Study of Antibacterial Properties of Ziziphus Mauritiana Based Green Synthesized Silver Nanoparticles Against Various Bacterial Strains

sustainability

Article Study of Antibacterial Properties of Ziziphus mauritiana based Green Synthesized Silver Nanoparticles against Various Bacterial Strains

M. Asimuddin 1, Mohammed Raﬁ Shaik 2 , Neeshat Fathima 1, M. Shaistha Afreen 1, Syed Farooq Adil 2 , Mohammed Raﬁq H. Siddiqui 2 , Kaiser Jamil 1,* and Mujeeb Khan 2,*

1 Center for Biotechnology and Bioinformatics, School of Life Sciences, Jawaharlal Nehru Institute of Advanced Studies (JNIAS), Hyderabad 500003, Telangana, India; [email protected] (M.A.); [email protected] (N.F.); [email protected] (M.S.A.) 2 Department of Chemistry, College of Science, King Saud University, P.O. 2455, Riyadh 11451, Saudi Arabia; [email protected] (M.R.S.); [email protected] (S.F.A.); raﬁ[email protected] (M.R.H.S.) * Correspondence: [email protected] (K.J.); [email protected] (M.K.); Tel.: +966-11-4670439 (M.K.)

Received: 27 January 2020; Accepted: 14 February 2020; Published: 17 February 2020

Abstract: Due to their low cost and environmentally friendly nature, plant extracts based methods have gained significant popularity among researchers for the synthesis of metallic nanoparticles. Herein, green synthesis of silver nanoparticles was performed using the aqueous solution of Ziziphus mauritiana leaves extract (ZM-LE) as a bio-reducing agent. The as-obtained silver nanoparticles were characterized by using UV-Vis spectroscopy, XRD (X-ray diffraction), TEM (transmission electron microscopy), and FT-IR (Fourier-transform infrared spectroscopy). In addition, the effects of the concentrations of the leaves extract, silver nitrate, and the temperature on the preparation of nanoparticles were also investigated. In order to determine the nature of secondary metabolites present in leaves extract, a preliminary investigation of phytoconstituents was carried out using different methods including Folin-Ciocalteu and AlCl3 methods. The results have indicated the presence of a considerable amount of phenolic and flavonoid contents in the leaves extract, which are believed to be responsible for the reduction of silver ions and stabilization of resulting nanoparticles. Indeed, the FT-IR spectrum of silver nanoparticles also confirmed the presence of residual phytomolecules of leaves extract as stabilizing ligands on the surface of nanoparticles. The antibacterial properties of as-obtained silver nanoparticles were tested against various bacterial strains including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis. The nanoparticles strongly inhibited the growth of S. aureus with a minimum inhibitory concentration (MIC) of 2.5 µg/ml and moderately inhibited the growth of E. coli with a MIC of 5 µg/ml.

Keywords: Ziziphus mauritiana; green synthesis; plant extracts; silver NPs; biological activity

1. Introduction Synthesis of metallic nanoparticles (NPs) is an area of active research as a result of their advance and fascinating accomplishments in several ﬁelds, including biomedical and engineering [1,2]. So far, due to their exciting properties, metal NPs have been successfully applied in drug delivery, imaging, bio-sensing, catalysis, etc. [3] Among various metallic NPs, silver (Ag) NPs have been widely applied for various biomedical purposes due to their remarkable biological properties [4–6]. For instance, the exceptional antimicrobial properties of Ag NPs have been exploited to develop a number of antimicrobial medical products, including wound dressings, surgical instruments, and contraceptive devices, etc. [7–9]. In many cases, the size and shape of NPs plays a crucial role in enhancing the

Sustainability 2020, 12, 1484; doi:10.3390/su12041484 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 1484 2 of 14 properties of Ag NPs. Generally, homogeneously dispersed, smaller size, and spherical-shaped Ag NPs are more effective in biological applications, compared to other morphologies of Ag NPs [10]. Therefore, considering their vast biological applications, economical accessibility, and biocompatibility of Ag NPs is utmost important [11]. However, majority of the physical and/or chemical methods which are commonly applied for the preparation of silver NPs either involve costly apparatus or hazardous chemicals and harsh conditions [12]. Therefore, alternative green methods involving biocompatible materials under facile conditions are more suitable for the synthesis of Ag NPs targeted for biological applications [13]. In this regards, plant extracts (PE) based synthesis of Ag NPs have gained respectable attention, which contain biologically active phytomolecules that act as bothSustainability reducing 2019 and, 11 functionalizing, x FOR PEER REVIEW agents [14]. Moreover, PEs are cheaply available, low cost,2 of 15 and are comparatively easy to apply for the eco-friendly preparation of nanomaterials [15]. contraceptive devices, etc. [7–9]. In many cases, the size and shape of NPs plays a crucial role in To date, a variety of PEs have been successfully applied for the preparation of different types of enhancing the properties of Ag NPs. Generally, homogeneously dispersed, smaller size, and metallicspherical nanomaterials-shaped Ag including NPs are moreAg NPs. effective For instance, in biological spherical applications, shaped comparedpolydispersed to other Ag NPs of differentmorphologies sizes ranging of Ag NPs from [10] 5. to 100 nm, were prepared using aqueous coffee and tea leaf extracts, whereasTherefore, in other studies considering fruit extract their vast of Terminalia biological chebula applications,, and stem economical bark of Callicarpa accessibility main-gayi, and were appliedbiocompatibility to obtain biocompatible of Ag NPs is Ag utmost NPs important [16,17]. Similarly, [11]. However, in our majority previous of study, the physical we have and/or successfully usedchemicalPulicaria methods glutinosa which, Origanum are commonly vulgare, and applied other for PE the for preparation the preparation of silver of diNPsfferent either sizes involve and shapes of AgNPscostly apparatus[18,19]. In or the hazardous present chemicalsstudy, we and have harsh employed conditionsZiziphus [12]. Therefore, mauritiana alternativleaf extracte green (ZM-LE), methods involving biocompatible materials under facile conditions are more suitable for the which is commonly referred as Jujube, for the synthesis of silver nanoparticles (ZM-AgNPs). Ziziphus synthesis of Ag NPs targeted for biological applications [13]. In this regards, plant extracts (PE) belongsbased to synthesisthe family of of AgRhamnaceae NPs have, gained and has respectable long been attent usedion in, traditional which contain medicines biologically due to active its nutritive andphytomolecules remarkable biological that act as properties both reducing [20]. and There functionalizing are more than agents 40 [14] diff. Moreover,erent species PEs ofareZiziphus, cheaply which are spreadavailable, throughout low cost, theand world are comparatively and usually easyrequire to applydry conditions for the eco to-friendly grow [21 preparation]. Its plant of extract is richnanomaterials in phytomolecules [15]. such as saponins, triterpenes, flavonoids, and alkaloids, along with proteins and a varietyTo date of, vitamins,a variety ofdue PEs tohave which been itsuccessfully exhibits excellent applied for antioxidant, the preparation antimicrobial, of different types antitumor, of and anticancermetallic activities nanomaterials [22,23 including]. Ag NPs. For instance, spherical shaped polydispersed Ag NPs of different sizes ranging from 5 to 100 nm, were prepared using aqueous coffee and tea leaf extracts, Interestingly, Z. mauritiana has been earlier applied for the preparation of other nanomaterials such whereas in other studies fruit extract of Terminalia chebula, and stem bark of Callicarpa maingayi were as zinc oxide and gold NPs [24,25]. Furthermore, very recently ZM-LE has also been successfully used applied to obtain biocompatible Ag NPs [16,17]. Similarly, in our previous study, we have to obtainsuccessfully AgNPs used [26 Pulicari]. However,a glutinosa the, NPsOriganum from vulgare this method, and other are PE prepared for the preparation applying aof photo-reduction different methodsizes usingand shapes sun lightof AgNPs as reaction [18,19]. In medium, the present which study, required we have employed long reaction Ziziphus time mauritiana of more leaf than two days.extract Moreover, (ZM-LE), the which AgNPs is commonly obtained referred from this as Jujube, method for are the irregularly synthesis of shaped silver nanoparticles and large in size. Therefore,(ZM-AgNPs). herein, Ziziphus we have belongs applied to the ZM-LE family for of theRhamnaceae, aqueous and synthesis has long AgNPs been used via in solution traditional reduction of Agmedicines ion in a dueshort to time its nutritive period and (cf. remarkableFigure1). Detail biological investigation properties [20] of the. There e ffect are of more time, than temperature, 40 anddifferent concentration species of of leaves Ziziphus extract, which on are the sprea propertiesd throughout of resultant the world NPs and is performed. usually require The dry NPs were conditions to grow [21]. Its plant extract is rich in phytomolecules such as saponins, triterpenes, identified using different microscopic and spectroscopic techniques. Furthermore, the antibacterial flavonoids, and alkaloids, along with proteins and a variety of vitamins, due to which it exhibits activityexcellent of phytomolecules antioxidant, antimicrobial, functionalized antitumor ZM-AgNPs, and anticancer were testedactivities against [22,23] various. bacterial strains.

FigureFigure 1. 1.Graphical Graphical representationrepresentation of ofgreen green synthesized synthesized silver silvernanoparticles nanoparticles (ZM-AgNPs) (ZM-AgNPs) using Z. using Z. mauritianamauritiana leavesleaves extract extract (ZM (ZM-LE).-LE).

Interestingly, Z. mauritiana has been earlier applied for the preparation of other nanomaterials such as zinc oxide and gold NPs [24,25]. Furthermore, very recently ZM-LE has also been successfully used to obtain AgNPs [26]. However, the NPs from this method are prepared applying a photo-reduction method using sun light as reaction medium, which required long reaction time of more than two days. Moreover, the AgNPs obtained from this method are irregularly shaped and large in size. Therefore, herein, we have applied ZM-LE for the aqueous synthesis AgNPs via solution reduction of Ag ion in a short time period (cf. Figure 1). Detail investigation of the effect of time, temperature, and concentration of leaves extract on the properties of resultant NPs is performed. The NPs were identified using different microscopic and spectroscopic techniques. Furthermore, the antibacterial activity of phytomolecules functionalized ZM-AgNPs were tested against various bacterial strains.

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2. Experimental

2.1. Materials

Silver nitrate (AgNO3) and all the reagents used for phytochemical screening tests were obtained from Sigma Aldrich. Mueller–Hinton agar (MHA) was purchased from Hi-Media Laboratories, Mumbai, India. Plastic wares were obtained from Tarsons Products Pvt. Ltd., India. All solutions were prepared in sterile Milli-Q water. Bacterial strains E. coli (MTCC, No. 1722), S. aureus (MTCC, No. 96), P. aeruginosa (MTCC No. 424), and B. subtilus (MTCC No. 441) were purchased from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India.

2.2. Collection and Preparation of Z. mauritiana Leaves Extract Fresh leaves of Z. mauritiana (Jujube) are obtained from the JNIAS campus, Hyderabad, Telangana, India. The leaves were washed twice with water and dried for three days before grinding into powder in an electric blender. The aqueous extract of leaves was prepared by mixing 10 g of leaves powder in 100 mL of Milli-Q (100 mg/mL) in a 300 mL conical ﬂask. The mixture was heated to ~100 ◦C for one hour while stirring. Thereafter, the mixture was allowed to cool down to room temperature. The leaves extract is isolated by centrifugation of resultant mixture at 7000 rpm for 20 mins. The residual powder was collected by simple decantation and stored in a refrigerator at 4 ◦C for further use. Hereafter, the resultant powder is referred to as leaves extract.

2.3. Leaves Extract Based Synthesis of Silver (Ag) Nanoparticles

At this stage, 10 mL of aqueous solution of ZM-LE (100 mg/mL) was added to 1mM of AgNO3 solution in 90 mL of water. The solution was heated to 90 ◦C for 30 minutes while stirring. Thereafter, the resulting mixture was incubated for 15 min at 85 ◦C. After incubation, the solution was centrifuged at 12000 rpm for 4 minutes, and the obtained precipitate mass i.e., Ag NPs, was washed twice with double distilled water and centrifuged at 12,000 rpm for 3 minutes. The mass was collected and left to dry in a hot air oven at 30–42 ◦C. After complete drying, the Ag NPs obtained were scraped and stored. In order to optimize the reaction condition for the synthesis of Ag NPs, several reactions were performed by varying different parameters including time, temperature, and concentration of precursors. For example, to optimize the reaction time different reactions were performed using same aforementioned conditions, except that the incubation time was varied between 15 to 120 minutes. Similarly, for the optimization of temperature, the incubation temperature was varied between 25 to 95 ◦C, while keeping the other parameters constant. Furthermore, to study the effect of the concentration of precursors on the quality of resulting Ag NPs, different reactions were performed by varying the concentrations of AgNO3 and leaves extract. For instance, in these reactions the concentration of AgNO3 was varied between 0.25 and 1.25 mM, whereas the leaves extract concentration was varied between 1 and 10 mL.

2.4. Phytochemical Screening Diﬀerent types of preliminary chemical tests were performed on the Jujube extract to investigate the presences of various secondary metabolites in the Z. mauritiana leaves extract.

2.4.1. Test for Alkaloids (Mayer’s Test) At this stage, 1 mL (100 mg/mL) of ZM-LE was added into 1 mL of Mayer’s reagent, which led to the formation of white precipitate indicating the presence of alkaloids. Similarly, 1 mL of Dragendroﬀ reagent was used, which formed brown precipitate in the presence of alkaloids. Sustainability 2020, 12, 1484 4 of 14

2.4.2. Test for Flavonoids (Shinoda Test) Afterwards, 1 mL of ZM-LE and few fragments of magnesium metal were added to the test tube followed by dropwise addition of 0.5 mL of 10% concentrated HCl. Formation of a reddish color indicates the presence of ﬂavonoids.

2.4.3. Test for Glycosides (Keller Killani Test)

A small amount of glacial acetic acid, one drop of 5% FeCl3 solution and concentrated H2SO4 was added into 2 mL of ZM-LE. Reddish brown color appears at the junction of the two liquid layers and the upper layer of bluish green indicates the presence of glycosides.

2.4.4. Test for Phenols

+2 Afterwards, 2 mL of ZM-LE was added to 1 mL of H2SO4 and a pinch of Mg metal. Formation of orange or pink color indicates the presence of phenols.

2.4.5. Folin-Ciocalteu Method The total phenolic content in ZM-LE was determined by using Folin-Ciocalteu colorimetric method based on oxidation-reduction reaction. Various concentrations of stock solution (Gallic acid solutions in methanol) 25, 50, 75, 100, and 200 µg/mL were prepared. In each of the test tube, gallic acid of diﬀerent concentrations was added and to that 5 mL of Folin-Ciocalteu reagent and 4 mL of 7% Na2CO3 were added to get a total volume of 10 mL by using distilled water. The blue colored mixture was shaken well and incubated for 30 minutes at 40 ◦C in a water bath. Then the absorbance was measured at 765 nm against blank. The blank used was distilled water along with FCR and Na2CO3 and did contain the stock solution. The average absorbance values obtained at diﬀerent concentrations of gallic acid were used to plot the calibration curve.

2.4.6. Test for Saponins (Foam Test) The 1 mL of ZM-LE was diluted with 2 mL of distilled water and was shaken vigorously in a measuring cylinder for 15 minutes. A 1 cm layer of foam indicates the presence of saponins.

2.4.7. Test for Sterols (Liebermann-Burchard Test)

When 1 mL of ZM-LE was mixed with 1 mL of acetic acid and 2 mL of H2SO4 reddish violet color was obtained, which indicated the presence of sterols.

2.4.8. Test for Tannins

Upon addition of 3-4 drops of 10% FeCl3 to 1 mL of ZM-LE, greenish or bluish color formed, which indicated the presence of tannins.

2.5. Characterization The as-prepared ZM-AgNPs were identified by using UV–visible spectroscopy, Fourier transform infrared (FTIR), X-ray diffraction analysis (XRD), and scanning electron microscopy (SEM). The bioreduction of Ag+ ions in solutions and the formation of ZM-AgNPs was monitored via UV–Vis spectrophotometer (LIUV-310, Lambda Scientific, Australia). The association of biomolecules with synthesized AgNPs was examined by using a FTIR spectroscope, the Spectrum One (Perkin Elmer, USA) with KBr pellets. The FTIR spectrum of washed and purified ZM-AgNPs was recorded over the 1 1 range of 400–4000 cm− and the resolution was kept as 4 cm− . The crystalline nature of synthesized ZM-AgNPs was analyzed on a XRD-6000 X-ray diffractometer model (Shimadzu, Japan) with 40 kV, 30 mA with Cu ka radiation at 2θ angle. The morphology of the synthesized ZM-AgNPs was examined by SEM using a SU1510 electron microscope (Hitachi, Japan). TEM images and EDX spectrum were recorded on a JEM 1101 transmission electron microscope (JEOL, Tokyo, Japan). Sustainability 2020, 12, 1484 5 of 14

2.6. Antibacterial Properties

2.6.1. Inoculums of Microorganism Fresh overnight bacterial cultures of four bacterial strains E. coli, S. aureus, P. aeruginosa, and B. subtilis were prepared in a sterile broth at a concentration of 0.5 McFarland (1.5 108 CFU/mL) of × UV–Vis spectrophotometer of 600 nm (LIUV-310, Lambda Scientiﬁc, Australia). This suspension was used at diﬀerent dilutions in the design’s proposed evaluation.

2.6.2. Disc Diffusion Assay To analyze the bactericidal activity of ZM-AgNPs, a disc diffusion method was used in line with the previously reported method using various bacterial strains E. coli, S. aureus, P. aeruginosa and B. subtilis [27]. Approximately 15-20 mL of Mulller Hilton Agar (MHR) was transferred into sterilized petri-dishes and the discs (Whatman No-1 filter paper) were utilized to impregnate the ZM-AgNPs solution of various dilutions (7, 14, and 28 µL) of (100µg/mL). Negative control consisted of the AgNO3 solution and Streptomycin (30 µg/mL) as standard. The plates were incubated at 37 ◦C for 24 hours and examined for the presence of zones of inhibition. The diameter of such zones of inhibition was measured and expressed in millimeter units.

2.6.3. Determination of Minimum Inhibitory Concentration (MIC) Determination of minimum inhibitory concentration (MIC) assay of different concentration of biosynthesized ZM-AgNPs was analyzed using the method described by reference [28]. The MIC assay was defined as the minimum concentration of a particular antimicrobial agent that can prevent the growth of bacteria. Afterwards, 100 µL of E. coli and S. aureus bacterial concentration was inoculated into plates containing Muller Hilton Agar and different concentrations of biosynthesized AgNPs i.e., ZM-AgNPs (0, 2.5 mg/mL, 5 mg/mL, 10 mg/mL, and control) suspension was added on plates. The agar plates were incubated at 37 ◦C for overnight in an incubator (Thermo Fisher Scientific Co., USA).

3. Results and Discussion

3.1. UV-Vis Analysis Z. mauritiana leaves extract (ZM-LE) was applied to prepare biologically active AgNPs under mild conditions. After adding LE to the aqueous AgNO3 solution and 15 min incubation, the color of the solution turned to light brown, indicating the successful preparation of ZM-AgNPs (Figure2). Contrarily, in the absence of LE, no such color change of AgNO3 solution was observed even after incubating the solution for several hours (24 hrs.). Typically, metal NPs including silver exhibit strong absorption in the visible range due to the surface Plasmon Resonance (SPR) phenomenon [29]. Due to this, the reduction of Ag+ ions can be monitored by UV analysis. In the case of the reduction of Ag ions using ZM-LE, the UV spectrum has exhibited a strong absorption peak at ~413 nm, which ultimately conﬁrmed the formation of ZM-AgNPs. Furthermore, the absorption peak at relatively lower wavelength (413 nm) also points towards the formation of smaller size nanoparticles, as a broader peak higher wavelength typically indicates an enhancement of particle size. For instance, in an earlier study of the photo-reduction of Ag ions using ZM-LE, the UV absorption peak appeared at ~445 nm, which reported the formation of large size (~85 nm) ZM-AgNPs [26]. Sustainability 2020, 12, 1484 6 of 14 Sustainability 2019, 11, x FOR PEER REVIEW 6 of 15

FigureFigure 2. 2.UV-Vis UV-Vis analysis analysis of of as-synthesized as-synthesized silversilver nanoparticlesnanoparticles (1(1 mLmL ZM-AgNPs)ZM-AgNPs) using ZM ZM-LE.-LE.

TheThe amount amount of ofreducing reducing agent agent (ZM-LE) (ZM- wasLE) optimized was optimized via preparation via preparation of AgNPs of usingAgNPs di ff usierentng concentrationsdifferent concentrations of ZM-LE, of such ZM as-LE, 1 mL, such 2.5 as mL, 1 mL, 5 mL, 2.5 mL, 7.5 mL, 5 mL, and 7.5 mL, 10 mL. and The 10 mL. results The were results monitored were usingmonitored UV (cf. using Figure UV3), upon (cf. Figure increasing 3), upon the concentration increasing the of concentration ZM-LE, the absorption of ZM-LE, bands the absorptionbroadened andbands shifts broadened towards the and higher shifts wavelength towards the which higher was indicative wavelength of whithech deterioration was indicative of the ofquality the ofdeterioration NPs [30]. Indeed, of the quality at higher of NPs concentrations [30]. Indeed, of at ZM-LE higher aboveconcentration 5 mL, thes of reactionZM-LE above ceased 5 tomL, occur the duereaction to the ceased presence to of occur excess due amount to the of presence bio-reductant. of excess Therefore, amount oflow bio concentration-reductant. Therefore, of ZM-LE, low i.e., belowconcentration 5 mL, was of suZMffi-cientLE, i.e. to, below obtain 5 high mL, qualitywas sufficient of NPs. to Besidesobtain high this, quality a comparative of NPs. Besides investigation this, a wascomparative also performed investigation to study was the influencealso performed of the concentrationto study the influence of silver precursorof the concentration (AgNO3), forof whichsilver variousprecursor experiments (AgNO3), werefor which performed various using experiments different were concentrations performed using of AgNO different3 (0.25 concentra mM, 0.5tions mM, 0.75of AgNO mM, 1.03 (0.25 mM, mM, and 0.5 mM, 1.25 mM), 0.75 mM, while keeping 1.0 mM the, and amount 1.25 mM), of ZM-LE while keeping constant the at 1amount mL. AgNPs of ZM were-LE notconstant formed at at 1 the mL. lowest AgNPs concentration were not formed of AgNO at the3 (0.25 lowest mM), concentration as substantiated of AgNO by the3 (0.25 UV spectrum. mM), as Whereassubstantiated upon increasingby the UV thespectrum. concentration Whereas of upon AgNO increasing3, a shift inthe the conce SPRntration band was of observed,AgNO3, a initiallyshift in itthe was SPR obtained band was at 406 observed, nm (0.5 initially mM) and it was shifted obtained to 413 at nm 406 (1.25 nm (0.5 mM), mM) indicating and shifted a slight to 413 increase nm (1.25 in particlemM), indicating size with a an slight increase increase in the in concentrationparticle size with of precursor.an increase Inin addition,the concentration the effect of of precursor. time and temperatureIn addition, on the the effect synthesis of time of AgNPs and temperature using ZM-LE on the is also synthesis investigated of AgNPs using using UV analysis ZM-LE and is also the datainvestigated is provided using in the UV supporting analysis and information. the data isIt was provided revealed in the that supporting temperatures information. above 65 ◦ ItC (best was resultsrevealed at 95that◦C) temperature and a times above of 15 minutes65 °C (best are results optimum at 95 to °C) prepare and a time a good of 15 quality minutes of are AgNPs optimum (data notto shown).prepare a good quality of AgNPs (data not shown).

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Figure 3. (aFigure) UV-Vis 3. (a) analysis UV-Vis analysis of as-synthesized of as-synthesized silver nanoparticles nanoparticles (ZM-AgNPs) (ZM-AgNPs) using various using various concentrationsconcentrations of ZM-LE of (1,ZM 2.5,-LE (1, 5, 2.5, 7.5, 5, and7.5, and 10 10 mL) mL) and (b) (b dispersion) dispersion images images of as-synthesized of as-synthesized silver silver nanoparticles using various concentrations of ZM-LE (1, 2.5, 5, 7.5, and 10 mL). nanoparticles using various concentrations of ZM-LE (1, 2.5, 5, 7.5, and 10 mL). 3.2. XRD analysis 3.2. XRD Analysis The crystallinity of Ag NPs obtained via ZM-LE was confirmed using XRD as shown in Figure 4. The crystallinityThe as-obtained of Ag ZM NPs-AgNPs obtained exhibited via cubic ZM-LE structure, was clearly confirmed reflected using by the XRD presence as shown of five in Figure4. characteristic peaks in the XRD pattern such as, 37.50° (111), 44.13° (200), 63.91° (220), 76.89° (311), The as-obtainedand 81.13° ZM-AgNPs (222). Apart exhibitedfrom the distinct cubic peaks structure, of ZM-AgNP clearlys, the absence reflected of any other by the additional presence of five characteristicpeaks peaks clearly in indicated the XRD that pattern the lattice such of as-obtained as, 37.50 ZM◦-AgNPs(111), was 44.13 unaffected◦ (200), by 63.91 other molecules◦ (220), 76.89◦ (311), in the leaves extract. Furthermore, the average crystallite size was calculated using the Debye– and 81.13◦ (222). Apart from the distinct peaks of ZM-AgNPs, the absence of any other additional Scherrer formula, D = kλ/βcosθ, where D is the diameter of the particle, k is constant (1), λ = 0.1541 peaks clearlynm indicated (Cu Ka), β thatis FWHM the, latticeand ‘θ’ is of the as-obtained diffraction angle ZM-AgNPs corresponding was to the una latticeffected planeby (1 1other 1). The molecules in the leaves extract.average Furthermore,size is found to be the61 nm, average which is crystalliteslightly higher size when was compar calculatedes to the size using of the the particles Debye–Scherrer formula, D =obtainedkλ/βcos byθ , TEM. where ThisD couldis the be diameter due the slight of the deviation particle, from k theis ideal constant shape (1), (spherical)λ = 0.1541 of the nm (Cu Ka), particles considered in the Debye–Scherrer formula. β is FWHM, and ‘θ’ is the diffraction angle corresponding to the lattice plane (1 1 1). The average size is found to be 61 nm, which is slightly higher when compares to the size of the particles obtained by TEM. This could be due the slight deviation from the ideal shape (spherical) of the particles considered in the Debye–Scherrer formula. The morphology of the as-prepared ZM-AgNPs observed under TEM and the corresponding particle size distribution are shown in Figure5. The TEM results clearly indicated that the particles are 7 of spherical shape with small size ranging from 3–20 nm, retaining a narrow size distribution (Figure5). Moreover, the nanoparticles are largely well separated and only a few of them were agglomerated. The surface morphology of the as-synthesized ZM-AgNPs was also monitored using scanning electron microscopy (SEM), as shown in Figure6. The image obtained clearly demonstrated the spherical shape of the nanoparticles. Additionally, the elemental composition determined by using EDX also indicated the presence of Ag as major element in the sample (cf. Figure6). Apart from Ag, the EDX spectrum also pointed towards the presence of carbon and oxygen in the sample; this outcome could be attributed to the existence of residual phytomolecules on the surface of resulting ZM-AgNPs as stabilizing ligands. Sustainability 2019, 11, x FOR PEER REVIEW 8 of 15

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Figure 4. XRD analysis of as-synthesized silver nanoparticles (1 mL ZM-AgNPs) using 1 mL ZM-LE.

The morphology of the as-prepared ZM-AgNPs observed under TEM and the corresponding particle size distribution are shown in Figure 5. The TEM results clearly indicated that the particles are of spherical shape with small size ranging from 3–20 nm, retaining a narrow size distribution (Figure 5). Moreover, the nanoparticles are largely well separated and only a few of them were agglomerated. The surface morphology of the as-synthesized ZM-AgNPs was also monitored using scanning electron microscopy (SEM), as shown in Figure 6. The image obtained clearly demonstrated the spherical shape of the nanoparticles. Additionally, the elemental composition determined by using EDX also indicated the presence of Ag as major element in the sample (cf. Figure 6). Apart from Ag, the EDX spectrum also pointed towards the presence of carbon and oxygen in the sample; this outcome could be attributed to the existence of residual phytomolecules on the surfaceFigureFigure of4. XRD XRDresulting analysis analysis ZM of of as- as-synthesizedAgNPs-synthesized as stabilizing silver silver nanoparticles nanoparticles ligands ( (11. mL ZM ZM-AgNPs)-AgNPs) using 1 mL ZM ZM-LE.-LE.

Figure 5. TEM images of as-synthesized silver nanoparticles using 1 mL ZM-AgNPs (a,b) overview, (c) magniﬁed TEM image, and (d) particle size distribution graph. 8

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SustainabilityFigure2020 5., TEM12, 1484 images of as-synthesized silver nanoparticles using 1 mL ZM-AgNPs (a, b) overview, 9 of 14 (c) magnified TEM image, and (d) particle size distribution graph.

FigureFigure 6. 6.(a ()a) SEM SEM images images of of as-synthesizedas-synthesized silver nanoparticles nanoparticles using using 1 1mL mL ZM ZM-AgNPs-AgNPs and and (b) (EDXb) EDX analysisanalysis of of 1 1 mL mL ZM-AgNPs. ZM-AgNPs.

3.3.3.3. Preliminary Preliminary Identification Identification ofof PhytomoleculesPhytomolecules on the the Surface Surface of of ZM ZM-AgNPs-AgNPs InIn order order to to determine determine thethe active active phytomolecules phytomolecules responsible responsible for forthe thereduction reduction of silver of silver ions, ions,a a preliminarypreliminary qualitative qualitative analysis analysis of of ZM ZM-LE-LE was was performed performed which which helped helped to to detect detect the the secondary secondary metabolitesmetabolites present present in inZiziphus Ziziphus mauritiana mauritianaextract. extract. For For this this purpose, purpose, various various tests tests were were performed, performed, such assuch theMayer’s as the Mayer’s test, Shinoda test, Shinoda test, Keller test, Keller Killani Killani test, Foamtest, Foam test, Liebermann-Burchardtest, Liebermann-Burchard Test, Test etc.,, etc., which pointedwhich towardspointed towards the presence the presence of various of various secondary secondary metabolites metabolites including including alkaloids, alkaloids, phenols, phenols, tannins, saponinstannins, and saponins glycosides and glycosides in the ZM-LE. in the All ZM these-LE. testsAll these were tests successful were successful in ascertaining in ascertaining the presence the of thesepresence secondary of these metabolites secondary inmetabolites the ZM-LE. in the ZM-LE. Additionally,Additionally, the the determination determination of of total total polyphenol’s polyphenol’s contentscontents inin extractsextracts was made according to thetoFolin-Ciocalteu the Folin-Ciocalteumethod method by measuring by measuring the absorbance the absorbance at 765 nm at 765 against nm a against control a without control extract without [31 ]. Thisextract technique [31]. This has been technique reportedly has been used reportedly in several studies used in to several determine studies the totalto determine phenolic the contents total of phenolic contents of the plant extracts. For example, Kumar et al., have reported the total phenolic the plant extracts. For example, Kumar et al., have reported the total phenolic contents of 23 and 40 contents of 23 and 40 mg per gram in the leaves and stem extracts of Syzygium cumini plant, which mg per gram in the leaves and stem extracts of Syzygium cumini plant, which were employed for the were employed for the synthesis of Ag NPs. During this study, they suggested a direct correlation synthesis of Ag NPs. During this study, they suggested a direct correlation between the total phenolic between the total phenolic contents and the size of resulting NPs, which increases with the increase contents and the size of resulting NPs, which increases with the increase of phenolic contents [32]. of phenolic contents [32]. In another study, Folin-Ciocalteu method was used to determine the Inpresence another study,of phenolicFolin-Ciocalteu contents onmethod the surface was used of resulting to determine silver the NPs. presence Approximately of phenolic 7.7 mg contents of onphenolic the surface conte ofnts resulting were found silver as NPs. stabilizing Approximately ligands on 7.7the mg surface of phenolic of Ag NPs contents prepared were from found the as stabilizingPotentilla ligandsfulgens wall on the plant surface extract of [33] Ag.NPs In this prepared study, the from total the polyphenol’sPotentilla fulgens contentwall in plantthe ZM extract-LE (at [33 ]. Infinal this concentration study, the total 100 polyphenol’s mg/mL) was calculated content in from the a ZM-LE linear calibrat (at finalion concentration curve (y = mx+c), 100 established mg/mL) was calculatedwith gallic from acid a linear(0–100 calibrationµg/mL) as curvea standard (y = mxreference+c), established in the same with conditions gallic acid as (0–100the sampleµg/mL) [34] as. a standardMeanwhile reference, the AlCl in the3 method same conditionswas used to as determine the sample the [total34]. Meanwhile,flavonoids contents the AlCl in3 themethod ZM-LE was [35] used. toThe determine results thehave total revealed flavonoids a total contents phenolic in contents the ZM-LE quantity [35]. of The 32.1 results mg/g have of ZM revealed-LE, as awell total asphenolic 533.5 contentsmg/g of quantity flavonoid of 32.1 contents. mg/g of Indeed, ZM-LE, various as well as phytomolecules 533.5 mg/g of flavonoid belonging contents. to polyphenols Indeed, various and phytomoleculesflavonoids were belonging also found to polyphenols to be present and on flavonoids the surface were of also resultant found toZM be-AgNPs present which on the were surface ofpre resultantpared by ZM-AgNPs using ZM- whichLE extract. were prepared by using ZM-LE extract. ThisThis was was further further confirmed confirmed by byFT-IR FT- analysis,IR analysis, which which was was used used to identify to identify the presence the presence of residual of biomoleculesresidual biomolecules of ZM-LE of on ZM the-LE surface on the of surfaceZM-AgNPs of ZM as-AgNPs stabilizing as stabilizing agents. In agents. order to In remove order to the unboundremove phytomolecules, the unbound the phytomolecules, sample was washed the samp repeatedlyle was before washed FT-IR repeatedly measurement. before For instance,FT-IR freshlymeasurement. obtained ZM-AgNPs For instance, were freshly redispersed obtained in ZM DI- waterAgNPs through were redispersed sonication (30 in min), DI water and centrifuged through sonication (30 min), and centrifuged at 9000 rpm (30 min), and this procedure was performed twice at 9000 rpm (30 min), and this procedure was performed twice to obtain pure ZM-AgNPs for IR to obtain pure ZM-AgNPs for IR measurement. Figure 7 exhibits the IR spectra of purified NPs. The measurement. Figure7 exhibits the IR spectra of purified NPs. The FT-IR spectrum ZM-AgNPs FT-IR spectrum ZM-AgNPs exhibited several peaks, which indicates the presence of several exhibited several peaks, which indicates the presence of several important functional groups. Some of 1 the prominent peaks include the peaks at 3747, 3441,9 1761, 1629, 1407, 1255, 1070, and 512 cm− which can be attributed to the hydrogen bonded O-H group, C H, C C, and C O stretching of aromatics, − − − C O stretching of alcohols, carboxylic acids, and ester groups, etc. [36]. In addition, these peaks − Sustainability 2019, 11, x FOR PEER REVIEW 10 of 15

Sustainabilityimportant2020, 12functional, 1484 groups. Some of the prominent peaks include the peaks at 3747, 3441, 1761,10 of 14 1629, 1407, 1255, 1070, and 512 cm-1 which can be attributed to the hydrogen bonded O-H group, C−H, C−C, and C−O stretching of aromatics, C−O stretching of alcohols, carboxylic acids, and ester revealedgroups the, presenceetc. [36]. In of addition diﬀerent, these compounds peaks revealed containing the presenc carbone andof different oxygen, compounds thereby suggesting containing that silvercarbon nanoparticles and oxygen might, thereby be capped suggesting by organic that components silver nanoparticles of ZM-LE, might which be not capped only help by organic to stabilize the surfacecomponents of NPs, of but ZM also-LE, enhancewhich not the only biological help to stabilize properties the of surface these of materials. NPs, but also enhance the biological properties of these materials.

FigureFigure 7. FT-IR 7 FT- spectraIR spectra of of as-synthesized as-synthesized silversilver nanoparticles 1 1mL mL ZM ZM-AgNPs.-AgNPs.

3.4. Antimicrobial3.4. Antimicrobial Properties Properties The antibacterialThe antibacterial assay assay of ZM-AgNPsof ZM-AgNPs was was performed performed against antibiotic antibiotic resistant resistant gram gram positive positive bacterialbacterial strains strains such as suchS. aureus, as S. aureus, B. subtilus B. subtilus, and gram, and negativegram negative bacterial bacterial strains strains such as suchP. aeruginosa as P. and E.aeruginosa coli, using and the E. disccoli, using diffusion the disc method. diffusion Streptomycin method. Streptomycin was employed was employed as a control as a andcontrol the and zone of inhibitionthe zone value of wasinhibition calculated value forwas di calculatedfferent concentrations for different concentrations of ZM-AgNPs of ZM i.e.,-AgNPs 7 µL, 14 i.e.,µL, 7 andμL, 14 28 µL from (100μL, andµg/ 28mL). μL Thefrom di (100fferent μg/mL). zones The of different inhibition zones obtained of inhibition are given obtained in Figure are given8. in Figure 8. From the results obtained, it can be understood that the highest zone of inhibition of about 28 mm was recorded in the case of biosynthesized AgNPs at the dilution of 28 µg/mL against S. aureus, a gram positive bacterial strain, which is much higher than the zone of inhibition obtained using the control i.e., Streptomycin. Moreover, upon a decrease in the concentration of ZM-AgNPs, the zone of inhibition also decreased. The values obtained as a result are given in Table1 and can be seen in Figure8A. Furthermore, in case of activity against B. subtilis, a gram positive bacterial strain, the zone of inhibition obtained was found to be comparable to the one obtained using the control (Figure8B). When the study was extended to gram negative bacterial strains such as P. aeruginosa and E. coli, the zone of inhibition obtained was found to be 21 mm and 22 mm, respectively, which is much smaller than that obtained using the control i.e., 26 mm and 28 mm (Figure8C and D). From the above results, it can be concluded that the ZM-AgNPs display selective activity against S. aureus.

10 Sustainability 2020, 12, 1484 11 of 14

Sustainability 2019, 11, x FOR PEER REVIEW 11 of 15

A B S. aureus B. subtilis

Control 7 μL 7 μL Control

AgNO3 14 μL AgNO3 14 μL

28 μL 28 μL

C D E. Coli P. aeruginosa

Control AgNO3 7 μL Control 7 μL 14 μL AgNO3

14 μL

28 μL 28 μL

Figure 8.FigureImages 8. Images of zone of zone of of inhibition inhibition obtained obtained when when the disc the diffusion disc diﬀ methodusion is method employed is for employed the for the antibacterialantibacterial study study of biosynthesized of biosynthesized ZM-AgNPsZM-AgNPs against against gram gram-positive-positive bacterial bacterial strains: (a) strains: S. aureus; (A) S. aureus; B) B. subtilis and gram-negative bacterial strains; C) P. aeruginosa; D) E. coli. (B) B. subtilis and gram-negative bacterial strains; (C) P. aeruginosa;(D) E. coli. From the results obtained, it can be understood that the highest zone of inhibition of about 28 mm was recorded in the case of biosynthesized AgNPs at the dilution of 28 μg/mL against S. aureus, Table 1. Eﬀect of varying concentrations of biosynthesized ZM-AgNPs as antimicrobial agents against a gram positive bacterial strain, which is much higher than the zone of inhibition obtained using the S. aureuscontrol, B. i.e., subtilus Streptomycin., P. aeruginosa Moreover,, and uponE. coli a decreasebacterial in the strains. concentration of ZM-AgNPs, the zone of inhibition also decreased. The values obtained as a result are given in Table 1 and can be seen in Zone of Inhibition (mm) Figure 8A. Furthermore, in case of activity against B. subtilis, a gram positive bacterial strain, the AgNO ZM-AgNPs zone of inhibitionBacterial obtained Strains was found to be comparableControl to the one3 obtained using the control (Figure 8B). When the study was extended to gram negative Solutionbacterial strains7 µ suchL as 14P. µaeruginosaL 28 µL and GramE. coli, the zoneS. aureusof inhibition(MTCC obtained No. 96) was found 8 to be 21 mm 3 and 22 mm, 8respectively, 16 which is 28 much+ smallerve thanB. subtilusthat obtained(MTCC using No. 441) the control 26i.e., 26 mm and 4 28 mm (Figure 10 8C and 18 D). From 26 the aboveGram results,P. aeruginosait can be concluded(MTCC No. that 424) the ZM-AgNPs 26 display 4selective activity 9 against 19 S. aureus. 21 ve E. coli (MTCC No. 1722) 28 4 8 12 22 −

The high activity of ZM-AgNPs in case of S. aureus bacterial strain can be attributed to their excellent tendency of adsorbing on the bacterial cell wall and undergo dehydrogenation due to the respiration process which occurs at the cell11 membrane of bacteria. Due to the interaction with nanoparticles, the enzymes in the bacteria are deactivated, generating a hydrogen peroxide that eventually leads to microbial cell death [37]. However, similar nanoparticles were prepared with dragon fruit peel extract and were tested for microcidal activity against the same bacterial strains as the ones we obtained; however, it was found that Ag NPs prepared by the dragon fruit peel extract yielded a zone of inhibition of 9 mm, while the Ag NPs reported in this study yielded a signiﬁcantly higher zone of inhibition i.e., 22 nm [38]. Sustainability 2019, 11, x FOR PEER REVIEW 12 of 15

Table 1. Effect of varying concentrations of biosynthesized ZM-AgNPs as antimicrobial agents against S. aureus, B. subtilus, P. aeruginosa, and E. coli bacterial strains.

Zone of inhibition (mm)

AgNO3 ZM-AgNPs Bacterial strains Control solution 7μl 14μl 28μl

S. aureus (MTCC No. 96) 8 3 8 16 28

+ ve

Gram Gram B. subtilus (MTCC No. 441) 26 4 10 18 26

P. aeruginosa (MTCC No. 424) 26 4 9 19 21

- Gra E. coli (MTCC No. 1722) 28 4 8 12 22

Sustainabilitythe ones we2020 obtained;, 12, 1484 however, it was found that Ag NPs prepared by the dragon fruit peel extract12 of 14 yielded a zone of inhibition of 9 mm, while the Ag NPs reported in this study yielded a significantly higher zone of inhibition i.e., 22 nm [38]. Furthermore,Furthermore, thethe minimumminimum inhibitoryinhibitory concentrationconcentration (MIC)(MIC) valuevalue ofof biosynthesizedbiosynthesized ZM-AgNPsZM-AgNPs waswas calculatedcalculated andand thethe bacterialbacterial strainsstrains S.S. aureusaureus andand E.E. colicoli werewere selectedselected forfor thisthis study.study. InIn this study thethe bacterialbacterial strains,strains, thethe E.E. colicoli and S. aureus were subjected to didifferentﬀerent concentrationsconcentrations ofof ZM-AgNPsZM-AgNPs (0,(0, 2.5 2.5µ g μg/ml,/ml, 5 µ 5g /ml, μg/ml, 10 µ g 10/ml) μg/ml) and were and also were taken also as taken controls aswithout control ZM-AgNPs.s without ZM The-AgNPs. ZM-AgNPs The showedZM-AgNPs the highestshowed level the highest of activity level against of activityE. coli againstwith the E. MICcoli with value the of MIC 5 µg /valuemL, while of 5 μg/mL, for S. aureus while, thefor MICS. aureus value, the was MIC found value to was be 10 foundµg/mL, to asbe shown10 μg/mL in Figure, as shown9. in Figure 9.

FigureFigure 9.9. EEffectffect of varying concentrationsconcentrations (2.5 µμg/mL,g/mL, 5 µμg/mLg/mL,, andand 1010 µμg/mL)g/mL) ofof biosynthesizedbiosynthesized ZM-AgNPsZM-AgNPs againstagainst bacterialbacterial strainsstrains E.E. colicoliand andS. S. aureusaureus.. 4. Conclusions 4. Conclusion In brief, we have succeeded in achieving the green reduction of silver ions by Z. mauritiana leaves extract under ambient conditions in a short period12 of time. The characterization of green synthesized ZM-AgNPs was performed by UV–Vis spectroscopy, FT-IR, XRD, SEM, and TEM analysis. The results have revealed the formation of spherical shape Ag NPs with an average size of 12.5 nm. Moreover, the presence of phenolic compounds and the flavonoid contents in the ZM-LE extract were confirmed by Folin-Ciocalteu and AlCl3 methods, which were believed to be responsible for the reduction of silver ions. In this method, we have successfully reduced the preparation time from a couple of days using the photo-reduction method to 15 min by using ZM-LE. Additionally, the antibacterial properties, the zone of inhibition, and the minimum inhibitory concentration of biosynthesized ZM-AgNPs showed the inhibitory effect against all tested bacteria in a short time; however, the ZM-AgNPs were found to be selective against S. aureus, a gram positive bacterial strain.

Author Contributions: M.A., K.J. and M.K. contributed towards the designing of the project. M.A., M.R.S., N.F., M.S.A. and S.F.A. contributed towards the experimental and characterization. M.K., M.R.S. and S.F.A. contributed towards the drafting of the manuscript. K.J., M.K. and M.R.H.S. provided scientific guidance for successful completion of the project and also helped to draft the manuscript. All authors read and approved the final manuscript. Funding: Researchers Supporting Project Number (RSP-2019/60), King Saud University, Riyadh, Saudi Arabia. Acknowledgments: The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2019/60), King Saud University, Riyadh, Saudi Arabia. Conflicts of Interest: The authors declare no conflict of interest. Sustainability 2020, 12, 1484 13 of 14

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