Vol.:(0123456789)1 3 Journal of Inorganic and Organometallic Polymers and Materials https://doi.org/10.1007/s10904-019-01262-5 Green Synthesis and Characterization of Highly Stable Silver Nanoparticles from Ethanolic Extracts of Fruits of Annona muricata Yahaya Gavamukulya1,2   · Esther N. Maina1,3 · Amos M. Meroka3,4 · Edwin S. Madivoli1,5 · Hany A. El‑Shemy1,6 · Fred Wamunyokoli1,7 · Gabriel Magoma1 Received: 15 May 2019 / Accepted: 15 July 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Green synthesis of nanoparticles from plant materials opens a new scope in nanobiotechnology and discourages the use of expensive toxic chemicals. The aim of this study was to develop and optimize a method for the synthesis of Silver Nano- particles (AgNPs) from ethanolic extracts of fruits of Annona muricata as well as to characterize the green synthesized AgNPs. AgNPs were synthesized via AgNO3 solution. The AgNPs were characterized using spectroscopy and microscopy techniques. The formed AgNPs had an absorption maximum of 427 nm and were stable under different temperature, pH and storage conditions. Fourier Transform Infrared Resorption spectroscopy revealed the functional groups responsible for the synthesis and stabilization of the AgNPs. Scanning Electron Microscopy analysis revealed a spherical nature of the AgNPs. Energy Dispersive X-Ray spectroscopy showed presence of Ag, Cl, Ca, and Si with Ag having the highest composition at 80%. X-ray diffraction and dynamic light scattering revealed a crystalline nature of AgNPs with an average size of 60.12 nm and a polydispersity index of 0.1235 respectively. Transmission Electron Microscopy analysis further confirmed the crystal- line and spherical nature of the AgNPs. In this article, an efficient, eco-friendly and low-cost method for the synthesis and recovery of stable AgNPs using ethanolic extracts of Annona muricata fruits as both reducing and capping agents has been reported. The synthesized AgNPs could have many biomedical and clinical applications. Keywords  Annona muricata · Silver nanoparticles (AgNPs) · UV/VIS · FTIR · XRD · Fruit extracts Abbreviations AgNPs � Silver nanoparticles DLS � Dynamic light scattering EDX � Energy Dispersive X-ray Spectrometer EEAM � Ethanolic extracts of Annona muricata FTIR � Fourier Transform Infrared Resorption PDI � Polydispersity index SEM � Scanning Electron Microscopy SPR � Surface plasmon resonance TEM � Transmission electron microscopy UV/VIS � Ultraviolet visible spectrum XRD � X-ray diffraction analysis * Yahaya Gavamukulya gavayahya@yahoo.com 1 Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation (PAUSTI), P. O. Box 62000‑00200, Nairobi, Kenya 2 Department of Biochemistry and Molecular Biology, Faculty of Health Sciences, Busitema University, P. O. Box 1460, Mbale, Uganda 3 Department of Biochemistry, College of Health Sciences, University of Nairobi, P. O. Box 30197‑00100, Nairobi, Kenya 4 Department of Biochemistry, School of Medicine and Health Sciences, Kenya Methodist University, P. O. Box 267‑60200, Meru, Kenya 5 Department of Chemistry, College of Pure and Applied Sciences, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000‑00200, Nairobi, Kenya 6 Department of Biochemistry, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt 7 Department of Biochemistry, College of Health Sciences, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000‑00200, Nairobi, Kenya http://orcid.org/0000-0001-6031-1642 http://crossmark.crossref.org/dialog/?doi=10.1007/s10904-019-01262-5&domain=pdf Journal of Inorganic and Organometallic Polymers and Materials 1 3 1  Introduction Nanoparticles are materials that are small enough to fall within the nanometric range, with at least one of their dimensions being less than a few hundred nanometres. This reduction in size brings about significant changes in their physical properties with respect to those observed in bulk materials. A very interesting application of nanopar- ticles in the scope of life sciences is their use as ‘smart’ delivery systems where they are usually loaded with a drug or therapeutic agent [1]. The various developed chemi- cal and mechanical methods of producing nanoparticles include ball milling, thermal quenching, precipitation techniques, vapor deposition. However, these methods are often costly, and may result in toxic byproducts. Generally, nanoparticles are synthesized in three ways: physically by crushing larger particles, chemically by precipitation, and through gas condensation [2–6]. The commercial sig- nificance of nanoparticles is limited by the nanoparticle synthesis process, which is generally energy intensive or requires toxic chemical solvents and is costly. Biological approaches, including use of microorganisms or plant extracts to synthesize metal nanoparticles, have been suggested. However, synthesis of nanoparticles using microorganisms involves an expensive process requiring cell culture and multistep purification. An emerging field in nanotechnology is the synthesis of metal nanoparticles using herbal plants. Metal nanoparticles display improved and/or novel properties compared to their source materi- als. These properties may be derived from their size, mor- phology, or distribution. This method is referred to as the green approach and is environmentally friendly. Thus, the advancement of green syntheses of nanoparticles is pro- gressing as a key branch of nanotechnology; where the use of biological entities like microorganisms, plant extract or plant biomass for the production of nanoparticles could be an alternative to chemical and physical methods in an ecofriendly manner [7]. Annona muricata L. is a species of the Annonaceae family that has been widely studied in the last decades due to its therapeutic potential. Annona muricata is known as Soursop (English), Graviola (Portuguese), Guanábana (Latin American Spanish), Omusitafeli/Ekitafeli (Uganda), and other local indigenous names as has been enlisted [8, 9]. This plant is a species of the genus Annona with the following taxonomic classification. Kingdom: Plantae, Division: Angiosperms (Magnoliophyta), Class: Mag- nolids, Order: Magnoliales, Family: Annonaceae, Genus: Annona, Species: Annona muricata L. [10]. The Annona muricata tree is about 5–10 m tall and 15–83 cm in diam- eter with low branches [11–13]. It is widely distributed in the tropical regions of Central and South America, Western Africa, Central and Eastern Africa and South- east Asia [10, 14] at altitudes below 1200 m above sea level, with temperatures between 25 and 28 °C, relative humidity between 60 and 80%, and annual rainfall above 1500 mm. The fruit is an edible collective ovoid berry, dark green in color. Various medicinal uses have been reported across the globe ranging from the use of leaves, bark, roots, fruits and seeds of Annona muricata [15]. From the studies reported, the most widely used preparation in traditional medicine is the decoction of bark, root, seed or leaf, each having unique bioactive compounds [8]. On the other hand, the least stud- ied plant part are the fruits, despite their being the most widely used and eaten as a food, unlike the leaves, barks and seeds. Because of their use as a food, fruits can provide an easier and more acceptable way of delivering the requisite bioactive compounds to the human consumers. Nevertheless, a number of unique bioactive compounds have been reported in the fruits extracts including alkaloids such as anonaine, asimilobine, nornuciferine [8]; acetogenins such as muricin, montanacin [8]; phenols such as kaempferol 3-O-ruti- noside, myricetin [8]; and a number of other compounds such as vitamin C, carotenes, tocopherol [8], 1,3-dimeth- ylthiourea, (4-chlorophenyl)-[4-(3-chlorophenyl)-2-[(Z)-3- (dimethylamino)prop-1-enyl]quinolin-6-yl]-(3-methylim- idazol-4-yl)methanol [16], among others. These bioactive compounds are reported as having antioxidant, cytotoxic, antidiabetic, anti-hypotensive, antimalarial, and anticancer properties among others [8, 9, 16]. The effectiveness of many species of medicinal plants depends on the supply of active compounds. It has there- fore been widely proposed to combine herbal medicine with nanotechnology, because nanosystems can deliver the bio- active components at a sufficient concentration during the entire treatment period, directing them to the desired sites of action, and hence potentiating the action of the compounds, an aspect that conventional herbal treatments do not meet [17, 18]. Among several noble metal nanoparticles, silver nanoparticles have attained a special focus [7]. Silver nano- particles are of particular interest because of their antimi- crobial, anticancer and cytotoxic activities. Studies involv- ing the use of Annona muricata leaves [19, 20], bark [21], and fruits [22] utilizing aqueous extracts in the synthesis of nanoparticles have previously been reported [19–22]. Nev- ertheless, there had been no reported method or publication on the use of ethanolic extracts of Annona muricata to pre- pare nanoparticles from fruits, despite the known advantages of use of ethanolic extracts as an extraction solvent, which include high recovery rate [8, 9, 23]. The aim of this study was therefore to develop and optimize a method for the syn- thesis of AgNPs from ethanolic extracts of fruits of Annona muricata as well as to characterize the green synthesized AgNPs. Journal of Inorganic and Organometallic Polymers and Materials 1 3 2 � Materials and Methods 2.1 � Samples Collection and Authentication Ripe fruits of Annona muricata were collected from the wild in Eastern Uganda in the districts of Kaliro, Iganga and Mbale during the month of January 2018. A sample of the plant was collected, pressed, dried and the plant was identi- fied and authenticated in the Makerere University Botanical Herbarium (MHU) by Dr. Namaganda Mary and a voucher specimen was deposited in the herbarium with the acces- sion number MHU50860. The study was registered by the Uganda National Council for Science and Technology (Reg No. NS 43ES) as well as the PAUSTI Board of Examiners (MB400-0007/17). 2.2 � Samples Preparation and Extraction The Fruits of Annona muricata were washed with clean water and then peeled to remove the fresh pulp. The pulp was then cut into small pieces and placed in a hot air oven to dry at 50 °C for a week. The dried pulp was then milled into a powder using an electric grater. 50 g of powdered fruits were extracted using 250 ml of absolute ethanol for three days by the plant tissue homogenization method as previ- ously described [23]. The light brown Ethanolic Extracts of Annona muricata fruits was then filtered and kept at 4 °C until use. Figure 1 shows the samples collection, drying and extraction process. 3 � Chemicals and Reagents All chemicals and reagents were procured from certified suppliers and were of the highest analytical standard. 3.1 � Preparation of the 1 mM AgNO3 Solution Extra pure AgNO3 at a percentage purity of 99.7% was used for the preparation of the AgNO3 solution. 0.1699 g of AgNO3 were weighed on an ultrasensitive measuring balance and transferred to 1000 ml volumetric flask. Then distilled water was added to the volumetric flask with con- tinuous shaking until the 1000 ml mark was reached. The solution was then left to completely dissolve the salt. The 1 mM AgNO3 solution had been successfully prepared. 3.2 � Synthesis of Silver Nanoparticles AgNPs were synthesized by the following method. About 50 ml of the filtered fruits extract was mixed with about 450 ml of 1 mM AgNO3 solution in a 500 ml flask and mixed thoroughly, forming a uniform mixture. The mixture was then rested at room temperature in the dark storage cabinets for up to about 72 h, with continuous monitoring. After about 3 h, the mixture was observed to start changing from light brown to yellowish brown. After about 72 h, the mixture had completely changed colour to dark brown. This color change is visual evidence of formation of AgNPs or Fig. 1   Plate showing the samples collection, drying and extraction process Journal of Inorganic and Organometallic Polymers and Materials 1 3 reduction of silver ions into AgNPs due to the excitation of surface plasmon vibration [21, 24–28]. 3.3 � Characterization of the AgNPs 3.3.1 � UV/VIS Measurements to Confirm Formation of AgNPs The synthesis of AgNPs from the ethanolic extract of fruits of Annona muricata was further confirmed by ultravio- let–visible spectroscopy (UV/VIS) in the range of between 300 and 650 nm [24–27] and ethanol was used as a blank. 3.3.2 � Temperature/Heat Stability of the Synthesized AgNPs About 10 ml of the formed AgNPs suspension in boiling tubes were subjected to different temperature conditions by heating in a digital water bath for about 3 min each and measuring the absorbance spectra on the UV/VIS in a scan range of 350 nm to 650 nm [29]. The temperature tested included room temperature (25 °C), 35 °C, 45 °C, 55 °C, 65 °C, 75 °C, and 85 °C. 3.3.3 � pH Stability of the Synthesized AgNPs About 15 ml of the formed AgNPs suspension was aliquoted into 5 test tubes each containing about 3 ml of the AgNPs suspension. The suspensions in the test tubes were then adjusted to and subjected to different pH conditions ranging from about pH 2 to about pH 11. The suspension in each test tube was subjected to a different pH condition. The specific pH conditions tested were pH 2, 4, 7, 9, and 11. The pH were adjusted by either adding drops of 1 N NaOH or 1 N HCl until the desired pH was achieved as observed on the pH meter [29, 30]. The absorbance spectra of the suspen- sions were then measured on the UV/VIS in a scan range of 300 nm to 650 nm. 3.3.4 � Storage Stability of the AgNPs About 20 ml of the formed AgNPs suspension was aliquoted into four 15 ml universal tubes each containing about 5 ml of the AgNPs suspension. The suspensions in the tubes were then stored at different temperature conditions for a period of 3 months. The temperatures at which the storage was done included room temperature (which varied between at about 20 °C to 30 °C during the experimental period), 4 °C, − 20 °C and − 80 °C. At the end of the 3 months, the samples were retrieved from the different storage facilities allowed to thaw at room temperature and then their absorb- ance spectra were measured on the UV/VIS in a scan range of 300 nm to 650 nm. 3.3.5 � Recovery of the Synthesized AgNPs About 400 ml of the AgNPs suspension were transferred into different plastic bottles of about 250 ml capacity each and frozen in freezer at − 80 °C for a period of about 12 h. The frozen suspension was then removed from the freezer and allowed to completely thaw at room temperature. Upon thawing, the AgNPs were visibly observed spread through- out the now much clear suspension. The suspension with the dispersed AgNPs were then recovered by transferring them into 50 ml universal centrifuge tubes and centrifug- ing them at a speed of about 6000 RPM for a period of between about 20 min to about 45 min. After centrifugation, the supernatant in each of the tubes was poured off and the silver nanoparticles were retained as pellets at the bottom of the tubes. The pellets were then washed several times with distilled water (about 10 ml of distilled water were added to each tube and then centrifuged afresh for about 5 min to wash and dissolve any water-soluble impurities). The now clean AgNPs were then lyophilized and kept in airtight tubes at 4 °C until further use. A total of 1.2 g of AgNPs were recovered following lyophilization. 3.3.6 � Functional Groups Analysis FTIR measurements were carried out to identify the promis- ing biomolecules in the Annona muricata ethanolic extract accountable for the reduction of the silver ions and also the capping agents liable for the stability of the bio-reduced AgNPs. The functional groups present in the AgNPs were analyzed by a Bruker Tensor II FT-IR spectrophotometer model (Bruker, Ettlingen, Germany). The KBr pellets of samples were prepared by grinding 10 mg of samples, with 250 mg KBr (FT-IR grade). The 13 mm KBr pel- lets were prepared in a standard device under a pressure of 75 kN cm−2 for 3 min. The spectral resolution was set at 4 cm−1 and the scanning range from 400 to 4000 cm−1 [31]. The representative FTIR spectra of the recovered and dried AgNPs synthesized from ethanolic extracts of fruits of Annona muricata were recorded and the major and minor peaks were manifested and identified accordingly. 3.3.7 � SEM and EDX Measurements Scanning electron morphological analysis of Silver nanopar- ticles were performed using Scanning Electron Microscope FEI XL30 Sirion FEG (Oxford Instruments Plc, Abingdon, UK) operated at an accelerating voltage of 6 kV. The system was equipped with an Energy Dispersive X-ray Spectrometer (EDX) system from EDAX having a lithium doped silicon detector. Journal of Inorganic and Organometallic Polymers and Materials 1 3 3.3.8 � TEM Analysis TEM was employed to characterize the size, shape and mor- phologies of formed biogenic synthesized AgNPs. A drop of AgNPs suspension was deposited on carbon coated copper grids and the film on grid was then dried. The TEM was operated and the measurements were performed at accelerat- ing voltage of 100 kV. 3.3.9 � Crystalline Size Determination Using XRD XRD analysis was employed to determine the average crys- talline size of the AgNPs formed. The XRD (D8 Advance; Bruker Optik, Ettlingen, Germany) with CuKα radiation (λ = 1.5406 Å) and working at 40 kV/40 mA in the range of 10°–80° with a 2°-per-minute scanning rate was used. The XRD diffraction data was analyzed using the Match! Software (Crystal Impact, Bonn, Germany) and the average crystalline size of the AgNPs formed in the bio-reduction was determined using the Scherrer equation, with a constant of 0.94. 3.3.10 � Dynamic Light Scattering (DLS) Analysis The hydrodynamic size distributions and polydispersity index (PDI) of the silver nanoparticles were analyzed by using dynamic light scattering (DLS) instrumentation. The average particle size, size distribution by intensity as well as PDI were determined by injecting 1:20 dilution of silver nanoparticle resuspension into the U-shaped glass cuvette of the photon correlation microscope as previously reported [21, 26, 32]. 4 � Results Figure 2 indicates the color change which is a visual evi- dence of the formation of AgNPs or reduction of silver ions into AgNPs due to the excitation of surface plasmon vibration. The spectrum shown in Fig. 3 has a maximum absorp- tion peak at a wavelength of about 427 nm, which is in the range of the surface plasmon resonance for AgNPs which is reported to have an absorption maximum of between about 400 nm to about 450 nm. From Fig. 4 it is evident that at all temperatures tested, the AgNPs remained stable maintaining a characteristic absorp- tion maximum of about between 420 nm to about 430 nm which is within the AgNPs range. From Fig. 5, it is evident that at all pH conditions tested, the AgNPs remained stable maintaining a characteristic absorption maximum of about between 410 nm to about 420 nm which is within the AgNPs range. There was a nota- ble and strong relationship between AgNPs absorption spec- tra at extreme acidic and alkaline pH conditions of 2 and 11. From Fig. 6, it is evident that at all storage tempera- tures tested for the 3 months, the AgNPs remained stable Fig. 2   Photo showing colour of the green synthesized AgNPs relative to the ethanolic extract of Annona muricata fruits (EEAM-F) and Silver Nitrate solution (AgNO3) (Color figure online) 0 0.5 1 1.5 2 2.5 3 3.5 300 350 400 450 500 550 600 650 A bs or ba nc e Wavelength/nm Fig. 3   UV/VIS spectrum of fruits derived AgNPs at 72 h of incuba- tion Journal of Inorganic and Organometallic Polymers and Materials 1 3 maintaining a characteristic absorption maximum of about between 410 nm to about 430 nm which is within the AgNPs range. There was a notable increase in the absorption of the AgNPs at room temperature compared to other storage con- ditions, nevertheless, the absorption maximum was main- tained in the AgNPs range. Table 1 shows the functional group analysis of the FTIR spectrum of the biosynthesized AgNPs from ethanolic extracts of fruits of Annona muricata. As shown in Fig. 7 and Table 1 the functional groups responsible for the formation of the AgNPs included; alkanes and alkyls, aldehydes and esters, nitro groups, alcohol groups, carboxylic acids, amides, alkenes, acids and alkyl halides. As shown in Fig. 8, the AgNPs were approximately spherical in shape with smooth surface. These results are in agreement with the shape of SPR band recognized from the UV–visible spectrum with absorption maximum at 427 nm. From Fig. 9, the EDX spectra showed the presence of elements such as Ag, Cl, Ca, and Si. EDX quantitative analy- sis demonstrated that the highest concentration of a single element in the Annona muricata derived AgNPs was silver (Ag), at about 80%. Figure 10 shows the TEM micrographs of the AgNPs at different resolutions. The micrographs reveal a spherical nature of the monodispersed AgNPs as well as a crystalline structure. Particle size analysis using the Image-J software further revealed the AgNPs having an average particle size of about 51 nm. Figure 11 shows the typical XRD pattern of biosynthe- sized AgNPs derived from ethanolic extracts of fruits of Annona muricata. Nine prominent diffraction peaks were observed at 28.07°, 32.50°, 38.41°, 44.61°, 46.56°, 55.18°, 57.85°, 64.86°, and 67.85°. The average size of the AgNPs formed in the bio-reduction was determined using the Scher- rer equation and is estimated as 60.12 nm. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 350 400 450 500 550 600 650 A bs or ba nc e Wavelength/nm Room Temperature (25°C) 35°C 45°C 55°C 65°C 75°C 85°C Fig. 4   UV/VIS spectra showing temperature stability of AgNPs syn- thesized from fruits extract 0 0.5 1 1.5 2 2.5 3 300 350 400 450 500 550 600 650 A bs or ba nc e Wavelength/nm pH 11 pH 9 pH7 pH4 pH2 Fig. 5   UV/VIS spectra showing pH stability of AgNPs synthesized from fruits extract 0 0.5 1 1.5 2 2.5 3 300 350 400 450 500 550 600 650 A bs or ba nc e Wavelength/nm Room temperature (20°C - 30°C) Plus 4°C Minus 20°C Minus 80°C Fig. 6   UV/VIS spectra showing storage stability of AgNPs synthe- sized from fruits extract Table 1   FTIR functional group analysis of biosynthesized AgNPs from ethanolic extracts of fruits of Annona muricata  Type of Peak Frequency (cm−1) Bond Functional groups Major 2922.58 C–H stretch Alkanes and alkyls 2850 C–H stretch Alkanes and alkyls 1739.48 C=O stretch Aldehyde and esters 1500 N–O stretch Nitro group 1068.44 C–O stretch Alcohol group Minor 3400 O–H stretch Carboxylic acids 1650 C=O STRETCH Amide 1400 –C–H bend Alkane 1200 C–O stretch Acid 900 =C–H bend Alkenes 700 C–Cl stretch Alkyl halide 550 C–Br stretch Alkyl halide Journal of Inorganic and Organometallic Polymers and Materials 1 3 Table 2 shows the DLS analysis revealing the average particle size for the AgNPs as 103.5 nm with a polydisper- sity index of 0.1235. The bold values in the table indicate the mean values upon which quick reference maybe made in relation to the DLS and PDI results. 5 � Discussion It has been known for a long time that silver nanoparticles exhibit a yellowish/dark brown color in solution due to exci- tation of surface plasmon vibrations in AgNPs, and therefore reduction of the silver ion to AgNPs during exposure to the plant extracts could be followed by color change and thus UV/VIS spectroscopy [27, 33, 34]. In the current study, the AgNPs formation was confirmed by the change in colour of the mixture from light brown to dark brown indicating the successful green synthesis process. The UV/VIS maximum absorption spectra of the synthesized AgNPs was recorded at 427 nm which is in range with previously reported studies on synthesis on AgNPs from plant extracts. Various studies have reported synthesis of AgNPs with UV/VIS absorp- tion maxima at 435 nm [26], 430 nm [34], 420 nm [19, 21], 410 nm [35] among others. The current results further provide, for the first time, a confirmation on the use of the Annona muricata fruits extracts in the green synthesis of AgNPs as a cheap and eco-friendly approach. The importance and use of any substances greatly depend on its stability under different conditions. In the current study, the temperature and heat stability, pH and storage sta- bility of the biosynthesized AgNPs was studied and results have been presented. From the results on temperature stabil- ity, it is evident that at all temperatures tested, the AgNPs Fig. 7   FTIR spectra of func- tional groups from the AgNPs synthesized from fruits extract Fig. 8   SEM micrograph showing the shape of AgNPs synthesized from fruits extract Fig. 9   Energy Dispersive X-ray Spectrometer (EDX) spectra demon- strating the quantitative amounts of different elements present in the AgNPs synthesized from the fruits extract Journal of Inorganic and Organometallic Polymers and Materials 1 3 remained stable maintaining a characteristic absorption maximum of about between 420 nm to about 430 nm which is within the AgNPs range [25, 26]. This is very important implying that the AgNPs can be stable under various temper- ature/heating conditions without losing their effectiveness. It is further important to note that there was an observed gen- eral spike in absorbance in the 350–370 nm region, but later stabilized and followed the normal trend for AgNPs. The possible explanation for this behavior is that these spikes would be due to the conditions of the synthesis process being slightly altered with the initial change in temperatures, even when they not affect the overall stability. In relation to pH stability, it is evident that at all pH conditions tested, the AgNPs remained stable maintaining Fig. 10   TEM micrographs of the AgNPs at different resolutions Fig. 11   XRD diffraction pattern spectra of AgNPs synthesized from fruits extract Journal of Inorganic and Organometallic Polymers and Materials 1 3 a characteristic absorption maximum of about between 410 nm to about 420 nm which is within the AgNPs range [25, 26]. This is very important implying that the AgNPs can be stable under various pH conditions without losing their effectiveness. This property is very important especially of the AgNPs are going to be delivered via the gastrointestinal tract which has gradients of pH conditions. The reported stability plays a critical role in ensuring maintenance of effectiveness of the AgNPs and thus helps overcome one of the obstacles encountered by many conventional crude extracts from plants which lose effectiveness in vivo due to the changing pH gradients as previously reported [17]. Accordingly, the strong relationship between AgNPs absorp- tion spectra at extreme acidic and alkaline pH conditions of 2 and 11 could be attributed to these conditions have nearly similar effects on the AgNPs. Generally, extreme changes in pH affect the shape and size of the particles because of the pH’s ability to alter the charge of biomolecules, which might affect their capping as well as stabilizing abilities. These observations are in line with earlier studies that showed that extreme pH conditions lead to a shift in the peak wavelength indicating a slight increase in size of the particles [29, 30]. As far as storage stability is concerned, it is evident that at all storage temperatures tested for the 3 months, the AgNPs remained stable maintaining a characteristic absorption maximum of about between 410 nm to about 430 nm which is within the AgNPs range. This is very important imply- ing that the AgNPs can be stable under different storage temperature conditions without losing their effectiveness for long periods of time. The notable increase in the absorption of the AgNPs at room temperature compared to other storage conditions, could probably be attributed to the continuous exposure to the same conditions as those used in the syn- thesis process thereby allowing the process of formation of the AgNPs to continue throughout the storage period, albeit at very low rates. Recovery of the biosynthesized AgNPs is of critical importance in the synthetic process. Various methods have been reported about the recovery of AgNPs [25]. These however are not optimal for all plants. In the current study, we developed a blended method for quick and fast recov- ery of the AgNPs. We introduced a step where the AgNPs suspension is frozen for a period of 12–48 h followed by thawing, centrifugation, washing and then drying. The freez- ing step allows for the particles to aggregate and thus easy sedimentation when the centrifugation step is conducted. This is the first study to report on such an optimization in the recovery of AgNPs. FTIR measurements are used to elucidate the functional groups responsible for the biosynthesis as well as stabili- zation and capping of the AgNPs. It is important to note that peaks in FTIR spectra can be divided into two regions: 4000–1500 cm−1 (the functional group region) and the 1500–400 cm−1 (the fingerprint region). Peaks in the func- tional group region arise from complex deformations of the molecule and they may be characteristic of molecular sym- metry, or combination bands arising from multiple bonds deforming simultaneously. On the other hand, peaks in the fingerprint region are characteristic of specific kinds of bonds, and therefore can be used to identify whether a spe- cific functional group is present. FTIR results showed that the functional groups responsible for the formation of the AgNPs from ethanolic extracts of fruits of Annona muricata included; Alkanes and alkyls, aldehydes and esters, nitro groups, alcohol groups, carboxylic acids, amides, alkenes, acids and alkyl halides. These are probably due to the pres- ence of most of the secondary metabolites reported much earlier in the plant [9, 21, 23, 36, 37]. Notably, the narrow band at 1650 cm−1 can be attributed to C=O stretching prob- ably due to the presence of amides which may be account- able for the reduction of Ag+ ions to AgNPs. The AgNPs were approximately spherical in shape with smooth surface. These results are in agreement with the shape of SPR band recognized from the UV–visible spec- trum with absorption maximum at 427 nm. Many previous studies reported different shapes of AgNPs including spheri- cal, conical, cuboidal, hexagonal, pentagonal among oth- ers [7, 25–27, 38]. The spherical AgNPs synthesized in the current study are therefore in line with the expected shapes for AgNPs. Similarly, EDX elemental analysis revealed that the AgNPs were composed of various elements as reported much earlier, with Ag taking the highest percentage compo- sition at 80%. These results indicate the high purity of the AgNPs albeit with a few contaminants at the different subtle concentration which are probably due to the environmental conditions used during the synthesis process. Earlier stud- ies on had also reported elemental compositions of AgNPs having Ag as the principle component [38–40]. From the XRD diffraction patterns, the 2θ peaks observed at 38.41°, 44.61°, and 64.86° corresponds to (111), (200), and (220) reflection planes representing the face centered spherical structure of silver respectively [26, 41]. The extra Table 2   DLS analysis results Counts Intensity (kCnt/s) Attenuation level (%) Diameter (nm) PD index 1 1361 95.1 103.3 1.268e−01 2 1331 95.1 103.9 1.047e−01 3 1378 95.1 103.7 1.105e−01 4 1360 95.1 103.3 1.349e−01 5 1321 95.1 103.1 1.406e−01 Mean 1350 95.1 103.5 1.235e−01 S 24 0.0 0.3 1.547e−02 S2 559 0.0 0.1 2.394e−04 Journal of Inorganic and Organometallic Polymers and Materials 1 3 peaks near to 28.07°, 32.50°, 46.56°, 55.18°, 57.85°, and 67.85° are due to the presence of bio-organic phase on the surface of particles. Generally, the broadening of peaks in the XRD patterns of solids signifies smaller particle size and reflects the effects of the experimental conditions on the nucleation and growth of the crystal nuclei [26, 39, 42]. In comparison to the other eight peaks, the strong reflec- tion at 32.50°, may perhaps signify the growth path of the nanocrystals or presence of other related intermediate com- pounds. The average size of the AgNPs formed in the bio- reduction was estimated as 60.12 nm. TEM analysis further confirmed the crystalline and spherical nature of the mono- dispersed AgNPs. The average particle size as determined by TEM analysis was on average 51 nm, which is within range with that calculated using XRD. Dynamic light scattering is a method that depends on the interaction of light with particles and the method can be used for measurements of narrow particle size distributions especially in the range of 2–500 nm [43]. The AgNPs size was larger as presented by DLS (103.5 nm) as compared to XRD (60.12 nm) and TEM (51 nm). This difference could be explained by the fact that the size measured by DLS is based on a combination of the particles as well as the hydro- dynamic radius which is not a true size of the AgNPs due to the hydration layer around the particles as well as the presence of capping and stabilizing agents as previously explained [21, 32]. Polydispersity Index measures the homogeneous nature of nanoparticles, the smaller the PDI the more homoge- neous nanoparticles. It is basically a representation of the distribution of size populations within a given sample. The numerical value of PDI ranges from 0.0 (for a perfectly uni- form sample with respect to the particle size) to 1.0 (for a highly polydisperse sample with multiple particle size populations). Values of 0.2 and below are most commonly deemed acceptable in practice for polymer-based nanopar- ticle materials, while nanoparticles with PDI smaller than 0.3 is considered acceptable for drug delivery [32, 44]. The synthesized AgNPs had an average PDI of 0.1235, which is a great indication that they are highly homogenous and would be effectively used in various applications. 6 � Conclusions We have reported and optimized for the first time an efficient, eco-friendly and low-cost method for the synthesis and recov- ery of AgNPs using ethanolic extracts of fruits of Annona muricata. The synthesized AgNPs are stable under different temperature, pH and storage conditions. The method used resulted into formation and recovery of spherical crystalline monodispersed AgNPs with an average size of about 60.12 nm and a polydispersity index of 0.1235. With the successful synthesis of AgNPs in the current study, we do recommend further studies aimed at testing the synthesized AgNPs from this method for different biomedical and clinical bioactivi- ties such as Antimicrobial, Anticancer, Anti-inflammatory, Antimalarial, Antidiabetic, Toxicities among others as a step towards the pharmaceutical utilization of these green synthe- sized AgNPs. Acknowledgments  The Authors thank The Sino-Africa Joint Research Centre (SAJOREC), the Uganda Natural Chemotherapeutics Research Institute and The Uganda National Crops Resources Research Insti- tute (NaCRRI) for the support that enabled part of the work to be conducted in their respective Institutions. We also wish to thank Mr Atwijukire Evans, Wembabazi Enoch, Mr Mukasa Yusuf, and Dr Nuwamanya Ephraim of NaCRRI for their professional and techni- cal support rendered during the time of the study. Last but not least, we thank the management of the Microanalytical System at the Earth Science Department, at the University of Fribourg, Switzerland for the additional support offered in the characterization of the AgNPs. Finally, we thank everyone that supported the process of samples col- lection in the different districts of Eastern Uganda, especially Naigaga Maureen, Mukwantampola George and Gaati Joweria (Kaliro District); Naikoba Macklyn and Lulenzi Jalia (Iganga District); as well as Kasajja Anthony, Salya Fred and Kirenzi Juma (Mbale District). Author Contributions  All Authors contributed substantively towards the work. “Conceptualization, YG, HAE, FW and GM; Data curation, YG, ENM, AMM and ESM; Formal analysis, YG and ESM; Fund- ing acquisition, YG, ENM, FW and GM; Investigation, YG and ESM; Methodology, YG, AMM, ESM, HAE, FW and GM; Project admin- istration, YG, ENM, FW and GM; Resources, YG, ENM, ESM, FW and GM; Software, YG and ESM; Supervision, ENM, HAE, FW and GM; Validation, YG, ENM, AMM, ESM, HAE, FW and GM; Writing – original draft, YG; Writing – review & editing, YG, ENM, AMM, ESM, HAE, FW and GM.” Funding  This research was funded by the Pan African University Insti- tute for Basic Sciences, Technology and Innovation Doctoral grant to YG, MB400-0007/17. Data availability  Sufficient data associated with this research and enough to draw the results and conclusions has been provided within the manuscript. However, all datasets have deposited in the public repository, Mendeley Data and is accessible via the link http://dx.doi. org/10.17632​/jkj2x​782wh​.1 [45]. Compliance with Ethical Standards  Conflicts of interest  Part of the work reported in this manuscript has been filed for a grant of patent at the African Regional Intellectual Property Organization (ARIPO) under the title: “Synthesis of Silver Nanoparticles from Extracts of Annona muricata and Use Thereof”. ARIPO Patent Application number: AP/P/2019/011514. The above information notwithstanding, we further declare that the patent appli- cation cannot in any way affect the outcome of this manuscript submis- sion. http://dx.doi.org/10.17632/jkj2x782wh.1 http://dx.doi.org/10.17632/jkj2x782wh.1 Journal of Inorganic and Organometallic Polymers and Materials 1 3 References 1. P. Gonzalez-Melendi, R. Fernandez-Pacheco, M.J. Coronado, E. Corredor, P.S. Testillano, M.C. Risueo, C. Marquina, M.R. Ibarra, D. Rubiales, A. 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Mendeley Data. v1 (2019). https​:// doi.org/10.17632​/jkj2x​782wh​.1 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1016/j.jrras.2015.11.001 https://doi.org/10.1016/j.jrras.2015.11.001 https://doi.org/10.4102/jomped.v2i1.39 https://doi.org/10.3390/pharmaceutics10020057 https://doi.org/10.1007/s00449-008-0224-6 https://doi.org/10.4014/jmb.1610.10019 https://doi.org/10.1016/j.arabjc.2016.01.004 https://doi.org/10.3390/ijms160715625 https://doi.org/10.3390/ijms160715625 https://doi.org/10.1038/srep32539 https://doi.org/10.1038/srep32539 https://doi.org/10.1080/13102818.2018.1448301 https://doi.org/10.1080/13102818.2018.1448301 https://doi.org/10.1016/J.REFFIT.2017.07.002 https://doi.org/10.1016/J.REFFIT.2017.07.002 https://doi.org/10.1016/j.partic.2015.05.005 https://doi.org/10.1088/2043-6262/3/2/025008 https://doi.org/10.1155/2013/313081 https://doi.org/10.1155/2013/313081 https://doi.org/10.17632/jkj2x782wh.1 https://doi.org/10.17632/jkj2x782wh.1 Green Synthesis and Characterization of Highly Stable Silver Nanoparticles from Ethanolic Extracts of Fruits of Annona muricata Abstract 1 Introduction 2 Materials and Methods 2.1 Samples Collection and Authentication 2.2 Samples Preparation and Extraction 3 Chemicals and Reagents 3.1 Preparation of the 1 mM AgNO3 Solution 3.2 Synthesis of Silver Nanoparticles 3.3 Characterization of the AgNPs 3.3.1 UVVIS Measurements to Confirm Formation of AgNPs 3.3.2 TemperatureHeat Stability of the Synthesized AgNPs 3.3.3 pH Stability of the Synthesized AgNPs 3.3.4 Storage Stability of the AgNPs 3.3.5 Recovery of the Synthesized AgNPs 3.3.6 Functional Groups Analysis 3.3.7 SEM and EDX Measurements 3.3.8 TEM Analysis 3.3.9 Crystalline Size Determination Using XRD 3.3.10 Dynamic Light Scattering (DLS) Analysis 4 Results 5 Discussion 6 Conclusions Acknowledgments References