Green synthesis, characterizations, and antibacterial activity of silver nanoparticles from Themeda quadrivalvis , in conjugation with macrolide antibiotics against respiratory pathogens

: The current study has portrayed the synthetic mixtures of Themeda quadrivalvis using gas chromato - graphy – mass spectrometry ( GCMS ) , the combination of green silver nanoparticles ( AgNPs ) formed with macro - lide antimicrobials. The counter microbial e ﬀ ects were investigated with various concentrates of plant com - pounds, AgNPs, and macrolide - formed AgNPs against respiratorymicroorganisms. GCMS examination has shown the presence of various substances that intensi ﬁ es the chloroform concentrate of T. quadrivalvis . A total of 51 mix - tures were distinguished, and furthermore, the most severe zone of restraint was found in chloroform removal and against Klebsiella sp. ( 18 ± 4.7 mm ) . It has been demon - strated that the green mixture of AgNPs containing macro - lide anti - toxins, such as azithromycin, erythromycin, and clarithromycin, demonstrates extensive antibacterial activ - ities against a wide range of microorganisms. In contrast, the green union of AgNPs also demonstrates their e ﬃ cacy against a wide range of respiratory microbes. The particles containing numerous relatively small fragments that were observed in the scanning electron microscopy analysis were found to be 20 nm in size. Previous studies have focused on phytochemicals and green amalgamations of AgNPs, but not much detail has been provided on T. quad - rivalvis . It has been reported that the two concentrates ( a plant concentrate in combination with consolidated green nanoparticle macrolide anti - toxins ) . The present study aims to treat respiratory microorganisms with a green methodology approach using nanotechnology; this analysis primarily focuses on o ﬀ ering creative approaches to make drugs against respiratory microbes.


Introduction
Irresistible sicknesses are a huge burden on general wellbeing, driven to a great extent by financial, natural, and biological elements. Around 15 million of 57 million deaths per year overall are assessed to be caused by irresistible illnesses, generally because of bacterial microorganisms [1,2]. The prairie biological system comprises 20% of the vegetation of the earth. Themeda quadrivalvis (L.) Kuntze (grader grass) is a grass weed of Poaceae family, which grows generally on farming land and no man's land. It is found mostly in India and South-East Asia. Poaceae is the most financially significant plant family, used as scavenge, building materials (bamboo, cover), fuel (ethanol), and food [3]. The Poaceae species (lemongrass, kangaroo grass, wheatgrass, etc.) is usually used as medication all over the world. They have cell reinforcement and antimicrobial properties, in light of their phytochemical compounds: aldehydes, alkaloids, saponins, terpenes, alcohols, ketone, flavonoids, which have different restorative properties.
Green nanoparticles are overall dynamically more utilized in numerous areas of the economy; there is a growing interest in organic and ecological security. The green combination of nanoparticles has gained attention because of the possibility and low ecological effect; green nanoparticles have an expansive use in numerous enterprises, including clinical, pharma, horticulture, and climate businesses [4]. Regular combination procedures of immaculate metal and metal oxide of nanoparticle amalgamation lead to sediments that are harmful to human beings and different species. Plant separates rapidly decrease metal particles chelated bacterial particles, which prompts the metallization of bacterial particles [5]. Hence, analysts are presently looking for a green blend way to eliminate the harmful synthetics during the synthesis of nanoparticles [6]. Macrolides are among the most clinically critical anti-infective agents used to treat wide varieties of respiratory microorganisms. The wide use of these anti-microbes has driven inescapably the spread of safe strains [7]. The current examination portrayed the synthetic mixtures of T. quadrivalvis using gas chromatography-mass spectrometry (GCMS) and a combination of green silver nanoparticles (AgNPs) and the formation of macrolide anti-infective agents that are more effective (mixture compounds have high efficiency when compared to single macrolide) against respiratory microorganisms. A green methodology approach to treat respiratory microorganisms using nanotechnology is discussed in this study. These approaches give the baseline structure for drug discovery.

Extraction of powdered plant material
Dried sample of 200 g was taken and was exposed to cold and hot extraction. The dried sample was dissolved in methanol, chloroform, oil ether, and water for these examinations. All the concentrates were dried using a revolving evaporator. The phytochemical examination (alkaloids, saponins, tannins, polyphenols, terpenoids, glycosides, and protein) was then completed ( Table 1) [8][9][10].

Green synthesis of nanoparticles
Around 20 g of leaves was washed with de-ionized water to eliminate dust particles and air-dried at room temperature. Then, the leaves were homogenized and added to 100 mL of de-ionized water and mixed for 20 min at 60°C. In the wake of heating up, the leaf extraction was cooled at room temperature and sifted conferring 75 mL of straightforward yellow variety leaf stock, which was stored at 4°C. For the green union of AgNPs (Nice Chemicals, Kochi, India), 0.01 M of a fluid arrangement of AgNO 3 was used. Leaf stock of 5 mL was added to 45 mL of 0.01 M AgNO 3 watery arrangement and kept under the encompassing condition to respond. After various time stretches, the changes of response blend are seen from straightforward yellow to dull earthy colour, which shows the arrangement of AgNPs. The arrangement of AgNP was collected and UV-Vis spectroscopy was used to investigate the reduction process of silver particles into nanoparticles within the solution. The AgNP arrangement was moved to the rotator and the excess fluid was removed using a dryer, yielding dark hued silver nanopowder.

Synthesis of AgNPs coated with macrolide antibiotic
Macrolide-coated AgNPs such as azithromycin, erythromycin, and clarithromycin (HiMedia, India) (Azi-AgNPs, Ery-AgNPs, and ClR-AgNPs) were integrated. Momentarily, every 5 mL (0.1 mM) of azithromycin, erythromycin, and clarithromycin watery arrangement was mixed with 5 mL (0.1 mM) of green amalgamation silver nitrate fluid arrangement, which was mixed for 10 min. A 20 μg of 5 mM newly pre-arranged sodium borohydride fluid arrangement (HiMedia, India) was added to the above blending response combination. When a diminishing specialist was added, the shade of the arrangement changed from clear to yellowbrown, showing that silver particles were decreased and macrolide-formed AgNPs were formed [11].

UV-vis spectrometer
Bio-decreased silver nitrate nanoparticle arrangement tests were carried out for optical absorbance in the range of 190-800 nm with a UV-Vis spectrometer (Thermo Fisher Scientific, India).

Fourier-transform infrared (FT-IR) spectra analysis
FT-IR (Benchtop Thermo Fisher Nicolet iS50, India) analysis of the sample was done to find the potential biomolecule and practical gatherings. FT-IR spectra were estimated by Perkin Elmer spectrometer with a goal of 1 cm −1 in the frequency range of 500-4,000 cm −1 .

X-ray diffraction (XRD)
XRD estimation (Bruker, Germany) was done with Cu-Kα radiation of 0.154 nm frequency to find out the development, translucent way of behaving, and the nature of the blended AgNP powder. The checking was completed in the range of 2θ from 30°to 80°at 0.03/min and the steady time was 2 s. The size of the AgNP was determined by the Debye-Scherrer condition.

Scanning electron microscopy (SEM) analysis
The surface morphology and molecule size of orchestrated green AgNPs were investigated by SEM analysis (Sneckner Elliot, California).

Antibacterial action
The antimicrobial action of green-synthesized AgNPs and the impact of green combination AgNPs and the unadulterated concentrate were examined by applying the standard agar plate dissemination measure [11]. The test microorganisms were obtained from Bioline Lab, Coimbatore, Tamil Nadu, India. The test microorganisms (Escherichia coli K7, Streptococcus sp. B1, Staphylococcus aureus A21, Pseudomonas aeruginosa S43, Proteus sp. M 34, and Klebsiella pneumonia X9) were consistently spread on Mueller Hinton Agar plates using a clean spreader, 20 µg of plant concentrate of plant extricates, green combination silver nanomolecule, and AgNPs coated with macrolide anti-infective agents (azithromycin, erythromycin, and clarithromycin) with different fixations (25, 50, 75, and 100 μg·mL −l ) were vaccinated done in a disinfected circle. After incubating the plates at 37°C for 18 hours, the bacterial growth inhibition zone was measured and recorded.

GCMS analysis
GCMS investigation of the chloroform concentrate of T. quadrivalvis was performed using a THERMO gas chromatography TRACE ULTRA VER: 5.0. The stove temperature is at 220°C at a pace of 6°C·min −l ; the transporter gas is at a stream pace of 1 mL·min −l . The split testing method was used to infuse the example in the proportion of 1:10. The maintenance records (RI) were verified by comparing maintenance periods of a series and identifying each component through an analysis of the maintenance file with relevant information from the literature. Any discrepancies were noted [12,13].

Statistical analysis
An antimicrobial analysis was performed in triplicate and the results (zone of inhibition) were statistically analysed and expressed as mean (n = 3) ± standard deviation.

Phytochemical analysis
The phytochemical analysis is the prominent identifying source of therapeutically and industrially valuable compounds in all plants by chemical methods [14]. In this present investigation, secondary metabolites were analysed using T. quadrivalvis (grader grass), by chemical methods, and the results are given in Table 1.
A variety of plant secondary metabolites have been reported to act as antioxidants and antibacterial agents, and among them phenolic compounds are the major groups [15]. Most important classes of phytochemicals present in the plants are phenolic polyphenols, terpenoids, essential oils, alkaloids, rectins, and polypeptides [16]. Tannins might be monomeric or polymeric and can be separated into strong and hydrolysable tannins. Two of the most important extractable components are waxes and phenolic aldehydes and coumarins, the group of phenolic compounds; it also includes the chemical families of flavonoids and tannins [17]. The present study correlated with the result that the chemical compounds of T. quadrivalvis have significance in all the solvents ( Table 1).
The visible colour change from green to brown within 7 days indicates the formation of AgNPs, with pH 8.0 which was confirmed by UV-Vis spectrophotometer and FT-IR results. After 7 days, there was a significant colour change to dark brown due to increase in the reaction time, which enhances the formation of AgNPs. It is well known that AgNPs exhibit yellowish-brown colour in water due to surface plasmon vibration [18]. Figure 1 shows the absorption peak of the synthesized AgNPs at various time intervals (first to seventh day) through UV spectrophotometer. The peak is centred at 376 nm, which is associated with an absorbance of AgNPs. The intensity of absorption peak at 376 nm increased with increasing period of the aqueous component.  Absorption peaks at 1,300-1,000 cm −1 frequency are related to C-N stretching for amine compounds. Absorption peaks at 1,450-1,375 cm −1 frequency are assigned to CH 3 bend for a methyl group. A frequency with 3,000-2,850 cm −1 is correlated with C-H, alkane stretch vibrations ( Figure 3).

XRD analysis
The XRD analysis was carried out to determine the crystalline nature of AgNPs in the range of 30-70°at 2θ angles. The intensity peaks of AgNP synthesized using quercetin were observed at around 38°, 44°, 64°, and    average particle size of AgNPs under the most favourable conditions for preparation according to Scherrer's equation calculated using the width of the (111) peak is estimated to be 20.3 nm, which was nearly in consonance with the particle size obtained from SEM image and particle size analyser AgNPs. A few passionate unassigned peaks were noticed at 36.52°, 43.17°, 55.64°, 66.41°, 82.65°, and 89.07°. These Bragg peaks might have resulted from some bioorganic compounds/proteins present in the T. quadrivalvis extract [19,20].

SEM analysis
Formation of green AgNPs of T. quadrivalvis and its morphological dimensions were studied using the SEM. The study demonstrated that the average size of the NPs was in the range of 20 nm and the formation of the sphericalshaped AgNPs is shown in Figure 5. The modern highresolution scanning electron microscope can identify the morphology of AgNPs below the level of 20 nm. All these compounds are proven to have excellent antibacterial activity against a wide range of pathogens. Silver has a broad microbial activity and is also used for many years  in the medical field, and has even shown to prevent binding of HIV to host cells [21,22].
The AgNP has a specific surface zone, which induces novel biochemical activity, enzymatic activity, and atomic behaviour compared with large synthetic, biological particles having a chemical composition. SEM analysis was used to confirm the presence of nanoparticles by identifying small particles when electrons pass through the sample. Using different magnification powers in an electron microscope, the shape and size of the nanoparticles could be captured. SEM is a high-resolution microscopic technique that can be used to study nanomaterials at various scales ranging from 0.1 to 5 μm [21][22][23]. In this study, SEM analysis was used to further confirm the presence of nanoparticles at various magnifications. The results showed that 20 nm particles were present in T. quadrivalvis. From the SEM image, the formation of green AgNPs of T. quadrivalvis was confirmed ( Figure 6(a)-(d)).

Characterization of AgNPs from T. quadrivalvis (grader grass) conjugated with macrolide antibiotics
The UV-Vis spectra of both green-synthesized silver-drug nanoconjugates of macrolide antibiotics such as Azi-AgNPs, Ery-AgNPs, ClR-AgNPs showed a characteristic surface plasmon resonance band in the range of 400-460 nm. Atomic force microscopy (AFM) images were confirmed to determine the morphology of these nanoparticles. The representative topographical images of Azi-AgNPs, Ery-AgNPs, and ClR-AgNPs revealed the formation of spherical nanoparticles (Figure 7

Brunauer-Emmett-Teller analysis
The surface of particle size distribution of Azi-AgNPs, Ery-AgNPs, ClR-AgNPs was evaluated by DLS and is shown in Figure 8(a)-(c). The peak with the highest scattering intensity is centred at 2.3 nm with smaller amounts of aggregated particles.

GCMS analysis of T. quadrivalvis extract
GCMS chromatogram of the chloroform extract of T. quadrivalvis showed 51 compounds in the chromatogram, and the compounds were categorized based on the retention time; among them high retention time was found in the test sample: 3-methyloctadecane. 3-Methyloctadecane is an organic compound known as acyclic alkanes (Figure 9). These are acyclic hydrocarbons consisting only of n carbon atoms and m hydrogen atoms [24]. Octadecane has antibacterial and antiviral activity against gram-positive,   gram-negative bacteria, and viruses [8,14]. Moreover, GCMS data could be used to find a bioactive component present in the plant extract.

Antimicrobial activity
Different concentrates from T. quadrivalvis (grader grass) were tried against six respiratory microorganisms, and the outcome is given in Table 2. The largest growth inhibition zone was observed in chloroform extract against all organisms at a concentration of 100 μg·mL −1 . The zone sizes were as follows: E. coli K7 (10 ± 1.5 mm), P. aeruginosa S43 (9 ± 1.1 mm), Streptococcus sp. B1 (9.6 ± 1.6 mm), S. aureus A21 (8.3 ± 1.4 mm), Proteus sp. M 34 (8 ± 2.1 mm), K. pneumonia X9 (18 ± 4.7 mm) ( Table 2). Extracts from the Poaceae family in chloroform have been shown to exhibit broad antimicrobial activity against microorganisms due to the solubility of all chemical compounds from the plants [2,[25][26][27]. It is evident that when concentrations are increased under 100 µg·mL −1 , the results show extremely low activity against all microorganisms.  It is confirmed that as the concentrations are grouped, microbial movement is accompanied by an increase in activity. It was found that 51 bioactive mixtures were found in the GCMS outline, and octadecane compound makes up the significant mixtures and is responsible for causing the movement. The following are the most significant research findings on this plant (T. quadrivalvis).

Antimicrobial activity of green AgNPs
Nanotechnology holds immense potential in the field of biomedicine, particularly in the areas of diagnosis and drug delivery. It permits helpful specialists to be conveyed to designated cells and receptors. Gold, silver, and iron oxide are the most incessant metal transporters for nanoparticle-based drug conveyance frameworks because of their idleness and biocompatibility [28]. Biosynthesis of nanoparticles utilizing plant separates is the most acknowledged practice because of the greatest antibacterial action of nanoparticles and simple decrease of their salts [29,30]. In the current review, AgNPs were combined with T. quadrivalvis (grader grass). The green combination particles were tried against human microorganisms in the convergence of 10, 30, 50, and 70 µg as given in Table 3. Most extreme zone of restraint was tracked down in K. pneumonia X9, 20 ± 1 mm (70 µg focus), and the least zone of hindrance was tracked down in Streptococcus sp. B1, 6 ± 2 mm (70 µg fixation) ( Table 3). Green AgNPs can divert the bacterial cells in this manner causing film separation [25,31].
3.9 Bactericidal effects of greensynthesized AgNPs conjugated with macrolide antibiotics: azithromycin, erythromycin, clarithromycin, and their antimicrobial assay against respiratory pathogen Drug conveyance frameworks in view of nanomaterials can possibly further develop pharmacokinetics and pharmacodynamics [32]. Nanoparticles give an enormous surface region to most extreme medication stacking and high openness for explicit focuses because of their small size. Different medications formed from nanoparticles have of late been made to combat sickness caused by microscopic organisms [28]. The system of AgNPs on microscopic organisms is to cause morphological and primary adjustments in bacterial cells, with the high surface region considering better take-up by microorganisms  4 Transmission electron microscopy micrograph of AgNPs Figure 13(a) and (b) shows a TEM micrograph of AgNPs at 15 and 20 nm. It represents the particle size with a variable shape. In addition, most of them showed a spherical shape in nature [40][41][42]. The majority of the nanoparticles were observed under the TEM micrograph. AgNPs extracted from T. quadrivalvis have been shown to have a strong antimicrobial activity against various respiratory pathogens. The application of AgNPs in conjugation with macrolide antibiotics can lead to improved efficacy against these pathogens and also reduce the risk of antibiotic resistance [43,44].
One novel application of this combination is in the treatment of "respiratory infections" caused by antibiotic-resistant bacteria including, methicillin-resistant S. aureus, and multidrug-resistant Streptococcus pneumoniae [45,46]. The combination of AgNPs and macrolides can enhance the antibacterial activity against these pathogens and provide an effective alternative treatment option [47,48].
Another potential application is in the prevention and treatment of respiratory tract infections in individuals with compromised immune systems, such as those with cystic fibrosis or those undergoing chemotherapy [49,50]. The combination of AgNPs and macrolides can improve the efficacy of the antibiotics and provide a more effective treatment option for these individuals.
Additionally, AgNPs extracted from T. quadrivalvis have shown strong antiviral activity against various respiratory viruses such as Influenza A and human respiratory syncytial  virus. The combination of AgNPs and macrolides can potentially be used in treating respiratory tract infections induced by these viruses, providing a new approach to the treatment of these infections [51].
Overall, the novel applications of AgNPs extracted from T. quadrivalvis in conjugation with macrolide antibiotics have the potential to provide new and effective treatment options for respiratory tract infections caused by both bacteria and viruses.
Thus, the current study demonstrated the formation of macrolide anti-infective agents from synthetic mixtures of T. quadrivalvis using GCMS and a combination of green AgNPs and the formation of macrolide antiinfective agents that have higher efficacy (mixture compounds have higher efficacy when compared to single macrolides) against respiratory microorganisms [52]. In addition, this study discusses a green methodology approach for treating respiratory microorganisms with nanotechnology, which provides a foundation for drug discovery [53].

Conclusions
The current study demonstrated the formation of macrolide anti-infective agents against respiratory microorganisms using the GCMS technique and a synthesized mixture of green AgNPs. Green-synthesized AgNPs are conjugated with macrolide antibiotics. In the green synthesis, AgNPs also showed their antibacterial efficacy against all respiratory pathogens. Moreover, green-synthesized AgNPs conjugated with macrolide antibiotics also significantly reduced the host cell cytotoxicity, but the specific mechanism of action of these nanoparticles is unknown, and future research will focus on that as well as testing their potential in vivo. The present study gives the baseline report for future prospects.