Green biosynthesis of stable silver nanoparticles bound with macaranga indica plant extracts for the purification of drinking water

Plant-assisted green biosynthesis of silver nanoparticles (NPs) has become the most powerful technique to prepare stable NPs. Macaranga indica is a medicinal plant widely available in the western ghats. It has verities of medical properties like antibacterial, antioxidant, antidiabetic, cytotoxicity, and antidysentery and is commonly used in the traditional medicine system. This plant also contains polyphenols, flavonoids, and other plant constituents. Here, in this study, macaranga indicia leaf extract is used as a capping agent for the synthesis of silver NPs by using the microwave irradiation technique. Synthesized NPs are characterized by using Uv-Vis Spectra, XRD, EDX, FESEM, and FTIR. Interpretation of characterization data reveals that synthesized NPs are in a spherical shape, monodispersed, and have particles the size of ~15-20 nm. These bicapped silver NPs have shown potential thermotolerant bacterial inhibition activity and are also efficient in methylene blue dye removal and hence can be used in the purification process of drinking water at its source.


Introduction
The ever-green plant species widely available in western ghats called Macaranga Indica (M.indica) has been incorporated into traditional Indian medicine for many decades for the treatment of anemia, paralysis, tumors, cuts, wounds, colon cancer, and other related disorders (1)(2)(3).The family of this plant species is rich in a range of phenolic compounds, most notably prenylated flavonoids, and stilbenes (4).M. indica is a species of tree that grows to about 25 meters in height and has big leaves.various people have reported the phytochemistry of M. indica various part extraction and isolation of flavonoids, isoflavones, and their prenylated derivatives in the plant (1,5).Although plant extracts are used in traditional Indian medicine, the lack of sustained potency of the extract becomes a major obstacle in drug formulation (6), hence novel methods are essential.Bio-synthesized AgNPs offer a wide range of mutation-resistant antibacterial and antifungal properties and are used as filter beds in water treatment units, ointments for wounds, HIV drugs-prevention and treatment, food packaging, cosmetic products as well as coatings on medical devices (7)(8)(9).Extracts obtained from the various parts of the plants like leaves, bark, seeds, flowers, and fruits have demonstrated various advantages as both reducing and bio-capping agents in the synthesis of NPs (10)(11)(12).Phytochemicals, the bioactive molecules of the plant extract are known for their role in reducing Ag + ions to Ag 0 and producing stable NPs in a green manner and can be used for sustainable environmental applications.Now a days, photocatalysis has been used as an alternative, cost-effective method to eliminate hazardous chemicals and environmentally friendly synthesis (13).The photocatalytic method of retro synthesis and reduction of organic pollutants has attracted a great deal of interest due to its exceptional advantages.Hence, it is most important to choose a suitable photocatalyst or bio-reducing agent to execute the degradation process.The performance of a photo decolorization reaction is affected by the quality and properties of the photocatalyst, which is typically a semiconducting material with the ability to generate electron-hole pairs in the presence of light (14,15).There were no findings in the biosynthesis of AgNPs using the aqueous leaf extract of M. Indica a conventionally used ethnomedicinal plant as described in this report.The aim of this present study was to prepare stable AgNPs by a sustainable approach using the medicinally important M. Indica plant extract and screen for thermotolerant bacterial colony inhibition present in the drinking water at its source.Further, the application of synthesized bio-capped Ag NPs in the removal of Methylene Blue (MB) dye under sunlight absorption is performed.The rate constant in the photocatalytic degradation process with respect to MB dye concentration was also investigated.

Experimental
2.1.Materials M. Indica plant leaves are collected from the village of Siddapur Taluk, Bilgi in Karnataka, India in the Western Ghats and dried at room temperature.Hi-media Laboratory supplied analytical grade silver nitrate (AgNO3) (Mumbai, India) is used for synthesis.The M. indica leaf extract was prepared in distilled water using a microwave oven (LG MJEN326UH, 2.45 GHz) for subsequent AgNP synthesis with monitoring time.

Plant-mediated sustainable synthesis of AgNPs
For extracting the phytochemicals, 10 g of dried, chopped and finely powdered M. Indica leaves were stirred with 250 mL of deionized (sterile) water in a flask.The mixture was then microwave-irradiated for around 300 seconds.In a heated environment, it was filtered using a 0.45-micron filter.After cooling to room temperature (RT), it was used further for the preparation of AgNPs.In this method of synthesis of AgNPs, 50 mL of a 0.001M aqueous solution of AgNO3 was mixed with 30 mL of leaf extract.The mixture was exposed to microwave radiation for different intervals of time.The pale yellow to dark brown color within 300 seconds reveals the formation of AgNPs.The prepared stock was stored for two days for stabilization prior to characterization.

Characterization of filtered solid AgNPs
The Ultraviolet and visible (UV-vis) spectral plot was recorded with a systronics spectrometer (UV-2205, Systronics India).The magnitude and structural geomorphology of AgNPs were exhibited by using scanning electron microscopy (SEM) (JEOL-JSM 6701-F, Japan) driven at an accelerated voltage of 200 kV.The elemental composition was determined using the Energy dispersive X-ray (EDX) analysis.The X-ray diffraction (XRD) study is carried out with an X-ray diffractometer (D8 Focus, Bruker, Germany).To identify the potential biomolecule capping on AgNPs, an Fourier Transform Infra-Red (FTIR -Perkin Emler) spectrum was obtained in the KBr pellet.

Thermotolerant bacterial inhibition Activity AgNPs
The thermotolerant bacterial colony inhibition ability of AgNPs was determined using the agar well diffusion method, which is described below.(16).Test pathogens included E. coli (ATCC 25922), and S. aureus (ATCC 11682).Mueller Hinton Agar and Potato Dextrose Agar were used for antimicrobial assays.A cork borer was used to create 6mm holes in the agar plates.In the holes, 100 µL of AgNPs (50 µg/mL) were added.Ciprofloxacin is used as the standard to check the antibacterial activity of synthesized AgNPs.DMSO is fixed as a control.The bacterial cultures are kept under incubation at 37 °C for 24 hours in an incubator shaker.The zone of inhibition (ZOI) was tabulated with a measuring scale fitted to an inverted microscope after the incubation period.All tests were performed in triplicate and the range is tabulated.
The photocatalytic degradation ability of the plant bio capped AgNPs was studied for the MB color dye exposure to sunlight.5mg synthesized AgNPs were added to 0.001 M, pH 6.5 aqueous solution of MB in distilled water.Then, the reaction was carried outside under the aid of sunlight.The improvement of their catalytic degradation was recorded by measuring the highest absorbance peak of the reaction solvents at intervals of 0,10, 20, 30, 40, 50, 60, and 70 mins after the initiation of the reaction under sunlight.The dye adsorption percentage is calculated using the following formula:  In figure 1B SEM image of as-synthesized AgNPs is presented.The majority of obtained AgNPs are spherical, mono-dispersed particles with a diameter () in the range 15 and 20 nm.The SEM image shows a type of foam on the particles, which is most likely the result of the capping of hydrocarbon structures from the plant extract.A typical XRD obtained for AgNPs is shown in the inset of figure 1B.The peaks obtained in the XRD pattern can be recognized as a face-cantered cubic (fcc) structure of Ag and meets the JCPDS No. 89-3722 (20).The powder XRD pattern exhibits the presence of diffraction peaks corresponding to (111), ( 200), ( 220), (311), and (331) planes.The typical particle size determined with the help of Scherrer calculation at the prominent (111) diffraction peak is 18±2 nm, agreeing with the size of evolved NPs observed in the SEM analysis.The EDX profile (figure 1C) confirms the occurrence of Ag as the main element with 88.18 weight %.A trace amount of C, O, and Cl is also seen in the picture due to the presence of plant extract residue and other organic molecules with AgNPs which mainly act as bio-capping agents.The AgNPs are stabilized by bioencapsulating, and these bio-encapsulating AgNPs, especially in combination with phytochemical plant extracts, have shown improved biological, medicinal and photocatalytic activities (21,22).The potent biomolecules of M. indica leaf extract involved in AgNO3 reduction and AgNPs stabilization is recognized by FTIR spectroscopy.The FTIR spectral plot in figure 1(D) shows the major bands at 3255, 2925, 1562, and 1282 cm −1 .The bands at 3255 cm −1 and 2925 cm −1 related to the O-H stretching of the polyphenols group and the C˗H stretch, respectively.The bands at 1562 cm −1 and 1282 cm −1 may be assigned to the C˗H bending of the aromatic and the C˗N stretching of the amine group or the O-H bending of the phenol moiety respectively.A band appears at 1707 cm −1 corresponding to carbonyl bond formation (23).This may be due to the oxidation process of the O-H group to the C=O group (8).This confirms the reduction of Ag +1 to Ag 0 by polyphenols of plant extract.The formation of the major bands also confirms the bio-capping of plant biomolecules on AgNps.

Bacterial inhibition activity of AgNPs
The bio-encapsulated AgNPs ability to prevent the growth of thermotolerant bacteria present in the water is tested.An invitro antimicrobial test was performed by using the agar well diffusion assay (24) and the results showed high inhibition activity of AgNPs against all tested pathogens.Table 1 shows the results for the mean zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) for all thermotolerant pathogens tested.Dimethyl sulfoxide (DMSO) as a negative control has shown no inhibition.According to the ZOI, gram-negative bacteria show the greatest antibacterial activity.The raw leaf extract of M. indica showed very little inhibitory activity against the pathogens tested.Using broth dilution methods, the MIC for AgNPs is determined to be most effective against E. coli, followed by S. aureus (figure 2A).ZOI and MIC indicate that AgNPs restrict the development of both thermotolerant bacteria.The percentage of colonies formed on agar plates as an indication of AgNP concentration used is shown in figure 2B.In comparison to the control, the number of colonies decreases significantly with the further addition of bicapped AgNPs.S. aureus has a thick peptidoglycan layer in its cell wall, which accounts for its low susceptibility (25).The microbial inhibition activity dispels the notion that cell membrane rupture is caused by electrostatic interactions (32,33).The magnitude and morphology of NPs are considered important parameters for microbial inhibition activity.The bio-encapsulated AgNPs accumulate in different parts of the cell and make the membranes of the thermotolerant bacterial colony less stable.This leads to cell death via the formation of a vacuole and the accumulation of NPs in the cell cytoplasm.Additionally, AgNPs that are 15-20 nm in size or smaller come into contact with cells more frequently than larger NPs, which causes the internalization of NPs and the subsequent release of toxic molecules (28).Therefore, the magnitude of the NPs plays a vital role in their bacterial inhibition activities and hence better inhibition activities arise due to the small size of NPs formed by combining with the M. indica leaf extract.In this study, the extent of MB dye degradation calculated from the absorption spectra was 92.2%.Previous studies have reported that AgNPs prepared by using the morinda tinctoria leaf extract method achieved 95% MB degradation after 72h of sunlight exposure (34).Furthermore, Roy et al. observed that 75% of methyl orange dye was degraded in 480 minutes by AgNPs derived from Solanum tuberosum infusion (35).Liang et al. reported that no significant orange G dye degradation activity was observed using AgNPs prepared from Lemon fruit extract (36).This evidence conclusively shows that the AgNPs synthesized from M. indica extract in the present study exhibit stronger photocatalytic activity than AgNPs synthesized from other sources.Since there are more active sites on the larger surface area of NPs, a wider variety of dye molecules can be degraded by electron transfer.figure 3C shows the possible route of dye degradation in the presence of M indicia leaf extract bonded AgNPs.When irradiated with Sunlight, the electrons in Ag ions become excited and transfer from the valence band (VB) to the conduction band (CB).This forms electron-hole pairs that serve as effective oxidizing and reducing agents.These excited electrons create free radicals such as HO•, O•2 2˗, and HOO• on the catalyst surface the active sites of AgNPs when they combine with the O2 present in the reaction mixture (37).The rate of degradation is significantly influenced by the structure of the dyes and the structure of the polycyclic dyes used in this study is shown in figure 3C.The rate of dye degradation is slowed down by the presence of sulfonyl and nitro groups.Since MB dye does not have a sulfonyl group, it degrades faster.It is imperative that the catalyst is reusable.As a result, AgNPs obtained from M. indica leaf extract were collected after each run and reutilized five times to determine their potency after each path.As shown in figure 3D, the activity of the catalyst slightly decreased after it was successfully used three times.This result demonstrates that AgNPs can be successfully regained and recycled, making them suitable for wastewater purification.

Conclusion
A sustainable green route for the preparation of stable silver nanoparticles is achieved by adopting the leaf extract of M. indica plant and a microwave-promoted rapid reaction technique.The identification and nature of obtained silver nanoparticles are fixed by Uv-Vis spectrophotometer, SEM, EDX, XRD, and FTIR techniques, and found that synthesized nanoparticles are spherical, monodispersed, and of size 15-20 nm.The encapsulating of plant molecules to AgNPs is evident by the FTIR spectra.These bio-encapsulated NPs have shown potent inhibition activity against thermotolerant and colonyforming bacteria.Our previous work with M.indica bark extract AgNPs also showed similar kind of results.The photocatalytic activity of AgNPs with MB dye has shown a potent degradation process and up to 92% dye removal can be achieved at 70 min contact time in sunlight.These properties of M.indica AgNPs can be used for the purification of drinking water at various sources like surface and groundwater.

Declaration of Competing Interest
No known competing Interests.

3 .
Results and Discussion3.1.Green reaction and nature of AgNPsAfter 10˗300 seconds of microwave irradiation, the reaction mixture (50 mL of 1 mM AgNO3 plus 30 mL of leaf extract) changed color from pale yellow to brown in about 70 seconds.With increasing irradiation time, the intensity of the solution increased until it reached saturation in about 300 seconds.The broad peak occurring in the region of 440-450 nm (figure1A) is related to contact time in the Surface Plasmon Resonance (SPR) of AgNPs(17).The inset figure shows the picture of the colour change of the aqueous leaf extract and AgNPs formation.This colour change reveals that M. Indica leaf extract reduces Ag + to Ag 0 .The formation of particles and yield of AgNPs with exposure time is likely the reason for the redshift of SPR from 440 nm to 450 nm.A similar UV-Visible spectrum was observed for AgNPs synthesized from plants Cucumis prophet arum aqueous leaf extract(18) and Cannabis sativa leaf extract(19).

Figure 1 .
Figure 1.(A) UV-Vis spectra of AgNPs formation at various time intervals (0-300 sec): inset shows the reaction mixture with M. indica leaf extract at 10 and 300 seconds, (B) SEM image of AgNPs: inst show the XRD pattern, (C) EDX profile of AgNPs inset is percentage weight and (D) FTIR analysis of AgNPs.In figure1BSEM image of as-synthesized AgNPs is presented.The majority of obtained AgNPs are spherical, mono-dispersed particles with a diameter () in the range 15 and 20 nm.The SEM image shows a type of foam on the particles, which is most likely the result of the capping of hydrocarbon structures from the plant extract.A typical XRD obtained for AgNPs is shown in the inset of figure1B.The peaks obtained in the XRD pattern can be recognized as a face-cantered cubic (fcc) structure of Ag and meets the JCPDS No. 89-3722(20).The powder XRD pattern exhibits the presence of diffraction peaks corresponding to (111), (200), (220), (311), and (331) planes.The typical particle size determined with the help of Scherrer calculation at the prominent (111) diffraction peak is 18±2 nm, agreeing with the size of evolved NPs observed in the SEM analysis.The EDX profile (figure1C) confirms the occurrence of Ag as the main element with 88.18 weight %.A trace amount of C, O, and Cl is also seen in the picture due to the presence of plant extract residue and other organic molecules with AgNPs which mainly act as bio-capping agents.The AgNPs are stabilized by bioencapsulating, and these bio-encapsulating AgNPs, especially in combination with phytochemical plant extracts, have shown improved biological, medicinal and photocatalytic activities(21,22).The potent biomolecules of M. indica leaf extract involved in AgNO3 reduction and AgNPs stabilization is recognized by FTIR spectroscopy.The FTIR spectral plot in figure1(D) shows the major bands at 3255, 2925, 1562, and 1282 cm −1 .The bands at 3255 cm −1 and 2925 cm −1 related to the O-H stretching of the polyphenols group and the C˗H stretch, respectively.The bands at 1562 cm −1 and 1282 cm −1 may be assigned to the C˗H bending of the aromatic and the C˗N stretching of the amine group or the O-H bending of the phenol moiety respectively.A band appears at 1707 cm −1 corresponding to carbonyl bond formation(23).This may be due to the oxidation process of the O-H group to the C=O group(8).This confirms the reduction of Ag +1 to Ag 0 by polyphenols of plant extract.The formation of the major bands also confirms the bio-capping of plant biomolecules on AgNps.3.2.Bacterial inhibition activity of AgNPsThe bio-encapsulated AgNPs ability to prevent the growth of thermotolerant bacteria present in the water is tested.An invitro antimicrobial test was performed by using the agar well diffusion assay(24) and the results showed high inhibition activity of AgNPs against all tested pathogens.Table1shows the results for the mean zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) for all thermotolerant pathogens tested.Dimethyl sulfoxide (DMSO) as a negative control has shown no inhibition.According to the ZOI, gram-negative bacteria show the greatest antibacterial activity.The raw leaf extract of M. indica showed very little inhibitory activity against the pathogens tested.Using broth dilution methods, the MIC for AgNPs is determined to be most effective against E. coli, followed by S. aureus (figure2A).ZOI and MIC indicate that AgNPs restrict the development of both thermotolerant bacteria.The percentage of colonies formed on agar plates as an indication of AgNP concentration used is shown in figure2B.In comparison to the control, the number of colonies decreases significantly with the further addition of bicapped AgNPs.S. aureus has a thick peptidoglycan layer in its cell wall, which accounts for its low susceptibility(25).

Figure 2 .
Figure 2. (A) MIC of Silver NPs against S. aureus, and E. coli and (B) the number of colonies vs concentration of AgNPs (µg/ml).

3. 3 .
Photocatalytic degradation ability of AgNPs Methylene Blue (MB) is a well-known water-soluble dye widely used in industrial entities like textile, paint, paper, and pharmaceuticals.The photodegradation of this dye in the manifestation of green synthesized AgNPs was studied by using a UV-Vis double beam spectrophotometer.The highest absorption peak of MB is measured at 665 nm.The degradation of MB dyes upon solar irradiation catalyzed by AgNPs is reflected by a gradual decrement in the absorption peak of the coloured solutions.From figure 3A the absorption band of MB at 665 nm decreases with time and eventually approaches the baseline, showing the complete degradation of dye promoted by silver NPs.figure 3(B) shows the course of the Ln (A0/At) Vs time for MB.The rate of degradation constant calculated for MB is 30 x 10 −3 /min.The decrease in Ln (A0/At) value with time confirms the reaction follows pseudo unimolecular kinetic reactions.

Figure 3 .
Figure 3. (A) UV-Vis spectra of MB dye degradation by AgNPs, (B) Ln (A0/At) Vs time plot, (C) Possible mechanism of MB dye degradation by AgNPs and (d) MB dye degradation by AgNps upto 5 replicates.Since there are more active sites on the larger surface area of NPs, a wider variety of dye molecules can be degraded by electron transfer.figure3Cshows the possible route of dye degradation in the presence of M indicia leaf extract bonded AgNPs.When irradiated with Sunlight, the electrons in Ag ions become excited and transfer from the valence band (VB) to the conduction band (CB).This forms electron-hole pairs that serve as effective oxidizing and reducing agents.These excited electrons create free radicals such as HO•, O•2 2˗, and HOO• on the catalyst surface the active sites of AgNPs when they combine with the O2 present in the reaction mixture(37).The rate of degradation is significantly influenced by the structure of the dyes and the structure of the polycyclic dyes used in this study is shown in figure3C.The rate of dye degradation is slowed down by the presence of sulfonyl and nitro groups.Since MB dye does not have a sulfonyl group, it degrades faster.It is imperative that the catalyst is reusable.As a result, AgNPs obtained from M. indica leaf extract were collected after each run and reutilized five times to determine their potency after each path.As shown in figure3D, the activity of the catalyst slightly decreased after it was successfully used three times.This result demonstrates that AgNPs can be successfully regained and recycled, making them suitable for wastewater purification.

Table 1
Area of ZOI (mm) and MIC (μg/mL) of AgNPs synthesized from M. indica leaf extract for tested strains.

Table 2
(26)(27)(28)tionship list of the antibacterial activity of AgNPs synthesized using M. indica leaf extract with that of other plant extracts in the previously reported studies.The MIC results of AgNPs synthesized with Banana peel extract are lower than those observed for AgNPs synthesized from M. indica, while the MIC of Solanum tricobatum and Terminalia arjuna bark extract are comparable to our results(26)(27)(28).This demonstrates that AgNPs synthesized from M. indica exhibit superior antibacterial activity towards thermotolerant bacterial colonies present in the drinking water source.

Table 2
Comparison of the antimicrobial activity of prepared AgNPs with various plant parts