Comparison of biogenic silver nanoparticles formed by Momordica charantia and Psidium guajava leaf extract and antifungal evaluation

Exploiting plant extracts to form metallic nanoparticles has been becoming the promising alternative routes of chemical and physical methods owing to environmentally friendly and abundantly renewable resources. In this study, Momordica charantia and Psidium guajava leaf extract (MC.broth and PG.broth) are exploited to fabricate two kinds of biogenic silver nanoparticles (MC.AgNPs and PG.AgNPs). Phytoconstituent screening is performed to identify the categories of natural compounds in MC.broth and PG.broth. Both extracts contain wealthy polyphenols which play a role of reducing agent to turn silver (I) ions into silver nuclei. Trace alkaloids, rich saponins and other oxygen-containing compounds creating the organic corona surrounding nanoparticles act as stabilizing agents. MC.AgNPs and PG.AgNPs are characterized by UV-vis and FTIR spectrophotometry, EDS and TEM techniques. FTIR spectra indicate the presence of O-H, C = O, C-O-C and C = C groups on the surface of silver nanoparticles which is corresponded with three elements of C, O and Ag found in EDS analysis. TEM micrographs show the spherical morphology of MC.AgNPs and PG.AgNPs. MC.AgNPs were 17.0 nm distributed in narrow range of 5–29 nm, while the average size of PG.AgNPs were 25.7 nm in the range of 5–53 nm. Further, MC.AgNPs and PG.AgNPs exhibit their effectively inhibitory ability against A. niger, A. flavus and F. oxysporum as dose-dependence. Altogether, MC.AgNPs and PG.AgNPs will have much potential in scaled up production and become the promising fungicides for agricultural applications.


Introduction
The development of nanotechnology has designed various nanoparticles with multi-function for many applications such as photovoltaic devices, catalysts, sensors and biomedical/pharmaceutical/antimicrobial products [1]. Metallic  In this study, the green methods biosynthesizing AgNPs were performed with the aqueous extracts of M. charantia and P. guajava leaves (MC.broth and PG.broth). Phytoconstituent screening was carried out to recognize the main phytochemical categories as well as to estimate qualitatively the difference between two these extracts. The biogenic silver nanoparticles (MC. AgNPs and PG.AgNPs) fabricated by MC.broth and PG.broth were characterized by UV-vis and FTIR spectrophotometry, EDS and TEM techniques. Furthermore, the formation reactions and stabilizing mechanism were suggested based on phytochemical compositions and the physicochemical properties of these green AgNPs. In order to demonstrate the antifungal activities of MC.AgNPs and PG.AgNPs, three fungi including Aspergillus niger, Fusarium oxysporum and Aspergillus flavus were cultured on the agar dishes containing different concentrations of these biogenic AgNPs.
Plant broth preparation. Momordica charantia and Psidium guajava leaves without diseases and contamination were carefully chosen. After washing with deionized water three times, each type of leaves was dried at 60˚C in oven. After chopping, Momordica charantia and Psidium guajava leaves (5 g) were added into two different erlenmeyer flasks. DIW of 100 mL was added into each flask. These flasks were heated to 60˚C for 1 hour. Then these aqueous extracts of Momordica charantia and Psidium guajava leaves were filtered by porcelain Buchner filter funnels lined by two Whatman No.1 filter paper sheets, being symbolized as MC. broth and PG.broth respectively ( Fig 1A). These broths were poured in brown bottles stored at 4˚C for other experiments.

Phytoconstituent screening
To test alkaloids, Wagner's reagent was utilized. Three mL of extract was put into test tube. Three mL of concentrated H 2 SO 4 was added into extract. Wagner's reagent (2.5 g I 2 in 250 mL KI solution (5 wt%)) was dropped into an acidified extract. If alkaloids were present, the redbrown precipitate was observed.
To qualitatively analyze saponins, the frothing test was performed. Five mL of extract was put into test tube. One test tube containing MC.broth and another tube containing PG.broth were shaken at the same time for few minutes. The presence of saponins was confirmed if the appeared froth was stable more than 10 minutes.
To identify phenolics and tannins, FeCl 3 test was used. The extract (3 mL) was added into test tube followed by dropping FeCl 3 (5 wt%) of 1 mL. A bluish-black color was produced [19] that could confirm the presence of tannins and phenolics.
To test steroids and triterpenes, Liebermann Burchard test was carried out. Three mL of extract was mixed with acetic anhydride (few drops) in the test tube. One mL of concentrated H 2 SO 4 was added. The steroid was present when there was an appeared green color. If the pink color was seen, there was a presence of triterpenoids.

Green synthesis of AgNPs using Momordica charantia and Psidium guajava leaf extracts
Aqueous solution 1 mM of silver nitrate was prepared for the AgNP biosynthesis. Two beakers contained 4.5 mL of silver nitrate. Then 0.5 mL of MC.broth and PG.broth was separately added into these silver nitrate beakers. These mixtures were stirred for 8 hours at room temperature (RT). The AgNPs fabricated by MC.broth and PG.broth were named as MC.AgNPs and PG.AgNPs respectively. After completing reaction, MC.AgNPs and PG.AgNPs were centrifuged to collect the suspension in the bottom of tube. Adding DIW for washing, then the centrifugation was performed to obtain MC.AgNPs and PG.AgNPs. The washing step was repeated 3 times. The MC.AgNPs and PG.AgNPs were stored in vacuum conditions after lyophilized.

Characterization of MC.AgNPs and PG.AgNPs
The MC.broth, PG.broth, and two reacted mixtures of MC.AgNPs and PG.AgNPs after 8 hours were fivefold diluted with DIW to collect their UV-vis spectra using UV-vis spectrometer (Shimadzu UV-1800, US). The UV-vis spectra were scanned from 350 to 750 nm.

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The lyophilized MC.broth, PG.broth, MC.AgNPs and PG.AgNPs were respectively crushed with KBr at the weight ratio of 1:10. These samples were pelleted and analyzed by FTIR spectrophotometer (Perkin Elmer, US). The scanning wavenumber was in the range of 4000-500 cm -1 .
The size and morphology of MC.AgNPs and PG.AgNPs were observed by TEM (JEOL model JEM-1400, Japan). The elemental composition of MC.AgNPs and PG.AgNPs was confirmed with EDS (Horiba H-7593, UK).

Antifungal activity of MC.AgNPs and PG.AgNPs
PDA (39 g) was dissolved in DIW (1 L). PDA solution was sterilized at 121 o C for 15 minutes. The sterilized PDA solution was poured into the Petri dishes and cooled to form the agar dishes which were shortly called PDA dishes. The sterilized PDA solution was cooled to around 55 o C to mix with tested agents including 0.5 mL of MC.broth, 0.5 mL of PG.broth, MC.AgNPs (20 and 40 ppm) and PG.AgNPs (20 and 40 ppm). The mixtures of PDA solution and each tested agent were poured directly into Petri dishes. The code of each agar dish was explained in Table 1.
Three pathogenic organisms including Aspergillus niger, Fusarium oxysporum, Aspergillus flavus were utilized for antifungal tests of MC.AgNPs and PG.AgNPs. Each fungal strain was spotted in the center of each dish. Each experiment was replicated three times. The incubation temperature was 30˚C. After 24, 48, 72, and 96 hours, the diameters of fungal zones were recorded and averaged. The values were presented as mean ± standard deviation (SD).

Statistical analysis
The experiments were replicated three times and represented as mean ± standard deviation. Student's t test was utilized to analyze all experimental data. P < 0.05 implied that two compared results were statistically significant. P > 0.05 indicated non-statistical difference.

Qualitative tests of phytoconstituents' MC.broth and PG.broth
In aqueous extracts of MC.broth and PG.broth, the phytoconstituent screening tests revealed the presence of some secondary metabolites (Fig 2 and Table 2). Very small amount of redbrown precipitation was observed in MC.broth, but not occurred in PG.broth (Fig 2A) that confirm the trace alkaloids in MC.broth. Fig 2B showed stable froth in both MC.broth and PG. broth, the height of foam layer observed in MC.broth was significantly higher than that in PG. broth. This phenomenon could estimate the saponin amount in MC.broth being 2.5-fold higher than saponin amount in PG.broth. The MC.broth and PG.broth had bluish dark color

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with high intensity when adding FeCl 3 that implied the abundant presence of tannin and polyphenols ( Fig 2C). Indeed, many researches confirmed that the phenolics and saponins (e.g. gallic acid, p-coumaric acid, chlorogenic acid, tannic acid, rutin, naringin, quercetin, epicatechin (-), genistein, naringenin and daidzein) distributed in various M. charantia tissues including leaves [20][21][22]. Diaz-de-Cerio et al. studied about polar compounds in guava leaves, they found 13 ellagic and gallic acid derivatives, quercetin and its derivatives, catechin, gallocatechin, gallic acid, naringenin and many guavinosides [17]. In case of Liebermann Burchard test, no reaction was happened that demonstrated an absence of steroids and triterpenes in MC. broth and PG.broth. Through phytochemical screening results, it was realized that the MC. broth contained more compound categories and especially richer saponins than PG.broth ( Table 2).

Synthesis of MC.AgNPs and PG.AgNPs
The MC.broth had brown color while PG.broth showed light yellow color. After dropping Ag + solution and reacting for 8 hours at room temperature (RT), the color of two mixtures turned reddish brown. This color change indicated the AgNP formation. In MC.broth and PG.broth, many phenolic compounds were present. Phenolic compounds theirselves could play a role of reducing agents to reduce Ag + into Ag o [7,23]. Then Ag nuclei were coagulated to grow into  Fig 3 showed the UV-vis spectra of MC.AgNPs (Fig 3A-i) and PG.AgNPs (Fig 3A-iii) in comparison with MC.broth (Fig 3A-ii) and PG.broth (Fig 3A-iv). The absorption peak of 420 nm was appeared in both MC.AgNPs and PG.AgNPs that indicated the occurrence of AgNPs [24], while two spectra of extracts didn't show that peak. Besides, the MC.AgNPs exhibited another broaden peaks at 540 nm tangled with the 420-nm peak that implied the aggregation behavior. Owing to the higher phytoconstituents of

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MC.broth, the organic compounds were attached on the surface of MC.AgNPs more than PG. AgNPs, that induced the secondary aggregate due to the interaction of nanoparticle organicshells [25]. The FTIR spectrophotometer was operated to identify the organic functional groups on the surface of two AgNP types (MC.AgNPs and PG.AgNPs) in comparison with two extracts (MC. broth and PG.broth) (Fig 3B). In all spectra, it was observed the same main troughs of 3455, 2934, 1623, 1388 and 1048 cm -1 respectively assigned to-OH stretching,-CH stretching,-C = O or-C = C-stretching,-CH 3 bending,-C-O-C-stretching vibrations. The FTIR results revealed that the functional groups of MC.AgNPs and PG.AgNPs were similar together and corresponded with MC.broth and PG.broth. From this fact, it was inferred that the phytoconstituents of extracts attached on the surface of their biosynthesized AgNPs. However, all peaks of each FTIR spectra in the fingerprint regions (500-1500 cm -1 ) were not the same that showed the difference in compound structures of MC.broth, MC.AgNPs, PG.broth and PG.AgNPs. From FTIR results, considering about element components, it was also realized that the MC. AgNPs and PG.AgNPs contained C and O. The EDS analysis was applied to confirm this conclusion as well as demonstrate the presence of Ag. As predicted, the EDS spectra of MC.AgNPs and PG.AgNPs (Fig 3C-i and 3C-ii) indicated the presence of C, O and Ag. The strange occurrence of Cu and Si was explained by contaminating from the grid holder [4,26]. Taken together, the formation mechanism of MC.AgNPs and PG.AgNPs was suggested that the natural compounds belong to phenolics have mainly four types such as phenol, catechol, meta-benzenediol and pyrogallol groups which could donate electron to silver (I) ions to generate silver nuclei. In the reacting mixture, residue of unreacted phenolics, oxidizing forms of phenolics after reaction, saponins and others containing oxygen which can bind with AgNP surface through coordination bonds.

The size and morphology of MC.AgNPs and PG.AgNPs
The morphology and particle size of MC.AgNPs and PG.AgNPs were examined by TEM technique. The MC.AgNPs (Fig 4A) were almost spherical shape and distributed separately together, but some MC.AgNPs trapped in the blurred membrane were observed as clusters. This TEM image of MC.AgNPs was agreed with their above UV-vis spectrum showing the secondary aggregation. Fig 4B exhibited the graph of MC.AgNPs' size distribution which were in the range of 5-29 nm with the average diameter of 17.0 nm. The TEM micrographs of PG. AgNPs (Fig 4C) showed their spherical morphology. Also the blurred membrane kept some PG.AgNPs together but the number of PG.AgNPs in cluster was less than MC.AgNPs. Fig 4D  exhibited the PG.AgNP size range of 5-53 nm, the average diameter was 25.7 nm. Making the comparison, the MC.AgNPs were in narrower size distribution and smaller diameter than PG. AgNPs. Specically, MC.AgNPs had over 75% of small nanoparticles being less than or equal 17 nm which PG.AgNPs achieved only 60%. Other 25% of MC.AgNPs was 23 and 29 nm, while 40% of PG.AgNPs was 29, 41 and 53 nm. Considering the phytoconstituent screening results, it was realized MC.broth with the presence of alkaloids and richer saponin than PG.broth that leads to create the smaller MC.AgNPs than PG.AgNPs. The explanation might be due to high content of capping agents (saponins, alkaloids and other phytoconstituents) inhibiting the silver seed growth into bigger AgNPs. Taken together, all MC.AgNPs and PG.AgNPs were obtained with the nano-size less than 53 nm. This dimension could completely meet the requirements for antimicrobial applications which need the nanoparticles being less than 100 nm. At this scale, the nanoparticles can penetrate into the microorganism membranes to inhibit their growth through interacting with many kinds of proteins, DNA and enzymes [7,8].

Antifungal activity of MC.AgNPs and PG.AgNPs
To examine the antifungal activity of MC.AgNPs and PG.AgNPs, three fungal strains including A. niger, A. flavus and F. oxysporum were seeded in the center of PDA agar dishes in which MC.broth, PG.broth, MC.AgNPs and PG.AgNPs were fortified at various amounts. The mycelium zones of each dish were measured every 24 hours (Table 3). The fungal diameter on each dish was compared to that of PDA dishes, the inhibition taken place when the mycelium zones was smaller than PDA media.
On PDA dishes (control sample), after 24, 48, 72 and 96 hours, the A. niger mycelium diameters respectively were 24.5, 44.6, 65.1 and 89.5 mm; the A. flavus diameters were 20.6, 32.8,  [4,26], the diluted concentration of leaf extracts was not enough strong activity to inhibit the fungal growth.
In case of MC.AgNP20 and PG.AgNP20, as presented detail in Table 3, the A. niger, A. flavus and F. oxysporum zones were significantly smaller than that on PDA dishes (P < 0.05). For example, after 96-hour incubation, the mycelium zones on MC.AgNP20 were 66.5, 60.1 and 49.1 mm; and the mycelium zones on PG.AgNP20 were 67.1, 61.3 and 43.5 mm for A. niger, A. flavus and F. oxysporum respectively ( Table 3). The similarity of fungal diameters on MC. AgNP20 and PG.AgNP20 (P >0.05) implied that two these green AgNPs achieved the same antifungal ability. It was explained by their nanosize being less than 100 nm which dimension was easily able to penetrate inside fungal cells. This penetration of AgNPs could cause the wounds or leakages on fungal membranes. In the biological environment, AgNPs interacted with oxidizers to sustainably generate silver (I) ions. Then Ag + ions could strongly bind with proteins, enzymes and DNA of fungi to break their bio processes that leads to fungus death [27]. Besides, AgNPs were also effectively anchored on the cell membranes or inside fungal cells to disturb the metabolism or proliferation. When increasing the green AgNP concentration to 40 ppm, the radial growth of A. niger and A. flavus was decreased one fourth-fold, F. oxysporum was a half fold deduction. As a result, MC.AgNPs and PG.AgNPs could show the effective antifungal ability in dose-dependence. Looking at Kim et al.'s study, they utilized the commercial AgNPs to test with 18 different fungal strains [28]. The commercial AgNPs exhibited antifungal properties at various concentrations of 10, 25, 50 and 100 ppm [28]. About dose as well as inhibitory effect, the MC.AgNPs and PG.AgNPs were corresponded with those commercial AgNPs. In case of an antimicrobial mechanism, silver nanoparticles were

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degraded in biologically reducing media to release Ag + ions interacting the proteins of cell membranes or inside cell walls that led to inhibit cell division or cause cell dead [8]. Thus these MC.AgNPs and PG.AgNPs covered with naturally organic functional groups might achieve the long-term biodegradation as well as the sustainable release of Ag + ions in vivo. As a result, MC.AgNPs and PG.AgNPs might exhibit the long-term antimicrobial activities. Indeed, surface coating of nanoparticles has been become the strategy for controlling the biodegradation of metallic nanoparticles in physiological conditions. Polysaccharides and poly(ethylene glycol) could modify the metallic nanoparticle surface to prolong their half-life in vivo [8,29]. However, poly(ethylene glycol)-modified nanoparticles become more vulnerable than bare ones both in vitro and in vivo [29] because the generation of anti-polymer antibodies caused the accelerated clearance for poly(ethylene glycol)-modified nanoparticles [30]. The same situation also occurred for other synthetic polymers [30], so they should be not considered in surface modification. Taken together, the natural compounds outside MC.AgNPs and PG.AgNPs might become a promising strategy to stabilize silver nanoparticles for long-term activities and avoid the anti-polymer antibody generation. Nowadays, metallic nanoparticles have been applied prevalently in consumer products, agriculture, medical and high-tech fields that induced the worrisome consequence of nanoparticle pollution. However, silver has been considered as a metal possessing least toxicity even in the accumulation state [31]. In spite of that, the rapid development of silver nanoparticles led emerging concerns related to nano-sized silver toxicity on ecosystem and humans [32]. In case of using AgNPs for agricultural applications, AgNPs will accumulated on soil and/or in sludge that might exhibit the bioactivities in next crops [33]. However, the transport and fate of AgNPs are complicated to fully understand [32,33], it might be leaked into water. In addition, the useful species might be influenced by silver nanoparticles, also the Ag + resistance might happen. Thus the aquatic environment must be controlled to achieve the silver content in acceptable level.

Conclusion
In summary, both aqueous Momordica charantia and Psidium guajava leaf extracts could be successfully applied to fabricated green AgNPs. MC.broth possessed richer phytoconstituents, especially trace alkaloids and more wealthy saponins, than PG.broth that led to form two types of AgNPs being different in dimension and size distribution. UV-vis spectra of MC.AgNPs revealed that the two entangled peaks of 420 nm and 540 nm indicated the formation of AgNPs with secondary aggregation due to physical interaction of organic corona, while PG. AgNPs had only a sharp peak centered at 420 nm. By using TEM technique, the spherical morphology of MC.AgNPs and PG.AgNPs were observed. MC.AgNPs were 17.0 nm distributed in narrow range of 5-29 nm, while PG.AgNPs were 25.7 nm in the nanoscale from 5 to 53 nm. FTIR and EDS spectra confirmed both two these AgNPs were capped with the functional groups originated from leaf extracts. Thus MC.AgNPs and PG.AgNPs were able to be stabilized without any additional steps. Due to their size less than 100 nm, MC.AgNPs and PG. AgNPs could show their highly antifungal efficiency against A. niger, A. flavus and F. oxysporum. So these green AgNPs were synthesized by ecofriendly method overcoming the disadvantages of traditional ones. In the future, MC.AgNPs and PG.AgNPs could be scaled up production and become the promising fungicides for protection of crops.