Synthesis of biogenic silver nanoparticles from the seed coat waste of pistachio ( Pistacia vera ) and their e ﬀ ect on the growth of eggplant

: Herein, the synthesis of silver nanoparticles using extracts of pistachio seed coat waste is investi -gated. The surface plasmon resonance peak at 443 nm was observed in the nanoparticles by using ultraviolet visible spectroscopy ( UV - Vis ) . To identify potential bio molecules involved in the bio - reduction of silver ions, Fourier - transform infrared spectroscopy ( FTIR ) was used. Scanning and transmission electron microscopy ( SEM and TEM ) show irregular shapes with an average size of ∼ 20 nm. The active surface determined by Brunauer, Emmett, and Teller analysis was 22 m 2 /g. The e ﬀ ect of silver nanoparti cles on eggplants sprayed with a nanoparticle suspension of 75 mg/L led to increased plant growth and chlorophyll and carotenoid contents. The ﬂ y ash addition to the soil pro moted plant growth. The highest increase in plant growth occurs when plants were sprayed with 75 ppm AgNPs in 20% ﬂ y ash amended soil.


Introduction
Nanotechnology has emerged in recent years as one of the most important areas of modern science associated with green synthesis [1]. The use of modern techniques to synthesize nanoparticles has become a popular application for a variety of products in the biomedical and healthcare fields [2]. Richard Feynman coined the term "nanotechnology" for the first time in 1959, which is widely regarded as the start of modern nanotechnology [3]. The green synthesis approach has been subjected to a reliable, sustainable, and eco-friendly protocol to synthesize a variety of nanoparticle metal oxides and metal oxides in some cases under exposure to solar radiation to accelerate the synthesis of nanoparticles. A great deal of attention has been paid in recent years to the exploration of waste materials in different areas of human application. One of these areas where scientists focus with zeal [4,5] is the use of plant waste materials with different names such as shell, seed coat fruit peel, skin, hull, etc., containing bioactive compounds [6]. These waste materials are a prime example of an undervalued and unused energy source that can be used as a reducing agent in nanoparticle synthesis. The list of waste materials of plants used for silver nanoparticles synthesis and their particle size is given in the Supplementary Information (Table S1). Due to their unique characteristics, nanoparticles are used in many industrial sectors. Of the various types of nanoparticles, silver nanoparticles (AGNPs) are the most commonly used [7][8][9]. Synthesis of nanoparticles from plant parts is preferred because it is simple, environment friendly, and safe [10,11]. The plant materials are more advantageous, feasible, and simple for nanoparticles synthesis than other materials [12,13]. The use of nanoparticles to enhance plants growth is currently being researched all over the world [14]. Silver nanoparticles (AgNPs) have antimicrobial, catalytic, physiochemical, and optical properties [15]. Brinjal (Solanum melongena L.) is a popular nightshade crop grown all over the world. It is one of the most common and widely consumed vegetables in India. In this country, 730,000 hectares of land are under eggplant cultivation with an annual production of 12,801 million tonnes [16]. Metalbased nanomaterials are being developed by scientists to aid plant growth and development. Nanotechnology has the potential to be a useful tool for delivering pesticides and fertilizers to the target site with accuracy and precision. Nanoparticles injected into plants could have a significant impact, and thus, could be used to improve plant growth and yield in agricultural applications [17]. However, more research work is needed to fully comprehend the molecular mechanism of action of nanoparticles in plant physiology [18]. Plants have been found to be capable of producing the natural mineralized nanomaterials required for their growth under certain conditions [19]. To the best of our knowledge, this is the first study to use aqueous seed coat extract of pista (Pistacia vera) to the biosynthesis of AgNPs as part of a green nanoparticle synthesis approach. The novelty of this article is therefore to describe the green synthesis of AgNPs from the waste part of the seed and to investigate their impact on eggplant plant growth capacity, carotenoid, and chlorophyll content.

Biosynthesis of silver nanoparticles and characterization
Silver nanoparticles were synthesized using the same protocol as in prior research [10,20]. All other chemicals such as sodium hypochlorite (NaOCl), silver nitrate (AgNO 3 ), and the solvents and chemicals used in this study were provided by Merck India Ltd. UV-Vis spectral analysis was used to validate the production of AgNPs using a Shimadzu spectrophotometer UV-Vis 1800 (Japan). Cu K radiation (λ = 0.15405 nm) was used for X-ray diffraction (XRD, PANalytical, X'pert PRO-MPD, The Netherlands). SEM (NOVA nano FE-SEM 450 FEI) and TEM (TECNAI-G-20). were used to examine the morphology and the size of the prepared AgNPs. FT-IR spectra of the plant extract and biosynthesized AgNPs were collected utilizing the KBr pellet technique with PerkinElmer Spectrum 2000 in the 4,000-400/cm range. The specific surface area of biogenic silver nanoparticles using nitrogen adsorption-desorption isotherms (Brunauer-Emmett-Teller (BET)) was calculated with the Nova 3200e.
The seed coat extract of pista (Pistacia vera) was used to synthesize silver nanoparticles (AgNPs). The material was cleaned with distilled water to remove contaminated contents and air-dried in sunlight. The material was ground into a fine powder, and 20 g of the fine powder was placed in a flask with 200 mL double distilled water, which was then refluxed for 30 min. For the next step, the plant extract was cooled and filtered through Whatman filter paper no. 1.
As such, silver nitrate GR was used, which was obtained from Merck (India). In an Erlenmeyer flask, 100 mL of silver nitrate at a concentration of 1 mM was prepared. Then, various concentrations of the seed coat extract (2.5, 5.0, and 10 mL) were added to 90 mL of silver nitrate solution to optimize the rapid reduction of AgNO 3 and left at room temperature to observe the color change; the formation was also recognized by using UV-Visible spectroscopy in the range of 443 nm. Centrifugation at 10,000 rpm for 20 min was used to collect biosynthesized AgNPs, which were then carefully washed with doubledistilled sterile water. The collected nanoparticles synthesized using 10 mL of the extract based on the rapid reduction of AgNO 3 to AgNPs were further investigated by using FT-IR, XRD, UV-Vis, SEM, and TEM spectroscopic techniques.

Plant culture, total leaf chlorophyll, and the carotenoid content
Eggplant seeds were sterilized for 5 min with 0.1% sodium hypochlorite and then washed twice with distilled water. Fifteen days old seedlings of eggplant were sprayed with biosynthesized AgNPs and one set of plants was left without spraying as control (without AgNPs). Each treatment had five replicates. The experiment results were terminated 100 days after spraying of AgNPs. The total chlorophyll and carotenoid content of photosynthetic pigments of eggplants were approximated using the method given by Mackinney [21] and Maclachlan and Saul [22], respectively.

Fly ash
In this experiment, we used loam soil, which was obtained from the fields of Jaipur, India.
Before planting, the soil was autoclaved at 137.9 kPa for 20-25 min, while the fly ash was sun-dried for 10-15 days and mixed (vol:vol) in four proportions with soil: 0 (100), 20, 50, and 100% fly ash. About 3 kg of fly ash and soil mixtures were placed in pots. Fly ash was purchased from a local market in Jaipur, India.

Statistical analysis
The data were analyzed using ANOVA in GraphPad Prism software, and means were compared using mean and standard deviation including the Tukey post-hoc test and Dunnett's multiple comparisons test at the p < 0.05 probability level. The mean and standard deviation presented in the graph was designed using Excel software.

Results and discussion
Previous research studies have reported on the biosynthesis of silver nanoparticles from plant parts [10,11,20]. Therefore, in continuation with our interest in the preparation of silver nanoparticles from the seed coat waste extract of pista, in the current research, AgNPs have been prepared and their impact on eggplant plants is studied.

Fourier infrared spectroscopy analysis (FTIR)
FT-IR spectra of the biogenic synthesis of Ag-nanoparticles and the aqueous extracts of Pistachio shells are displayed in Figure  and AgNPs were used to identify the metabolites responsible for Ag ion reduction ( Figure 1b). Typical infrared absorption bands at 3446.08 and 3436.70/cm showed an indication of the presence of phenol and alcohol with a free functional group (-OH) [23]. The wide infrared bands of the OH group, generally as shown in Figure 1, were overlaid with the NH stretching band [24]. The absorption bands reflected the presence of alkanes (methyl and methylene groups) in the lipids indicating asymmetric stretching bands at 2,926 and 2930.78/cm [23]. The presence of carboxylic ion in the biosynthesized solution [25] was revealed by the bands at 2434.90/cm. The OH and CO functionality of the extract plays a responsible role in reducing and stabilizing the nanoparticles [26].

XRD
The X-ray diffraction (XRD) pattern of the green synthesized AgNPs was recorded in the 2θ range 20-80°, as shown in Figure 2.  Figure 2) are attributed to the silver metal corresponding to hkl values at 210, 111, 200, 220, and 311 silver planes. These peaks are related to the face-centered cubic (fcc) crystal lattice structure of silver (JCPDS Nos. 87-0720, 03-0921, and 04-0783). This indicates that the biosynthesized AgNPs are polycrystalline. Similar peak patterns have also been reported [8,10,20] for AgNPs. The average crystallite size of the synthesized AgNPs was found to be around 20 nm using Scherrer's formula. The XRD pattern shows that the silver nanoparticles synthesized by the seed coat extract are crystalline [27,28]. In addition, the extra peaks appearing in the diffractogram may be due to the biomass residue capping of AgNPs [29].

UV-Vis spectroscopy
To confirm the green synthesis of AgNPs and their stability, UV-Vis analysis was performed. Figure 3b depicts the UV-Vis spectrum of the synthesized AgNPs. It had a ∼443 nm absorption band, which is a characteristic band of AgNPs due to the surface plasma resonance (SPR) of silver. The UV-Vis spectrum of the extract of the seed coat ( Figure 3a) indicated the presence of polyphenols and flavonoids as reported elsewhere [30]. The absorption peaks at about 260 and 290 nm were attributed to the transition from π to π* located within the polyphenols as the antioxidant content of the shell.

Mechanism and stabilization
The polyphenols and flavonoids of the shell play an essential part in the biogenic synthesis of silver nanoparticles by reducing and stabilizing the nanoparticles. They also have other unique properties of adhering to the metal surface to form a coat over AgNPs and thus protect them from aggregation. Based on the GC-MS of pistachio shell extracts published earlier [31][32][33], featuring important biomolecules such as polyphenols, flavonoids, glycosides, terpenoids, and anthocyanins, these biomoleculesin particular phenolic compoundsplay a major role in the reduction of silver ions and prevent them from aggregation; the tentative mechanism ( Figure 4) of silver nanoparticle biosynthesis is drawn based on the screening of literature studies [23,34,35].

SEM, TEM, SAED, and BET studies of biosynthesized AgNPs
SEM data of biosynthesized silver nanoparticles have been used to identify the morphology; it reveals that silver nanoparticles are agglomerated due to induced dehydration but polydispersed with irregular shapes ( Figure 5). TEM data confirm the formation of nanoparticles (AgNPs) with irregular shapes, from rectangular to spherical shapes with an average size of 20 nm ( Figure 6). BET analysis of AgNPs was performed to know additional information on the surface of nanoparticles obtained from the Brunauer-Emmett-Teller (BET) analysis of the specific surface area. The BET analysis of silver nanoparticles displayed that the surface area of the biogenic AgNPs was 21.73 m 2 /g at ambient temperature, and the value obtained from BET correlated with the results obtained from TEM findings (Supplementary file S1). A static volumetric absorption analyzer was also used to calculate the isotherms of N2 absorption-desorption of the biogenic NPs. Figure 6b shows a selected area of the electron diffraction (SAED) pattern of the biogenic AgNPs, which confirms the crystalline nature of the synthesized nanoparticles in the sample, as evidenced by the presence of bright dots.      Figure 7). Concentrations of 150, 300, 500, and 1,000 ppm , fresh weight (g), dry weight (g), root dry weight (g), chlorophyll (mg/g), and carotenoid in fresh leaves (mg/g). Graph (b) with fraction data is constructed separately because the graph is not visualized when combined with graph (a) with large data values. There is no significant difference between values within a column and the same type of treatment followed by the same letter. The mean and standard deviation values were used to create the bar graph (Dunnett's test was used as a post hoc test). Statistically significant level: p < 0.05. were found to be toxic to the eggplant and decreased the plant growth parameters; the highest toxicity was found at 1,000 ppm for the eggplant ( Table 1 and Figure 8). The treatment with 1,000 ppm caused a 30.46% reduction in the plant length. The SEM image of the eggplant leaf sprayed with AgNPs is shown in Figure 9. In our experiments, different concentrations of silver nanoparticles were used to determine the effect of nanoparticles (NPs) on the plant growth parameters and photosynthetic pigments, and it was noticed that the eggplant function is adversely affected after a certain limit of the nanoparticle concentration. Eggplants grown in soil amended with 20% fly ash and sprayed with 75 ppm AgNPs showed a significant increase in the plant growth when compared to plants grown in soil without fly ash (Table 2 and Figure 10). Amendment of the soil with 20% fly ash with 75 ppm AgNPs caused a 10.66% increase in the plant length. However, amendments with 50 and 100% fly ash caused a reduction in the plant growth parameters, and 100% fly ash-amended soil with 75 ppm AgNPs caused a 14.96% decrease in the plant length. A high concentration of AgNPs and also a high concentration of fly ash are not beneficial for the growth of the eggplant. Table 2 shows the positive and negative effects of nanoparticles (NPs) at different concentrations on the plant growth parameters and photosynthetic pigments.

Effect of different concentrations of
Recent research reported that plant response to AgNPs is dependent on the concentration of AgNPs, which can either enhance or inhibit growth. Exposure to specific concentrations of AgNPs may enhance plant growth compared to nonexposed plants, while higher concentrations have a negative effect on plant growth. AgNPs have a concentration-based effect on eggplants, and a high concentration is toxic for plants. Biosynthesized AgNPs at concentrations of 25, 50, and 75 ppm were found beneficial for eggplant plants but 300, 500, and 1,000 ppm were found to be toxic. Higher concentrations of AgNPs resulted in a reduction of biomass in the Arabidopsis plant, according to Kaveh et al. [36]. AgNPs reduced the length of wheat shoots and roots in a dose-dependent manner, as mentioned in the literature [37]. Thuesombat et al. discovered that increased AgNP concentrations in jasmine rice reduced seed germination and subsequent seedling growth [38]. The effect of AgNPs on plant morphology and physiology is determined by their size and shape. Foliar application of AgNPs at different concentrations (20, 40, and 60 mg/L) was found to improve fenugreek plant growth parameters [39]. On mung beans, similar results of improving the role of AgNP treatments were obtained [40]. Growth-promoting AgNP activity on the wheat plant was also registered [41]. Brassica juncea, common bean, and corn all benefit from AgNPs in terms of plant growth and biochemical attributes such as chlorophyll, carbohydrate/protein contents, and antioxidant enzymes [42,43]. Silver is an impressive growth simulator as reported in the literature elsewhere [44]. Furthermore, Krishnaraj et al. [45] discovered that biosynthesized AgNPs influenced seed germination and induced protein and carbohydrate synthesis in Bacopa monnieri. In agriculture, AgNPs were developed as plant-growth stimulators [46] and the report of Khan and Siddiqui [47] states that 25% fly ash-amendment in the soil is better for plant growth, and 50% fly ash amendment and 100% fly ash are harmful to the eggplant plant growth. It is evident from previous studies that AgNPs can affect the plants' growth attributes and metabolic processes. It was reported that AgNPs improved seed germination in pearl millet [48]. Parveen and Rao [34] reported that AgNPs can increase the germination rate, vigor index, and germination index in Brassica juncea. Sadak [39] found that silver nanoparticles could improve fenugreek plant (Trigonella foenum-graecum) growth, yield, and some biochemical aspects. AgNPs can be used for the breeding of chrysanthemum plants. Treatment of AgNPs (20 ppm) could improve the carotenoid content and induce genetic and phenotypic variation in the chrysanthemum plant [49]. An in-depth investigation is needed to understand the actual mechanism of the mediating effects of AgNPs on crops. Danish et al. [50] synthesized silver nanoparticles (AgNPs) from plants and found that the 50 ppm AgNP treatment in Trachyspermum ammi L. plant improved plant growth, biochemical, and antioxidant enzyme activities. They also reported that the AgNP treatment reduced the nematode infection in treated plants.

Statistical analysis
The results were provided as mean ± SD and expressed as an average for the measurement. Test values, degrees of freedom, and p values were used to examine statistical differences and significant means in the experimental data. In all statistical studies, the significant difference was determined using the Dunnett test at a level of 0.05 (or 0.01), which is provided in the supplementary information under statistical analysis. Each experimental value was differentiated to its corresponding control value. In nanoparticles-treated plants, a differential correlation pattern was noted between various concentrations of nanoparticles and parameters for plant growth such as plant length (cm), plant fresh weight (g), shoot dry weight (g), root dry weight (g), chlorophyll in fresh leaves (mg/g), and carotenoid in fresh leaves (mg/g). The total chlorophyll in fresh leaves (r 2 = 0.9859), carotenoid in fresh leaves (r 2 = 0.9116), and root dry weight (r 2 = 0.9787) showed positive correlation at different concentrations of silver nanoparticles. However, different concentrations of fly ash with a fixed concentration of silver nanoparticles (75 ppm) show significant difference on various factors of plant growth as indicated by positive R squared values for the plant length (r 2 = 0.7246), plant fresh weight (r 2 = 0.9160), shoot dry weight (r 2 = 0.9551), root dry weight (r 2 = 0.9934), chlorophyll in fresh leaves (r 2 = 0.9508), and carotenoid in fresh leaves (r 2 = 0.9871). The plant length, plant fresh weight, and shoot dry weight did not have a significant difference mean with different concentrations of silver nanoparticles (see supplementary information S1).

Conclusion
In this study, 10 mL of the pista seed coat waste extract was found to be suitable for harvesting a larger quantity of AgNPs within 30 min. The results explained in this article are therefore novel, which could emerge as a better alternative to the synthesis of AgNPs using waste plant materials. Biologically active compounds such as alkaloids, amino acids, flavonoids, and glycosides were identified from the extracts by the study of functional groups: -OH, -NH, -C]O, -COOH, and -CONH using IR spectrum; previous reports of GC-MS and these phytochemicals may be responsible for the rapid reduction of Ag+, leading to the AgNP formation. Morphological and particle sizes were confirmed using various techniques, such as SEM, TEM, XRD, and BET, and were found to possess polydispersed irregular shapes from rectangular to spherical shapes with an average size of 20 nm. The BET study also confirmed the surface area of biogenic AgNPs to be 21.73 m 2 /g at ambient temperature. The effect of AgNPs on the eggplant has been shown to enhance the growth of the eggplant at low concentrations but higher concentrations are toxic to the eggplant plant: 20% fly ash amendment in soil with spraying 75 ppm AgNPs was found most effective in improving the growth of the eggplant and 50% fly ash amendment in the soil was found to be less effective. Further research is required to determine the degree of toxicity, and the mechanistic approach of AgNPs to plant growth for field applications is important for further use.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Conflict of interest:
The authors declare no conflict of interest.