1 Introduction

Biogenic method of synthesis of metal nanoparticles are increasingly becoming popular in present day world due to their simplicity, less toxic, effortless and eco-friendly nature. Copper, zinc and silver are mostly used metals for synthesis of nanoparticles because of their biomedical properties [1]. Silver nanoparticles is increasingly becoming popular and faces an annual demand of five hundred tons [2] due to its non-toxic, optical, catalytic, bio-sensing, drug delivery, antioxidant, cytotoxic and antimicrobial activities [3, 4]. Literature study reports several techniques including chemical reduction [5], thermal decomposition [6, 7], photochemical reduction [8], heat evaporation [9] and microwave irradiation [10] for the formation of silver nanoparticles. Most of these techniques are costly, toxic and requires chemical compounds which put harmful effect on living system, imposing an extra demand for finding alternative technique that utilizes nontoxic natural compounds for nanosilver production.

Biogenic method uses microorganism both living and dead [11, 12], fungi [13], leaf extract [14], root extract [15], fruit extract [16, 17], latex [18], enzymes [19,20,21] and many others for the preparation of bio-friendly silver nanoparticles. Plant extract gain extra advantage over microorganism, as isolation and maintenance of microbial culture under aseptic condition was not cost effective [22]. Besides the rate of production of nanoparticles using microorganisms was much slower than plant mediated synthesis [23]. Plant extracts that were reported recently in biosynthesis of silver nanoparticles included Datura stramonium [24], Moringa stenopetala [25], Cymbopogon citratus [26], Cassia roxburghii [27], Bergenia ciliata [28], Cardiospermum halicacabum [29], Carica papaya [30], Eclipta alba [31] and many others.

We have selected leaf extract of Morus alba (Mulberry), plant which bears not only economic importance but also having medicinal importance. Wild and cultivated species of mulberry are distributed throughout India [32], making their easy availability. Global importance of mulberry is the utilization of its leaves for the feeding of monophagous insect Bombyx mori. Mulberry is also used for its anti-diabetic [33], antimicrobial [34], antioxidant [35, 36], neuroprotective [37], anticancerous [38] and hepatoprotective [39] activity. Because of its high medicinal importance, mulberry leaves along with its root and stem are consumed directly as tea in different parts of world [40, 41].

Earlier workers have biosynthesizing silver nanoparticles using dried mulberry leaf extract, which was either sun dried [42] or shade dried [43, 44]. Oxidative change in phytochemical constituents may occur during drying process, which may put significant impact in reduction process during nano formation. To avoid such possibility, we aimed in using aqueous decoction of fresh mulberry leaves through refluxing for nanosilver formation.

Current work is based on the hypothesis that synthesized nanoparticles will bear effective antimicrobial and antioxidant activity. Present approach deals with biosynthesis of silver nanoparticles using mulberry leaf extract that will act as reducing and stabilizing agent. The synthesized nanoparticles will be characterized for determining nature, type, morphology, shape and functional groups involved in nanoparticles formation. The antimicrobial activity of synthesized nanoparticles will be screened by studying the zone of inhibition and MIC of both gram positive and gram negative bacteria and antioxidant scavenging potential will be screened through DPPH, ABTS+, nitric oxide, superoxide and metal chelating activity.

2 Materials and methods

2.1 Preparation of plant extract

Fresh, mature and disease free S1 genotype of mulberry leaves were collected from Matigara Sericulture Complex, Siliguri, West Bengal, India (26°70′40″N and 88°35′37″E). The leaves were surface cleaned with double distilled water several time to remove the debris and organic contaminants and were then air dried for 45 min to remove the water content at room temperature. About 10 g leaves were finely chopped and refluxed with 100 ml double distilled water for 60 min. The yellowish aqueous extract was filtered out with Whatman No. 1 filter paper (GE Healthcare UK Ltd, China make) and then centrifuged at 2000 rpm for 5 min to remove suspended impurities. The supernatant was used for biogenic synthesis of silver nanoparticles.

2.2 Synthesis of silver nanoparticles

For synthesizing silver nanoparticles, 10 ml plant extract was added drop wise to 90 ml aqueous solution of silver nitrate (SIGMA-ALDRICH Batch # 0000003756) with continuous uniform stirring for 10 min using magnetic stirrer (REMI EQUIPMENTS). The reducing and capping agents present in the extract changes the colour of the solution from transparent to reddish or blackish brown, indicating the formation of silver nanoparticles.

2.3 Characterization of silver nanoparticles

2.3.1 UV–visible spectra analysis

Initial characterization of reduction of Ag+ ion was done after 12 h of reaction by diluting the nano solution in 1:4 ratio and by plotting absorption spectra against wavelength range of 300–800 nm using UV–Vis Spectrophotometer (SYSTRONICS-2201).

2.3.2 Fourier transformed infrared spectroscopy (FTIR)

Detection of functional groups for predicting the involvement of organic molecules as reducing and capping agents for reduction of silver ion was done using Fourier Transformed Infrared Spectroscopy (THERMO NICOLET, AVATAR 370), with a wavelength range of 4000–500 cm−1 and a resolution of 4 nm. Dried nanoparticles and plant extract were incorporated directly on potassium bromide crystals to obtain the spectra in transmittance mode.

2.3.3 Scanning electron microscopy (SEM) and field emission scanning electron microscope (FESEM)

SEM and FESEM analysis was conducted for studying the shape and surface morphology of synthesized nanoparticles. SEM analysis was done using JEOL Model JSM-6390LV SEM machine. For analysis, drop of sample was dried on a carbon-coated copper grid and then images were taken at different magnification. FESEM was analyzed with JEOL Model JSM-7600F at an accelerating voltage of 10 kV and at 50,000× magnification.

2.3.4 Energy dispersive X-ray spectroscopy (EDX)

EDX analysis was performed for determining the elements present in the synthesized nanoparticles. EDX analysis was done on dry sample through Oxford-EDX system that uses 80 mm2 SDD detector that detects element under high resolution.

2.3.5 High resolution transmission electron microscopy (HR-TEM)

HR–TEM was analyzed was done using FEI TECNAI G2, F30 by operating at an accelerating voltage of 300 kV. HR–TEM analysis was done for determining the size, shape and morphology of silver nanoparticles. The size distribution range was obtained using Origin b9.5.5.409 software (Origen Lab Corporation, USA) by measuring the size of more than 350 particles. Percent polydispersity of synthesized nanoparticles was determined using the following formula: Polydispersity (%) = (σ/xc) × 100, where σ = standard deviation of particle size distribution and xc = average nanoparticles size.

2.3.6 X-ray diffraction analysis (XRD)

For determining X-ray diffraction pattern, centrifuged and dry crystals of silver nanoparticles were used. XRD analysis was done using BRUKER AXS D8 ADVANCE (BRUKER KAPPA APEX II) machine, operated at 30 mA current and at 40 kV voltage. For generating 2θ data the sample was Cu Kα radiated, operated at a speed of 5°/min. The result obtained was compared with standard JCPDS library for determining the crystalline structure. The average crystalline size has been estimated using Debye–Scherrer’s formula, \({\text{D}} = \left( 0.9\uplambda/\upbeta{\text{Cos}}\uptheta \right)\) where λ is the wavelength of the X-ray source, β is the angular FWHM of the XRD diffraction peak and θ is the Bragg angle. FWHM was calculated from Gaussian function using Origin b9.5.5.409 software. Inter planar spacing (d) was calculated from Bragg’s Law, \(2{\text{d}}{\text{Sin}}\uptheta = {\text{n}}\uplambda\) where n is the order of diffraction pattern. Lattice constant (a0) has been derived from the following formula, a0 = d × √ (h2 + K2 + l2), where d is inter planar spacing and h, k, l are plane direction.

2.3.7 Dynamic light scattering (DLS)

DLS analysis of synthesized nanoparticles was done to correlate relationship between particle size and number of particles. Through DLS study zeta potential and average particle size of synthesized nanoparticles was determined. Measurement was done through DLS analyzer (ZETASIZER NANO ZS90 ZEN3690) where water was used as dispersion medium with dispersion and material refractive index of 1.332 and 1.330 respectively, viscosity of 0.8872 cP, count rate of 343.7 Kcps and temperature of 25 °C.

2.3.8 Antimicrobial activity

Disk diffusion method was followed for screening the antimicrobial activity of synthesized nanoparticles using mulberry leaf extract. Antimicrobial activity was tested at seven different concentrations of silver nanoparticles (25, 50, 100, 200, 300, 400, 500 µg/ml). Gram positive (Bacillus megaterium ATCC 14581, Staphylococcus aureus ATCC 11632, Bacillus subtilis ATCC 11774) and gram negative (Escherichia coli ATCC 11229 and Salmonella typhimurium ATCC 25241) test organisms were grown for 6 h on nutrient broth prior to their application to obtain rapidly growing viable cells. 100 µl test organism from nutrient broth was mixed uniformly with nutrient agar plate and was allowed to solidify. After 30 min, paper disk soaked with appropriate concentration of nanosilver was placed in the nutrient agar plate. The zone of inhibition was calculated in millimetre scale after 24 h of incubation at 37 °C.

Antimicrobial activity of silver nanoparticles was also evaluated in terms of minimum inhibitory concentration (MIC). For estimation, different concentration of silver nanoparticles was added to 50 ml sterilized nutrient broth and to it 0.1 ml actively growing viable bacterial culture was added maintained at 106 CFU/ml. Microbial growth was measured using UV–visible spectrophotometer (SYSTRONICS-2201) at 600 nm after 24 h incubation at 37 °C and 120 rpm.

2.3.9 Antioxidant activity

Antioxidant activity of prepared nanosilver and plant extract was evaluated following standard protocol in terms of ABTS+ [45], DPPH [46], superoxide [47], nitric oxide [48] scavenging and metal chelating [49] activity. Scavenging and chelating activity was measured as percent inhibition using the following equation: Percent inhibition = [(A0 − A1)/A0] 100%, Where A0 is the absorbance of the control and A1 is the absorbance of the sample. Antioxidant activity was expressed as concentration where 50% reduction in free radical takes place referred to as IC50 value.

3 Result and discussion

3.1 UV–visible spectra analysis

Biogenic synthesis of silver nanoparticles was confirmed by colour change of silver nitrate solution from transparent to yellowish and finally to reddish or blackish brown (Fig. 1). The dielectric medium and the organic constituents of the plant extract are few of many factors responsible for change in colour during nano formation [50]. Further validation of nanoparticles formation was done using UV–Vis spectrophotometer. Sastry et al. [51] reported that appearance of surface plasmon resonance (SPR) spectra in the wavelength range of 400–500 nm confirms the formation of silver nano particles. Figure 2 shows SPR spectra of silver nanoparticles formed using different concentration (2.5, 5, 10, 15, 20 ml) of plant extract keeping the concentration of silver nitrate constant (10−3 M). The characteristic UV–visible spectra shows SPR band of formed nanosilver at ~ 429 nm when synthesized using 2.5 ml plant extract. The obtained peak gets shifted towards red region when nano was prepared with 5 ml (~ 432 nm) and 10 ml (~ 432 nm) plant extract. However decrease in SPR spectra (blue shift) was noticed with increase in concentration of extract to 15 ml (~ 426 nm) and 20 ml (~ 423 nm). The red or blue shift of SPR spectra mainly depends on size, shape and nature of organic constituents present in surrounding medium [52]. The decrease in SPR spectra at high concentration of plant extract takes place because excess biomolecules beyond a certain limit ceases nano formation [53].

Fig. 1
figure 1

Biogenic synthesis of silver nanoparticles mediated by mulberry leaf extract at different concentration of silver nitrate

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Fig. 2
figure 2

UV–visible spectra of silver nanoparticles synthesized at different concentration of leaf extract

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Nano synthesis was also monitored at different concentration of silver nitrate (10−1, 10−2, 10−3, 10−4, 10−5 M) keeping the concentration of plant extract constant in the ratio of 1: 9. The UV–visible spectrum (Fig. 3) shows SPR bands that range from 430 to 450 nm which confirms nano formation. It was observed that 10−4 M and 10−5 M concentration of silver nitrate was insufficient for nano formation, while at high concentration (10−1 M), formed nanoparticles in solution became hazy due to the reaction between excess concentration of silver nitrate with biomolecules, resulting in low intensity and broad SPR spectra. The broad and low intensity SPR band at high concentration appeared because of sedimentation of particle with time. Similar result was also reported by Balavijayalakshmi and Ramalakshmi [54] using Carica papaya peel. Nanosilver formed with 10−2 M and 10−3 M silver nitrate showed maximum wavelength peak at ~ 450 nm and ~ 435 nm respectively, indicating red shift with increase in concentration. SPR peak at high wavelength indicates large particle size [14]. Kaya et al. [55] reported that small size nanoparticles are biologically more active than large size nanoparticles. As nanosilver produced from 10−3 M silver nitrate shows SPR peak at lower wavelength than 10−2 M, so its particle size will be smaller than that produced by 10−2 M silver nitrate and thus is biologically more active.

Fig. 3
figure 3

UV–visible spectra of silver nanoparticles synthesized at different concentration of silver nitrate

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3.2 Fourier transformed infrared (FTIR) spectroscopy

FTIR analysis was conducted to determine possible involvement of functional groups participating in reduction and stabilization of silver nanoparticles. FTIR spectra of dried aqueous plant extract showed absorption band at 3425.1, 1627.7, 1386.6, 1066.4, 1020.2, 761.7, 572.7 and 518.7 cm−1 (Fig. 4a). After reduction, certain spectral peaks showed slight shift in wave number such as 3430.9, 1033.7 and 796.4 cm−1; bands at 1627.7, 1386.6, 572.7 and 518.7 cm−1 appeared at exact location, while two extra band appeared at 2919.8 and 2854.2 cm−1 (Fig. 4b). The spectral similarity between plant extract and nano silver with minor deviation due to reduction process [56] strongly supports the involvement of different components of plant extract in bioreduction of metallic salts into nanoparticles. Ganesh Babu and Gunasekaran [57] reported that interaction between metal salts and biomolecules for the production of nanoparticles takes place through the involvement of functional groups. The bands at 3425.1 cm−1 shifted to 3430.9 cm−1 corresponds to N–H vibration mode which was overlapped with –OH vibration stretching of alcoholic and phenolic compounds [22, 58]. The peaks at 2919.8 cm−1 and 2854.2 cm−1 represents vibrations of –CH2 and –CH3 functional groups. These peaks are not detected in plant extract probably due to interference of –OH vibration stretching. Strong intense peak at 1627.7 cm−1 corresponds vibration of primary and secondary amines [59, 60] and 1386.6 cm−1 corresponds to C–N vibration stretch, probably representing amide I band of proteins found in leaf extracts [61]. The intense band at 1066.4, 1020.2 cm−1 of plant extract and 1033.7 cm−1 of nano silver represents strong C–O– and C–OH stretching vibration of carboxylic acid, alcohol, ester and ether bond of protein and carbohydrate present in the extract [62, 63]. Peak at 761.7 cm−1 of extract was shifted towards higher wave number at 796.4 cm−1 in prepared nanosilver indicates N–H vibration of primary aliphatic amines. Absorption band at 572.7 and 518.7 cm−1 indicates C–Cl stretching vibration and C–C skeleton vibration of branch alkenes respectively. Butt et al. [64] reported the presence of protein, carbohydrate, glycoprotein, phenols, flavonoids, aminoacids, carotene and anthocyanins in mulberry leaf extract. Liang et al. [65] through spectroscopic analysis detected the presence of glucose and sucrose as carbohydrate; alanine, asparagines, GABA and proline as amino acid; acetic acid and succinic acid as a mono and di-carboxylic acid respectively; trigonelline as alkaloid in mulberry leaf extract. Hunyadi et al. [66] reported the presence of secondary metabolites like chlorogenic acid, rutin, isoquercitrin in mulberry leaf extract. Thus the functional groups detected in IR spectra, defines the presence of carbohydrate, proteins and different secondary metabolites in the plant extract that are involved in reduction of silver ion and also acts as stabilizing agent.

Fig. 4
figure 4

FT-IR spectra of a mulberry leaf extract and b biosynthesized silver nanoparticles

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3.3 Scanning electron microscopy (SEM) and Field emission scanning electron microscope (FESEM)

SEM micrograph of biosynthesized silver nanoparticles was given in Fig. 5a. From SEM imaging it was observed that most of the silver nano particles were spherical while some are irregular in shape. The micrograph obtained in FESEM (Fig. 5b) also shows spherical nanoparticles, supporting the result obtained by SEM. Uniform alignment of silver nanoparticles was observed in FESEM with average particle size of 16.33 ± 5.14 nm. The particle size distribution ranges from 12 to 40 nm, some particles of size greater than 50 nm were also observed but are probably due to the overlapping of one particle with another. In some places particles were agglomerated due to cross linking [52] or may be due to evaporation of solvent during preparation of sample [67].

Fig. 5
figure 5

a SEM and b FESEM micrograph of biosynthesized silver nanoparticles

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3.4 Energy dispersive X-ray spectroscopy (EDX)

For identifying the elements involved in synthesis of silver nanoparticles, energy dispersive spectrum analysis was conducted. EDX analysis gives both qualitative and quantitative information regarding the participation of element in bioreduction. The elemental profile shows the presence of C, O, S, Cl and Ag (Fig. 6a), with strong intensity peak at 3 keV representing Ag. Due to surface plasmon resonance, silver shows intense peak at 3 keV confirming the formation of silver nanoparticles [68, 69]. The quantitative profile of elements in terms of percent weight indicated that silver accounting maximum portion ~ 70% followed by carbon and oxygen (Fig. 6b). Presence of carbon and oxygen indicates the presence of alkyl chain as stabilizing agent during the bioreduction of metallic silver [70], supporting the result obtained through FTIR analysis.

Fig. 6
figure 6

a EDX spectra and b elemental profile of biosynthesized nanoparticles

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3.5 High resolution transmission electron microscopy (HR-TEM)

From HR-TEM images it was observed that formed silver nanoparticles were spherical in shape and they vary in their size distribution. HR-TEM image of prepared nanosilver at 200 nm scale was represented in Fig. 7a. From HR-TEM study average particle size was estimated to be 14.95 ± 2.29 nm and size distribution ranges between 12 and 38 nm (Fig. 7b). The variation in size distribution was mainly due to clustering of nanoparticles at some places [67]. TEM analysis reveals that the periphery of the nanoparticles is thinner than centre, indicating the involvement of protein molecules as capping agent [71]. The polydispersity was found to be 11.35% indicating that most of the particles remain in monodispersed phase; similar finding had been reported by Ibharim [72] and Banala et al. [30] using banana peel and papaya leaf extract respectively. Application of nanoparticles largely depends on size, shape and polydispersity index of particles [73]. Agnihotri et al. [74] reported that bioactivity of silver nanoparticles, mainly antimicrobial activity is inversely proportional to the size of the nanoparticles. Present study represents smaller size nanoparticles, supporting their bioactive nature.

Fig. 7
figure 7

a HR-TEM micrograph of biosynthesized silver nanoparticles and b particle size distribution

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3.6 X-ray diffraction analysis (XRD)

For confirming the crystalline nature of biologically synthesized nanoparticles, XRD pattern of dried nanosilver was studied and was represented in Fig. 8. Four prominent diffraction peak were obtained at 38.14°, 44.26°, 64.46°, 77.41° corresponding to (hkl) values of (111), (200), (220), (311) Bragg’s reflections plane of face centered cubic silver. The obtained data was matched with standard JCPDS library file no: 04-0783 which also confirms face centered cubic structure of biosynthesized silver nanoparticles. The intensity of peak at (200), (220) and (311) are weak and are broad, while (111) represents sharp and intense peak indicating that nanocrystals are (111) oriented. By determining Debye–Scherrer’s equation at (111) Bragg’s reflection, the average crystalline size was found to be 10.65 nm. Similar XRD orientation was earlier reported by Awwad et al. [3] and Premasudha et al. [31] while synthesizing silver nanoparticles using Carob and Eclipta leaf extract respectively. Besides normal peaks of silver, three extra peaks at 27.86°, 32.26° and 46.11° were observed and these peaks represents the organic constituents of extracts that are responsible for reduction of silver ion [75].

Fig. 8
figure 8

XRD pattern of silver nanoparticles synthesized from mulberry leaf extract

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The crystalline nature of synthesized nanoparticles was further evaluated through selected area electron diffraction (SAED) pattern (Fig. 9). The bright diffraction spots corresponds to (111), (200), (220) and (311) Bragg reflection planes [76]. SEAD pattern reflects that the crystals are mostly oriented on (111) plane and due to which sharp and intense peak was generated at (111) XRD pattern.

Fig. 9
figure 9

SAED image of biosynthesized silver nanoparticles

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The FWHM value, crystalline size (D), dislocation density (δ), inter planar spacing (d), lattice constant (a0) and cell volume against Bragg’s reflections at (111), (200), (220) and (311) are represented at Table 1. The obtained lattice constant value of most intense peak (111) was 4.084 Å which almost matches exactly with the reported standard value of silver which is 4.086 Å (JCPDS file no: 04-0783). Similar finding on biogenic synthesis of silver nanoparticles was earlier reported by Anandalakshmi et al. [77] and Mehta et al. [78].

Table 1 Variation in crystalline size, dislocation density, inter planar spacing, lattice constant and cell volume of synthesized nanoparticles
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3.7 Dynamic light scattering

DLS analysis reveals that all the particles in the dispersion remain in nano-size with average zeta diameter of 29.68 nm and polydispersity index (PDI) of 0.441 (Fig. 10). The particle size obtained from DLS analysis is greater than that obtained through TEM and XRD analysis, this may be due to the fact that DLS analysis takes into consideration associated capping and reducing agents during size measurement [79]. Besides size for biological activity, stability of nanoparticles plays an important role [80]. Nano stability is measured in terms of zeta potential which measures the surface charge of nanoparticles. Nanoparticles bearing greater positive or negative zeta potential will repulse each other, which will prevent nano agglomeration and there by increases stability [81]. Particles bearing zeta potential greater than + 30 mV or less than − 30 mV are considered to remain stable without agglomeration for longer duration [82]. The zeta potential value of biosynthesized silver nanoparticles was + 37.4 mV, indicating that the particles are present in highly stable state. Positive value of zeta potential was due to development of electrostatic force of attraction between positively charged capping agents with the nanoparticles [83].

Fig. 10
figure 10

DLS size distribution pattern and zeta potential of biosynthesized silver nanoparticles using mulberry leaf extract

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3.8 Antimicrobial activity

Biosynthesized silver nanoparticles using mulberry leaf extract showed potent antimicrobial activity against both gram positive and gram negative bacteria as evident by measuring the diameter of inhibition zone (Fig. 11). The data tabulated in Table 2 indicates that at maximum concentration (500 µg/ml) silver nanoparticles showed highest activity against gram positive bacteria, Bacillus megaterium (22.03 ± 1.06 mm) while at lowest concentration (25 µg/ml) best activity was obtained against gram negative bacteria, S. typhimurium (10.51 ± 1.17 mm) than other tested microorganisms. This result was possibly due to the fact that metals at higher concentration can easily bind to the surface of gram positive bacteria [84], while at lower concentration thick peptidoglycan layer, consisting of linear polysaccharide chains cross linked by short peptides makes the cell wall of gram positive bacteria a rigid structure which creates difficulty for silver nanoparticles to penetrate the bacterial cell wall [72]. In Gram negative bacteria the cell wall possesses thinner peptidoglycan layer, due to which silver nanoparticles could easily release silver ion causing damage to membrane leading to bactericidal activity [85]. It has been reported earlier that silver nanoparticles prepared with extract of Aloe vera [86], Eriobotrya japonica [87], Sida acuta [88], Capparis spinosa [89], Lycopersicon esculentum [90], Artocarpus heterophyllus [67] showed strong antimicrobial activity against different strains of microorganisms.

Fig. 11
figure 11

Antimicrobial activity of silver nanoparticles at seven different concentrations (I—500, II—400, III—300, IV—200, V—100, VI—50, VII—25 µg/ml) against a, b Bacillus megaterium, c, d Staphylococcus aureus, e, f Bacillus subtilis, g, h Escherichia coli, i, j Salmonella typhimurium

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Table 2 Antimicrobial activity of silver nanoparticles against tested microorganisms
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On assessing MIC value of silver nanoparticles against different tested microorganisms (Fig. 12) it was found that silver nanoparticles showed highest bactericidal activity against gram negative bacteria, S. typhimurium followed by Escherichia coli with MIC value of 40 µg/ml and 60 µg/ml respectively. The MIC value of gram positive bacteria was found to be on higher site with maximum value of 160 µg/ml in Bacillus megaterium. MIC value against gram negative bacteria appeared at low nanosilver concentration probably because the thin peptidoglycan layer of gram negative bacteria easily permits nanoparticles to penetrate the cell wall [91]. Silver nanoparticles by releasing silver cations inhibit bacterial growth by damaging cellular organization [92]. Stoimenov et al. [93] reported that electrostatic force of attraction between positively charged nanoparticles and negatively charged microbial membrane was the driving force for bactericidal activity. Silver nanoparticles by interacting with negatively charged phosphorous and sulphur containing cellular constituents like DNA and proteins inhibit microbial growth [94, 95]. Although in recent years different mode of action of silver nanoparticles has been demonstrated by different workers but exact mode of action was not clear and requires further analysis.

Fig. 12
figure 12

Minimum inhibitory concentration of silver nanoparticles against tested microorganisms

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3.9 Antioxidant activity

Biosynthesized silver nanoparticles exhibit significant dose dependent scavenging activity against all the free radicals as shown in Fig. 13. The functional groups of leaf extract that are involved in reduction of silver ion for nanosilver formation are mainly responsible for its antioxidant activity [96].

Fig. 13
figure 13

DPPH, ABTS, nitric oxide, super oxide and metal chelating scavenging activity of biosynthesized silver nanoparticles in comparison with plant extract and respective standards (results are expressed as mean ± STDEV of triplicate determinations. Values with different letters (a, b, c, etc.) differ significantly (p ≤ 0.05) by Duncan’s multiple range test)

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The lipophilic radical DPPH was considered as model test for determining the free radical scavenging activity of synthesized nanoparticles and natural compounds because of its high stability [97]. In its radical form, DPPH shows maximum absorbance at 517 nm which gradually decreases by reduction caused by antioxidants [98]. In present study DPPH scavenging activity increases with increase in concentration of synthesized nanoparticles with maximum activity of 47.81% at highest concentration (100 µg/ml) which is ~ 56% of the activity showed by standard ascorbic acid at the same concentration, while plant extract showed considerably less scavenging activity of 36.22%. Our finding in the present study was supported by earlier workers who also observed almost similar outcome while working with silver nanoparticles prepared using extract of Iresine herbstii [99], Alpinia katsumadai [100].

ABTS scavenging activity of silver nanoparticles was evaluated using BTH as standard. Oxidation of ABTS with potassium persulfate generates ABTS+ cation, a blue chromophore that causes oxidative damage [27]. ABTS+ scavenging activity is mostly used to access the potential of hydrogen donors and antioxidant agents present in the biological samples [26]. Silver nanoparticles displayed maximum ABTS+ scavenging activity of 95.08% almost equivalent to that exhibited by BTH standard 95.51% at 100 µg/ml, representing strong activity. Strong ABTS+ scavenging activity of green synthesized silver nanoparticles was also reported by Shanmugam et al. [101].

Silver nanoparticles showed 64.04% nitric oxide scavenging activity at 100 µg/ml in comparison with 88.62% and 45.72% in gallic acid standard and plant extract respectively. Silver nanoparticles utilize electronegative property of nitric oxide radical for reducing by donating electron [102]. Nitric oxide plays vital role in bio-regulation, but excess production or accumulation of nitric oxide may cause several disorders [96]. Silver nanoparticles through its scavenging potential nullify the harmful effect of nitric oxide.

Superoxide scavenging activity was measured at 560 nm and reduction in absorption value indicates consumption of superoxide ions by antioxidants. Silver nanoparticles at 100 µg/ml concentration exhibit significant superoxide scavenging activity of 81.92% in comparison with 85.35% in tocopherol standard. Superoxide is a weak anionic radical but bears the capacity to generate two harmful radicals, hydroxyl radical and singlet oxygen that develops oxidative stress [103]. Inside living system superoxide directly reacts and damages DNA and protein [104], besides this hydroxyl radical generated by superoxide causes cellular damage by reacting with polyunsaturated fatty acid associated with phospholipids [105]. In present study silver nanoparticles showed high superoxide scavenging activity which was supported by Reddy et al. [104] showing 60% superoxide scavenging with silver nanoparticles prepared using fruit extract of Piper longum.

Metal chelating is the ability of the antioxidant to destabilize the formation of Ferrozine-Fe2+ complex [106]. Metal chelating activity was estimated at 562 nm and decrease in absorption in respect to control indicates the ability of antioxidant to bind with iron. Nanosilver at 100 µg/ml concentration exhibits 84.24% metal chelation, while at same concentration plant extract and ascorbic acid standard showed 68.62% and 90.81% scavenging activity respectively, indicating high metal chelation potential of biosynthesized nano particles with respect to standard.

IC50 value of synthesized silver nano particles, plant extract and respective standards against all the studied antioxidants are enlisted in Table 3. It was observed that silver nanoparticles showed maximum activity against ABTS+ cation radical with IC50 of 25.929 µg/ml, while least activity was against DPPH scavenging activity having IC50 of 97.27 µg/ml. In comparison with standard, it can be stated that IC50 of synthesized nanoparticles increased significantly with respect to plant extract from which it was prepared.

Table 3 IC50 value of silver nanoparticles, plant extract with respect to standards
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4 Conclusion

Mulberry leaves as an agricultural product in the field of sericulture has been successfully utilized for quick, simple, stable, eco-friendly and cost-effective production of silver nanoparticles using biogenic process. UV–visible spectral analysis reveals that the optimum concentration for nanosilver formation is 10−3 M silver nitrate and 5 ml plant extract. The synthesized nanoparticles were crystalline and spherical in nature with average particle size ranges from 10 to 16 nm as revealed through XRD, SEM and TEM analysis. FTIR analysis showed that the stability of silver nanoparticles was mainly due to the presence of proteins, carbohydrate and different secondary metabolites that acts as reducing and capping agents. Synthesized nanoparticles showed high potential to protect against damage caused by free radicals. The antimicrobial screening demonstrates that biosynthesized silver nanoparticles exhibit strong antimicrobial activity against both gram positive and gram negative microorganisms. From our study it may be recommended that synthesized silver nanoparticles can be explored as an alternative option in the prevention of diseases related with generation of free radicals and also in reducing microbial count in biological systems.