Biological synthesis and characterization of silver nanoparticles synthesized from Pometia pinnata and Diospyros discolor Fruits

The use of biological agents for nanoparticle biosynthesis is an alternative to the eco-friendly green synthetic method. In this study, fruit extracts from Pometia pinnata (Matoa) and Diospyros discolor (Bisbul) were used as reducing agents to produce silver nanoparticles (AgNPs). The ratio of silver precursors to water extracts from the fruit and the reaction time was observed to determine optimum reaction conditions. The characterizations were carried out using a UV-Vis spectrophotometer, Transmission Electron Microscope (TEM), and Particle Size Analyzer (PSA) to analyze the size and morphology of the AgNPs. With the increase in the volume ratio of extracts and silver nitrate, the absorption peak intensity tended to increase—these results were shown by the color of the colloid. Based on the comparison of these two extracts, the peak absorbance of the synthesis using D. discolor fruit extract was higher than P. pinnata. The result showed D. discolor fruit extracts had faster reaction times for AgNPs synthesis. The average size of silver nanoparticles from D. discolor was 32 nm and from P. pinnata was 51 nm. The use of D. discolor fruit extract tends to produce smaller AgNPs. This method can be developed for further application for antimicrobial nanoparticles and sensors.


Introduction
Phytochemical biodiversity compounds from plants are open to vast potential for their possible utilization and exploration. The role of phytochemical compounds is also very diverse among plants with many functions such as protection for the plants from pests, diseases, and environmental stress [1]. Some even have benefits for human health, such as medicine [2,3]. Diospyros discolor and Pometia pinnata are plants that can be found in the tropics. Diospyros discolor fruit is known to have high tannin compounds [2] and is also known to have saponin and alkaloid compounds [4]. The fruit has been used for medicine against diarrhea, and the infusion from the fruit can be used for stomatitis medication [5]. The fruit also contains an antioxidant compound to scavenge free radicals [6]. As for the fruit of P. pinnata, it is known to be rich in polyphenol compounds and is a known antioxidant [7,8]. Nowadays, phytochemical compounds play a role in nanotechnology applications. The synthesis of nanoparticles by utilizing phytochemical compounds is interesting to explore [9,10]. Phytochemical compounds act as reducing agents in the synthesis of AgNPs. The synthesis process using these biological agents affects sizes, shapes, and chemical compositions, which lead to specific functions for applications in the future. This method tends to be eco-friendly, cost-effective, easily ramped up for large-scale synthesizing, and there is no need for high temperature, energy, pressure, and toxic chemicals [11,12]. Silver nanoparticles can be applied in biomedical sciences for high sensitivity biomolecular detection and diagnostics, antimicrobial agents and therapeutics, and biosensors and bioimaging [13][14][15]. Among all of the metal nanoparticles, AgNPs have good electrical conductivity, chemical stability, catalytic, and antibacterial properties [14]. Phytochemical compounds in the phenol and flavonoid groups are known to have a role in the reduction process of Ag + ions to Ag 0 [11,16]. These compounds are known to be contained in D. discolor and P. pinnata fruit.
Several studies have shown the effectiveness of plant extracts for synthesis of AgNPs using specific plant parts such as leaves [17], roots [15], and stem barks [18]. Therefore, D. discolor and P. pinnata fruit act as silver ion reducing agents. This study explores the potential of D. discolor and P. pinnata extracts as reduction agents in AgNPs synthesis. Based on their chemical compounds, they have the requirement that acts as a reductant, yet no previous studies have been conducted using these plant parts. The variation between the plant extract and silver precursors will affect the reaction speed, size, and distribution of AgNPs [10].

Plant preparation and extraction
The fruits of Diospyros discolor and Pometia pinnata plants (figure 1) were taken from FMIPA UI plant collection. The fruit's flesh was washed to remove all the dirt then peeled. Next, the peeled part of the fruits was dried in an oven with a temperature of 40 o C. After drying, the fruits were ground into powder and filtered through T32 mesh. The powder of each fruit was weighed to make an aqueous extract with a concentration of 2% (w/v). The powder was boiled in double distillate water for 15 minutes. Then, the aqueous extracts were filtered through Whatman filter paper No.1. These filtrates can be used for the biosynthesis process and stored in the refrigerator at 4 o C for a maximum of 2 weeks.  theAgNPs. The size distribution of nanoparticles from TEM resulted in further analysis using ImageJ software. Figure 2 shows the synthesis results of the AgNPs with the ratio variations using the fruit extracts D. discolor (figure 2A) and P. pinnata ( figure 2B). The solution using the D. discolor extract at the1:20 ratio hada yellowish color while at a ratio of 1:2 it hada darker brown color. This result also supported by the absorbance spectrum, which shows AgNPs from a higher ratio of fruit extract showed a sharper and strong absorption band at a wavelength of 430 nm, with a range between 340-600 nm. This result showed thetrend to increase with the increasing of ratio until it reaches the higher absorbance value. Meanwhile, the AgNPs synthesis results using fruit extract from P. pinnata on the ratio 1:2 with showed absorbance value below 2, while at a ratio of 1:20 below 1.5 and the solution color showed no difference. It was also shown from the peak in the range 350-500 nm and all the solution having absorbance values below 1.5. Therefore, it caused the solution color looked similar in all extract and precursor ratios. The brown color formed is the unique characteristic of AgNPs spectrum Surface Plasmon Resonance (SPR). The formation of these colors indicates the formation of AgNPs and the colloid solution from AgNPs. The research conducted by Masum et al. [19], using the fruit section also obtained the color of the solution changing from yellow to brown. The solution color formed looks more concentrated as the ratio between the extract and the precursor of silver increases. Based on the absorption spectrum observation with the in the increasing of time, both solution with ratio of extracts and precursors 1:2 the color of the solution turned to yellow within 15 minutes. The absorption spectrum indicates that the reaction to reduce precursors in both extracts begin to occur (figure 3). After one hour of synthesis using D. discolor there was a very rapid change in color from yellow to brown, getting thicker and tend to be constant at 24 hours. That result shows that the reaction was slowing down due to the lack of availability of reducing agents and silver ions. Meanwhile, when the reaction used extracts from P. pinnata after 1 hour, the solution color was still yellow, then it starts to turn brown in 4 hours and becomes thicker in 24 hours. At some point, the

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The reaction may continue, due to the availability of the reductant and precursors. This result showed that D. discolor on that ratio extract still contain reductant to reduce Ag + ions faster than P. pinnata fruit. The P. pinnata fruit extract tends to have a slower reduction rate. AgNO 3 itself slowly reduced in water, but very low [10]. The speed of this reaction shows the strength of the reducing agent contained from each fruit extracts. Besides, the peak that increases by the time shows the number of nanoparticles formed, the higher the absorbance value can indicate more nanoparticles formed.   Table 1 shows the results of the AgNPs characteristic from PSA, including zeta potential value and polydispersity index. The results showed that the potential zeta value of the synthesis using P. pinnata extract lower (-15.7 mV) than using D. discolor extract (-21.4 mV). The result shows that the AgNPs formation using P. pinnata extract tend to be relatively stable in the range +10-20 mV, which supports dispersion and stability [20]. Based on the result of polydispersity index, AgNPs synthesized with P. pinnata tends to moderately dispersed and AgNPs synthesized with D. discolor tends to be highly polydispersity. The PSA results confirmed by TEM, where the AgNPs from P. pinnata tend to distribute and AgNPs from D. discolor tend to form clusters. The TEM results with 59,000x magnification showed that AgNPs synthesized with D. discolor fruit extract has the size average at 32 nm. Meanwhile, AgNPs from P. pinnata tends to be larger, with an average size of 51 nm ( figure 4). The AgNPs formed in all treatments has the spherical shape.

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The  Biosynthesis mechanism is based on a redox reaction, in which metal ions are reduced to stable the nanoparticles due to the compounds contain in the extract [21]. During the synthesis process plant extracts can have two roles, as reducing agents and as stabilizers to prevent aggregation so that the dispersion and size of nanoparticles can be maintained. Based on information from previous research, D. discolor and P. pinnata fruit extracts are known to contain groups of alkaloids, flavonoids, tannins, phenolics, and terpenoids compounds (table 2). Compounds such as terpenoids [22] and flavonoids [16] are the potential compounds that play a role as a reducing agent in the synthesis of AgNPs. This synthesis process is a bottom-up process, which can induce crystal growth, where atoms, ions, or molecules are assembled [11]. There are still not much full reports have been explaining the mechanism for each species of flavonoid or terpenoid role in reduction and stabilization of AgNPs. The mechanism of nanoparticle formation consists of mainly three stages: reduction of ions, clustering, and further nanoparticle growth. The features of each step depend upon the nature of the reducing agent and its concentration. Based on table 2, the phytochemical screening by Irawan et al. [8], did not detect any flavonoid content containing from P. pinnata fruit extract. This condition might cause a slow reduction to happen while using P. pinnata extract than D. discolor extract. The P. pinnata did not contain flavonoids or it on have a very small number. Therefore, its possible other compounds can act as the reducing agents. The possible reaction occurs is not a single reaction, and there is a possibility of any synergistic reaction which can involve several compounds as reductant from the extract.

Conclusion
Silver nanoparticles synthesis using D. discolor fruit extract have a faster reduction time than using P. pinnata extract, based on the comparison of AgNPs absorption peak. Diospyros discolor tend to have a higher peak than P. pinnata between 24 hours of reaction and tend to be constant. Meanwhile, P. pinnata has a slow reaction time, and the reaction yet still happened after 24 hours. The PSA and TEM results showed AgNPs from P. pinnata had smaller Polydispersity Index (PI), higher Z-average, and larger AgNPs size than AgNPs from D. discolor. Both extracts can be used for AgNPs synthesis and modify with a further process to get their best AgNPs characteristic based on size, shape, stability, and distribution.