics attractions, hydrophobic interactions, etc. change the metabolic action and NPs can easily penetrate the bacterial membrane. NPs' interactions with the bacterial components like DNA, ribo-  somes, enzymes etc. lead to the oxidative stress, electrolyte unbalancing, enzyme and protein disruption, etc., and ultimately cause the bacterial death [91]. In addition, the generation of reactive oxygen species (ROS), and other types of interactions of the Ag ions with the membrane (Figure 8) are also the possible reasons for the high anti-bacterial action of the AgO NPs [92]. The high activity against the gram-negative Escherichia coli than gram-positive Bacillus subtilis and Staphylococcus aureus is due to the thin cell wall of the Escherichia coli [93]. It has also been observed that a large amount of ROS generated in case of the gramnegative Escherichia coli, which produces the high activity oxidative stress over Escherichia coli [94,95]. In addition, the NPs prevent the production of bio-film by the microbes to resist the drug action and hence, better results are obtained in case of NPs [96]. In the light of above discussion, the mechanism of antibacterial action can be divided into three parts, namely, penetration of the cell wall, generation of ROS, and release of the metal ions. Along with the antibacterial action, the cytotoxic studies and NPs intracellular interactions with ROS are the important aspects that can be correlated to cell growth and a probable mechanism for antibacterial action of the AgO NPs on the different bacterial pathogens. Figure 9 picturised the MIC and MBC results, which are tabulated in Table

Conclusions
Ag NPs with tuneable characteristics for customised applications were synthesised using Aloe barbadensis miller and Ocimum tenuiflorum leaf extracts in the present work. The effect of precursor, AgNO 3 concentration on the chemical and biophysical properties of the nanostructures was also investigated. The XRD spectral studies confirm the formation of mixed Ag+AgO crystal phase at 50 mmol·kg −1 of AgNO 3 . The TEM analysis illustrated that the particles are mostly spherical and possess an average size in the range of 7-70 and 9-48 nm when synthesised from the Aloe barbadensis miller and Ocimum tenuiflorum leaf extracts, respectively. The nano-samples synthesised at 50 mmol·kg −1 of AgNO 3 have shown very high anti-bacterial inhibition property due to the presence of AgO in addition to Ag nano-structures. Further, the largest zone of inhibition was found in the Escherichia coli for all the prepared nano-samples. Thus, study revealed that the precursor concentration and leaf extract compositions play a key role in describing the features of the Ag NPs and their application as potent anti-bacterial agents. It can also be concluded that a very less research has been reported on AgO nanostructures, hence this field need to be explored particularly in terms of biomedical applications. This work creates an opportunity to a facile and low-cost bio-synthesis approach to synthesise highly bio-active Ag NPs. However, the supplementary studies are required to profoundly examine the cytotoxic effects of the prepared NPs. Moreover, the ability of the bio-synthesized NPs to alter the intracellular ROS levels will also be a new area to be assessed in future.

Acknowledgement:
The authors are grateful to the Department of Chemistry and Centre for Nano-Science and Technology, Career Point University Hamirpur (HP), India for providing the all required facilities to carry out the work.

Funding information:
This research received no specific grant from any funding agencies, all funds are settled by authors.

Authors' contribution:
The manuscript writing, conceptualization, and methodology contributed by Saurabh Sharma, under the supervision and project administration of Kuldeep Kumar and Naveen Thakur. The validation, visualization, review and editing executed by Kuldeep Kumar.

Conflicts of interests:
The authors declare no competing financial interest.

Ethical approval:
The conducted research is not related to either human or animal use.
Data availability: All data generated or analysed during this study are included in this published article.