Surgical Site Infection (SSI) are considered as the third most nosocomial infectious status as described by National nosocomial infection surveillance system [1, 2]. SSI is developed within 5-30 days after surgery which directly contributes the patients’ morbidity and mortality. The triangular relationship between host, pathogens and environment may determine the prevention or initiation of wound infection [3, 4]. The identification, differentiation and diagnosis of SSI are difficult that leads to improper diagnosis, inappropriate antibiotic prescription, evolving of antibiotic resistance microbes and some mild to severe side effects [5].

Risk of wound infection is majorly created by microbial contamination, thus originates from the patient [6, 7] or by exogenous/ endogenous microbial group of pathogens, the most common microbes involved in SSI are Streptococcus sp [8], Escherichia coli, Methicillin Resistant Staphylococcus aureus (MRSA), Bacillus sp and SSI are Streptococcus sp [5, 9, 10]. The increase in the emergence of antibiotic resistant strains lead to treatment failure is a major challenge in the healthcare industry, in the recent decades had to search a new approaches and method for finding a new compound against drug resistant pathogens. Now, scientists accepted that the metal nanoparticles from herbal plants are also a novel area that proved biophysical chemistry [11, 12, 13].

Even though, the remarkable development in the use of techniques used for surgery, antibiotic selection procedure, aseptic operative rooms and surgical site infections remains a serious issues [14, 15]. Antibiotic resistance gram positive isolates showed maximum sensitivity to vancomycin, teicoplanin, linezolid and gram negative isolates were highly sensitive against meropenem and amikacin [16, 17]. Combinational drugs may have a high chance of exhibiting bactericidal and bacteriostatic nature against drug resistant pathogens due to its synergestic approaches [18].

Apart from antibiotics, chemically synthesized and derived drugs, green mediated metal nanoparticles are more or less equal effect against microbial pathogens with minimal health related risks [19, 20]. In the last decades, plants are the major source of antimicrobial agent in various countries, nearly 60-90% of peoples in the developing countries majorly using plant derived medicine, thus to treat human infectious diseases [21, 22, 23, 24, 25].

Generally metals are basic constituent to all living cells and get nourished when in combination with other nutrients, but once it reaching the minimal level (in micron) which was highly toxic [26], because of this nature, metal have a capacity to destroy microbes when its reach very minute size. Basically, silver and copper plated materials were used on those days there were not aware about its microbicidal nature. Technology development which helps to synthesize metal nanoparticles from herbal plants to perform specific function when compare to the bulk form of metal alone [27].

Recently, Ocimum sanctum leaves were used to synthesize silver and gold nanoparticles [28, 29] because of the presence of eugenol compound in tulsi helps to the action of antimicrobial activity. Synthesis of O. sanctum mediated silver nanoparticles reveal excellent antimicrobial activity was a cost effective method [30]. Here, we simultaneously synthesis and characterize O. sanctum mediated silver and copper nanoparticles to analyze the antimicrobial activity of isolated from wounds.

The biomolecular analysis of herbal reduced metals whose particle sizes are exhibited as nanoscale has its own toxic effects to pathogenic microorganisms. “Lowering the molecular size attack the higher scale organism” according to this principle, the herbal based materials whose material size below the micron have enough reducing, capping ability and antimicrobial activity [34, 35]. The biggest question is about the environmental toxicity.

This study has its own manner of evaluating the bactericidal effect of un-processed metal solutions and crude herbal compound. As a result, in vitro aqueous solution of silver were analyzed for bactericidal nature and showed maximum inhibition towards S. aureus, K. pneumoniae and P. aeruginosa. The copper solution revealed the intrinsic bactericidal activity to S. aureus, K. pneumoniae, P. aeruginosaand Serratia sp. The detailed antibacterial activity of the test metal solutions against clinical pathogens was depicted in table 1. In this study, the concentration of 4% and 10^-3 concentration of copper and silver solutions showed maximum inhibition, thus this concentration was taken for consideration to evaluate the herbal mediated metal nanoparticles.

Table 1: In vitro antibacterial activity of metal solution

The antibacterial effect of crude aqueous extract of O. sanctum was methodologically done and the results revealed that the maximum inhibition was observed against S. aureus, P. aeruginosa, K. pneumoniae and E. coli at the concentration of 250µg (Figure 1).

Figure 1: In vitro antibacterial activity of O. sanctum

The silver and copper nanoparticle synthesis through herbs (O. sanctum) was analyzed thereby it was preliminarily characterized by color change from pale to dark color. The silver complex is colourless initially later changed to dark brown to black threads (Figure 2a and b); whereas copper was initially observed as pale blue and later changed to dark (Figure 2a and b). All these changes were recorded within 24 hours.

The synthesis of metal nanoparticles using herbs can be easily observed and preliminarily evaluated by the color change of the mixture, thus changed from light colour to yellowish to brown colour. This colour change arises from the excitation of vibrations of the surface plasmon resonance due to oscillation of free electrons accumulation occurrence [36, 37, 38, 39].

Figure 2a: Silver and copper control; Figure 2b: Silver and copper with test herb

The calorimetric value of the synthesized nanoparticles were evaluated by uv visible double beam spectrophotometric method, thereby the peak spectra showed decreased OD value verses time duration (Figure 3). The reduction in the OD value of the mixture indicated that the particles size were condensed and exacerbated as less concentrated molecules. By this we cannot confirm the molecule as nanoparticles, thus further extension of the work done to understand the enzymatic break down and physical characterizations (shape and size).

Figure 3: Calorimetric value of metal herbal mixture

The pH of the mixture was also supported by reducing from 7 to 5 and 3 among silver and copper – herbal mixture respectively. On comparing with silver, the copper – herbal mixture showed better results drastically after 60 minutes of interactions (Figure 4).The evaluation ofthe pH of solutes is the major factor for the synthesis thus influence on the size, shape and morphology of the nanoparticles.

Reaction of pH stimulate the electrical charge of the biomolecules which might be change the reducing and capping ability of the metal solution to synthesize biomolecules [40, 41, 42]. This study supported the same where pH was changed due to the reduction of metal ions with the compounds of herb.

Figure 4: Reduction of pH value of solution mixture (copper and silver with test herb)

The nitrate and sulphate are bio-reducted by the test herb – O. sanctum, thereby evidences of formation of nitrite and sulphide observed. The elevated OD with 0.8 and 0.11 was observed among Ag and Cu herbal solution respectively (Table 2). The major peaks of the calorimetric intensity at 460 nm corresponding the intensity maximum of 2,3-diaminonapthotriazole that are having the bactericidal properties. This reaction showed the nitrate reductase through the reaction of nitrite with 2,3 – diaminophthalene. It was observed that there is a reasonable reduction affinity for magnesium ions which are essential for assay of sulphate reductase activity.

The residual sulphate reductase activity was also done and comparing the assay reaction time, with enzymatic action revealed some observable reduction (Table 2). The bio-reduction was also characterized by the enzymatic analysis of nitrate and sulphate reduction revealed the theorem of enhanced antimicrobial properties. No evidences were so far created to proof this data [43]. Currently, very few methods can be adopted for the determination of metal reductase activity and the methodology has been optimized in order to confirm the enzymatic reduction and increase the efficacy of nanoparticles. There was high correlated phenomena thereby the study and the data will be correlated in future for standardizing the principles of nano-molecular synthesis [43, 44].

Table 2: Calorimetric analysis of nitrate and sulphate reduction

The scanning electron imaging revealed the better resolute of images thereby the particle size were analyzed as 15 and 26.3nm while magnified at 500X of Cu-O.s and Ag-O.s respectively (Figure 5). The characteristic particle agglomeration initiated from cluster size to size of the single particle was executed. The maximum of 86 clusters with 10mm sized and 72nm distance between the clusters were determined while evacuating O. sanctum mediated silver nanoparticles. One hundred and thirty seven (137) number of particle in one cluster was counted and the size of the single particle was approximately estimated as 36nm whose particle peak is 125nm height (Table 3).

Two different shapes were observed by X-ray diffraction where Cu-O.s and Ag-O.s exhibited cystalline cubic lattice (Figure 6 A) and polycrystalline Wurtzite structures (Figure 6 B) respectively. The inter-relationship analysis of effectiveness of light and electron microscopy considers substantial reach to synthesize and characterize newer biomolecules which are sub-cellular in nature and utilize the Applications and principles of physical and life sciences principles in order to obtain nano images with molecular specificity and spatial resolution. This phenomena has its own limitations to countenance the technical challenges for standardizing the sensitivity and specificity to achieve the reliable and efficient co-registration of optical and electron images. In this study, we characterize the images between 15 and 26nm by SEI, 36 and 63nm by AFM and 42 and 69nm by XRD for Ag-O. s and Cu- O.s respectively very few contaminations found but predominantly stable, spectrally-distinct cluster formation with controlled surface chemistry [45].

Figure 5: SEI of Cu-O.s (a) and Ag-O.s (b) at 500X

Table 3: Atomic force microscopic descriptions of Cu-O.s and Ag-O.s

Figure 6: A model crystalline cubic lattice (A) and wurtzite (B) [46]

[Source: glossary.periodni.com]

The results of in vitro antibacterial activity of O. sanctum mediated silver and copper nanoparticles were measured as zone of inhibition and data was compared. The maximum inhibition was observed against Proteus species (40mm) followed by Micrococcus species (35mm) while using Cu-O.s. The silver nanoparticles also have more or less effective bactericidal nature against Pseudomonas aeruginosa (40mm) followed by Klebsiellapneumoniae and E. coli with 39mm each. The detailed antibacterial potential of O. sanctum mediated metal nanoparticles were depicted in table 4. The synthesis of effective antimicrobial agents is the challenging task for overcoming the state of drug failure and antimicrobial resistance.

The byproduct synthesis and environmental compatible bio-molecular production are the choice of current and future herbonanoceutical industries [47]. Through the scientific method of biocompatible formation of newer therapeutics [48], this study may provide some sort of information that explore the antibacterial potential of silver nanoparticles induced by O. sanctum, and study on the molecular mechanism of copper nanoparticles potential against gram positive and gram negative bacterial pathogens are to evaluated [49]. Effective inhibition towards Proteus species, Micrococcus species, Pseudomonas aeruginosa, Klebsiellapneumoniae and E. coli were studied and exhibited (Table 4).

Table 4: Antibacterial activity of inhibition against bacterial pathogens

The synthesis, manipulation, characterization and biological activity are sermonized and advocated for the proof of evidence of constructing green nanomaterials using eco-friendly metals solutions by biological entities for potential applications [50]. The optimization of the laboratory techniques and appropriate factors for reactions including light intensity, pH of the solution, interaction time and temperature are enhancing the nanoparticle synthesis [33].

The observation of various shapes also currently attracts the scientists to identify the exact mechanism of reducing the aqueous metal ion and herbal extract mixture [50]. In various studies the triangular Ag [51] and nanoprisms [52] synthesized by chemical and photochemical methods are impregnated but in this study crystalline cubic lattice and polycrystalline wurtzite elevated with 41.6 and 69.2nm sized molecules. By this sub-cellular microscopic analysis, we can characterize the particle size distribution, agglomeration and aggregation state, number of clusters and particles, and nanomaterials’ surface characters [53].

Thus by this study, it was anticipated that the synthesis and characterization of O. sanctum mediated silver and copper nanoparticles initially and later confirmed the nano scale images through SEI, XRD and AFM. Further, it will be extended to analyze the exact nanochemistry of the synthesized bio-molecules for the pharmaceutical expansion.