PLOS ONE

RESEARCH ARTICLE

Comparison of biogenic silver nanoparticles

formed by Momordica charantia and Psidium
guajava leaf extract and antifungal evaluation
Dai Hai Nguyen1,2, Thanh Nguyet Nguyen Vo1,3, Ngoc Tung Nguyen4, Yern Chee Ching5,
Thai Thanh Hoang Thi ID3*
1 Institute of Applied Materials Science, Vietnam Academy of Science and Technology, Ho Chi Minh City,
Vietnam, 2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology,
Hanoi, Vietnam, 3 Biomaterials and Nanotechnology Research Group, Faculty of Applied Sciences, Ton Duc
Thang University, Ho Chi Minh City, Vietnam, 4 Center for Research and Technology Transfer, Vietnam
Academy of Science and Technology, Hanoi, Vietnam, 5 Department of Chemical Engineering, Faculty of
Engineering, University of Malaya, Kuala Lumpur, Malaysia

* [email protected]
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a1111111111 Abstract
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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
OPEN ACCESS
nanoparticles (MC.AgNPs and PG.AgNPs). Phytoconstituent screening is performed to
Citation: Nguyen DH, Vo TNN, Nguyen NT, Ching
identify the categories of natural compounds in MC.broth and PG.broth. Both extracts con-
YC, Hoang Thi TT (2020) Comparison of biogenic
silver nanoparticles formed by Momordica tain wealthy polyphenols which play a role of reducing agent to turn silver (I) ions into silver
charantia and Psidium guajava leaf extract and nuclei. Trace alkaloids, rich saponins and other oxygen-containing compounds creating the
antifungal evaluation. PLoS ONE 15(9): e0239360.
organic corona surrounding nanoparticles act as stabilizing agents. MC.AgNPs and PG.
https://doi.org/10.1371/journal.pone.0239360
AgNPs are characterized by UV-vis and FTIR spectrophotometry, EDS and TEM tech-
Editor: Raghvendra Bohara, National University of
niques. FTIR spectra indicate the presence of O-H, C = O, C-O-C and C = C groups on the
Ireland, Galway, IRELAND
surface of silver nanoparticles which is corresponded with three elements of C, O and Ag
Received: May 13, 2020
found in EDS analysis. TEM micrographs show the spherical morphology of MC.AgNPs and
Accepted: September 6, 2020 PG.AgNPs. MC.AgNPs were 17.0 nm distributed in narrow range of 5–29 nm, while the
Published: September 22, 2020 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. oxy-
Copyright: © 2020 Nguyen et al. This is an open
access article distributed under the terms of the sporum as dose-dependence. Altogether, MC.AgNPs and PG.AgNPs will have much poten-
Creative Commons Attribution License, which tial in scaled up production and become the promising fungicides for agricultural
permits unrestricted use, distribution, and
applications.
reproduction in any medium, provided the original
author and source are credited.

Data Availability Statement: All relevant data are

within the paper.

Funding: The author(s) received no specific Introduction

funding for this work. The development of nanotechnology has designed various nanoparticles with multi-function
Competing interests: The authors have declared for many applications such as photovoltaic devices, catalysts, sensors and biomedical/pharma-
that no competing interests exist. ceutical/antimicrobial products [1]. Metallic nanoparticles have gained high attention because

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

of their unique characteristics including surface plasmon resonance, thermo-optical properties

and inherently antimicrobial capacity [2]. Depending on particular needs, the nanoparticle
properties were tailored differently. The green synthesis of metallic nanoparticles have been
becoming the alternative routes of chemical and physical methods owing to lower costs,
environmentally friendly and abundantly renewable resources [3–5]. Among green methods,
using plant extracts to fabricate silver nanoparticles (AgNPs) has gained much attention because
of fast procedure, simplicity and feasibility of scaling-up production. Especially, it does not need
to maintain the complex conditions for microbial culture of the AgNP synthesis utilizing fungi,
bacteria and other organisms [6]. In addition, plant biodiversity has opened various topics for
worldwide researchers. To obtain the improved AgNPs with the desired and controllable prop-
erties, a myriad of tropical/herbal plants could be studied [7]. By exploiting this route, the bio-
genic AgNPs were able to be formed within the range less than 100 nm. These sizes facilitate
AgNPs to go through the bacteria/fungi walls and damage membranes as well as to release Ag+
to disturb some mechanisms in cells [7, 8]. In addition, phytochemicals not only could stabilize
AgNPs but also act as an effective molecules in antimicrobial activities [7].
From 1900s, the biogenic AgNPs fabricated by plant extracts have been reported [9]. The
general mechanism was mainly explained by the dual function of secondary metabolites which
could reduce silver ions and stabilize AgNPs in one-pot reaction [9]. Till date, various plant
species were exploited to fabricate green AgNPs and explore their physicochemical properties
and bioactivities. For example, to obtain the green AgNPs with diameter less than 30 nm, it
could be utilized the extracts of banana peel, Boerhaavia diffusa, olive leaf, Prunus japonica
leaf, dried grass, pomegranate juice, Taraxacum officinale leaf, Acalypha indica leaf, Juglans
regia bark, Cucumis sativus [10], Acacia leucophloea bark, Adiantum philippense fronds or
Bacopa monneria [9]. In contrary, the bigger green AgNPs (more than 100 nm) were formed
by Ficus carica latex, Euphorbia lacteal, Euphorbia milii latex, Euphorbia ingens latex, Euphor-
bia hirta leaf, Eucalyptus hybrid leaf, Citrus limon and Azadirachta indica leaf [9], Caltropis
procera fruit and leaf, Rheum palmatum root [10]. About nanoparticle shape, these plant
extract-mediated AgNPs were impacted by plant species, it could be spherical, cubic, hexago-
nal or multi-shaped morphology [9–11]. The size and shape of AgNPs were indicated to be sig-
nificantly impacted by reducing power and stabilizers of reaction mixture. These reagents
effected on the growing and agglomerating stages of AgNP formation. Thus the different com-
ponents in each extract were considered to vary the reducing and capping ability to fabricate
green AgNPs that would assist to finely tune the physicochemical properties of green AgNPs.
Herein, Momordica charantia and Psidium guajava were chosen.
Momordica charantia (M. charantia) belongs to the Cucurbitaceae family [12]. Possessing a
rich source of bioactive compounds, M. charantia exhibited the remarkably biological and
pharmacological activities in anti-cancer, anti-inflammation, anti-diabetes, anti-hyperglyce-
mia, anti-oxidant and hepatoprotection [12]. The recent studies on active compounds of M.
charantia reported the main ingredients were polysaccharides, proteins, saponins, phenolic
compounds (catechin, caffeic acid and gallic acid) [13–15]. Psidium guajava (P. guajava)
belongs to the Myrtaceae family. P. guajava possesses many medicinal activities [16]. It was
proved that P. guajava had positive effects on hepatoprotection, anti-inflammation, anti-oxi-
dant, anti-diarrhea, anti-stomachache, anti-cancer, antioxidant, anti-bacteria, anti-hyperglyce-
mia and antispasmodic [16]. The main components of P. guajava were found to contain
flavonoids, vitamins, phenolic compounds, tannins, essential oils, triterpenoid acids, caroten-
oids and sesquiterpene [16–18]. Although all parts of the tree were able to use for nanoparticle
synthesis, only leaf was chosen to fabricate AgNPs because it contains abundantly bioactive
ingredients. In addition, leaf harvest is easy, leaf regeneration is rapid that will bring an eco-
nomical effect.

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

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 reac-
tions 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 oxy-
sporum and Aspergillus flavus were cultured on the agar dishes containing different concentra-
tions of these biogenic AgNPs.

Materials and methods

Materials
Momordica charantia and Psidium guajava leaves were supplied by the medicinal plant garden
of Tra Vinh University (Tra Vinh province, Vietnam). Silver nitrate (AgNO3), sulfuric acid
(H2SO4), ferric chloride (FeCl3), potassium iodide (KI), potassium bromide (KBr) and iodine
(I2) were bought from Sigma-Aldrich (Merck, Darmstadt, Germany). Acetic anhydride
((CH3CO)2O) was purchased from Labkem (Casablanca, Morocco, US). F. oxysporum, A.
niger and A. flavus were taken from Institute of Applied Materials Science, Vietnam Academy
of Science and Technology (Ho Chi Minh city, Vietnam). Potato dextrose agar (PDA, compo-
sition: agar of 15 g/L, dextrose of 20 g/L, potato extract of 4 g/L) was bought from Sigma-
Aldrich (Merck, Darmstadt, Germany). Deionized water (DIW) was purified by Milli-Q HX
7150 machine (Merck Millipore, France).
Plant broth preparation. Momordica charantia and Psidium guajava leaves without dis-
eases 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 Buch-
ner 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 H2SO4 was added into extract. Wagner’s reagent (2.5 g I2 in 250 mL
KI solution (5 wt%)) was dropped into an acidified extract. If alkaloids were present, the red-
brown 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, FeCl3 test was used. The extract (3 mL) was added into
test tube followed by dropping FeCl3 (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

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

Fig 1. Extraction procedure of aqueous Momordica charantia and Psidium guajava leaf extracts (respectively symbolized as MC.broth and PG.broth) (A); Biosynthesis
procedure of silver nanoparticles using MC.broth and PG.broth (respectively symbolized as MC.AgNPs and PG.AgNPs). (Created with BioRender.com).
https://doi.org/10.1371/journal.pone.0239360.g001

H2SO4 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 temper-
ature (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 centrifuga-
tion 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 spectrome-
ter (Shimadzu UV-1800, US). The UV-vis spectra were scanned from 350 to 750 nm.

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

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 spec-
trophotometer (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 con-
firmed 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 oC 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 oC 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 com-
pared results were statistically significant. P > 0.05 indicated non-statistical difference.

Results and discussion

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 red-
brown 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

Table 1. The code of agar dishes utilized in antifungal tests.

Agar dish code Compositions of agar dishes
PDA Sterilized PDA solutions
MC.broth Sterilized PDA solution + 0.5 mL of MC.broth
PG.broth Sterilized PDA solution + 0.5 mL of PG.broth
MC.AgNP20 MC.AgNPs homogenized in a sterilized PDA solution at 20 ppm
MC.AgNP40 MC.AgNPs homogenized in a sterilized PDA solution at 40 ppm
PG.AgNP20 PG.AgNPs homogenized in a sterilized PDA solution at 20 ppm
PG.AgNP40 PG.AgNPs homogenized in a sterilized PDA solution at 40 ppm
https://doi.org/10.1371/journal.pone.0239360.t001

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

Fig 2. The Wagner’s test: adding solution of I2/KI into MC.broth (left) and PG.broth (right), an occurrence of very little red-brown precipitate in MC.broth indicated
tracing alkaloid presence (A); foam test: after shaking MC.broth (left) and PG.broth (right) test tubes for few minutes, the froth formation being stable for 10 minutes
showed the presence of saponins (B); FeCl3 test: after FeCl3 was respectively added into MC.broth (left) and PG.broth (right), the color was turned into bluish-black
color that implied the presence of polyphenols (C); Liebermann Burchard test: MC.broth (left) and PG.broth (right) were tested by Liebermann Burchard reaction,
green or pink color was not appeared that implied the absence of steroids and triterpenes (D).
https://doi.org/10.1371/journal.pone.0239360.g002

with high intensity when adding FeCl3 that implied the abundant presence of tannin and poly-
phenols (Fig 2C). Indeed, many researches confirmed that the phenolics and saponins (e.g. gal-
lic acid, p-coumaric acid, chlorogenic acid, tannic acid, rutin, naringin, quercetin, epicatechin
(-), genistein, naringenin and daidzein) distributed in various M. charantia tissues including
leaves [20–22]. 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, gallocate-
chin, 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 Ago [7, 23]. Then Ag nuclei were coagulated to grow into

Table 2. The phytoconstituent screening of MC.broth and PG.broth.

Tests MC.broth PG.broth Phytoconstituents
Wagner’s test + - Alkaloids
Foam test +++++ ++ Saponins
FeCl3 test +++ +++ Tannins and phenolics
Liebermann Burchard test - - Steroids and triterpenes

Notes: (-): absence; (+) trace amount; (++), (+++), (+++++): presence at low, medium and high level.

https://doi.org/10.1371/journal.pone.0239360.t002

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

Fig 3. UV-vis spectra of MC.AgNPs (i), MC.broth (ii), PG.AgNPs (iii) and PG.broth (iv): The absorption peak of 420 nm was appeared in both MC.AgNPs and PG.
AgNPs that indicated the presence of silver nanoparticles (A); FTIR spectra of MC.broth (i), MC.AgNPs (ii), PG.broth (iii) and PG.AgNPs (iv) showed the presence of
organic functional groups (B); EDS spectra of MC.AgNPs (i) and PG.AgNPs (ii) exhibited that silver, carbon and oxygen were contained in MC.AgNPs and PG.AgNPs,
while Si and Cu were strange elements contaminated by TEM grids (C).
https://doi.org/10.1371/journal.pone.0239360.g003

silver nanoparticles. Depending on the stabilizing agents (saponins, alkaloids and oxidized
compounds), silver nanoparticles were defined their stability, dimension and morphology. To
demonstrate the formation of silver nanoparticles, UV-Vis spectrophotometry was utilized.
Due to the local surface plasmon resonance (SPR), AgNPs could absorb the light in UV-vis
region. For clear observation, two reacted mixtures and two extracts were scanned by UV-Vis
spectrophotometer from 350 to 750 nm. The 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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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

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 organic-
shells [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,–CH3 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 phytocon-
stituents 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 con-
clusion 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 occur-
rence 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 natu-
ral compounds belong to phenolics have mainly four types such as phenol, catechol, meta-ben-
zenediol 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 tech-
nique. 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 sec-
ondary 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 sil-
ver 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].

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

Fig 4. The MC.AgNPs’ TEM micrograph (A) showed the spherical shape and individual nanoparticles, but some MC.AgNPs trapped in the
blurred membrane; the size distribution graph of MC.AgNPs (B) exhibited that MC.AgNPs’dimension was in the range of 5–29 nm; the PG.
AgNPs’ TEM micrograph (C) implied that the PG.AgNPs were less cluster than MC.AgNPs; the size distribution graph of PG.AgNPs (D)
indicated that PG.AgNPs were in the range of 5–53 nm.
https://doi.org/10.1371/journal.pone.0239360.g004

Antifungal activity of MC.AgNPs and PG.AgNPs

To examine the antifungal activity of MC.AgNPs and PG.AgNPs, three fungal strains includ-
ing 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 myce-
lium 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 diam-
eters respectively were 24.5, 44.6, 65.1 and 89.5 mm; the A. flavus diameters were 20.6, 32.8,

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

Table 3. The diameter of mycelium zones (mm) of A. niger, A. flavus and F. oxysporum proliferated on various agar dishes.
PDA MC.broth MC.AgNP20 MC.AgNP40 PG.broth PG.AgNP20 PG.AgNP40
A. niger 24h 24.5 ± 0.5 24.8 ± 0.2 18.8 ± 0.2 14.6 ± 0.5 24.5 ± 0.2 19.3 ± 0.5 12.8 ± 0.2
48h 44.6 ± 0.5 44.3 ± 1.1 28.5 ± 0.8 22.3 ± 1.1 44.8 ± 0.5 30.0 ± 0.5 18.8 ± 0.7
72h 65.1 ± 0.2 64.8 ± 0.2 45.8 ± 0.7 36.8 ± 0.2 64.2 ± 0.5 46.8 ± 0.2 34.1 ± 0.2
96h 89.5 ± 0.5 88.6 ± 1.1 66.5 ± 0.5 53.3 ± 0.5 89.5 ± 0.5 67.1 ± 0.2 48.3 ± 0.5
A. flavus 24h 20.6 ± 0.2 20.3 ± 0.5 16.8 ± 0.2 12.8 ± 0.2 20.7 ± 0.2 16.5 ± 0.5 11.8 ± 0.2
48h 32.8 ± 0.2 31.0 ± 1.0 24.0 ± 0.5 18.0 ± 0.5 32.3 ± 0.8 24.8 ± 0.2 16.5 ± 0.5
72h 57.5 ± 0.5 56.0 ± 1.0 41.1 ± 0.2 33.1 ± 0.2 54.7 ± 0.5 43.5 ± 0.5 27.8 ± 0.2
96h 89.3 ± 1.1 88.3 ± 1.5 60.1 ± 0.2 48.8 ± 0.2 88.8 ± 0.2 61.3 ± 0.2 43.1 ± 0.2
F. oxysporum 24h 11.1 ± 0.2 11.0 ± 0.8 7.8 ± 0.2 7.3 ± 0.2 10.9 ± 0.2 7.3 ± 0.2 5.8 ± 0.5
48h 24.0 ± 0.5 23.5 ± 0.5 17.8 ± 0.2 12.1 ± 1.0 24.5 ± 0.8 16.0 ± 1.0 11.0 ± 1.5
72h 52.0 ± 1.0 50.1 ± 0.7 35.7 ± 0.8 22.0 ± 1.0 52.0 ± 0.5 31.6 ± 0.7 16.5 ± 0.5
96h 73.3 ± 1.5 71.3 ± 1.5 49.1 ± 1.2 30.6 ± 0.5 72.8 ± 0.2 43.5 ± 0.5 23.1 ± 0.2
https://doi.org/10.1371/journal.pone.0239360.t003

57.5 and 89.3 mm; the F. oxysporum ones were 11.1, 24.0, 52.0 and 73.3 mm. After 96 hours, A.
niger and A. flavus were spread out full surface of dishes, but F. oxysporum achieved only near
edge of dishes. So F. oxysporum proliferation was slower than two others (P < 0.05). On MC.
broth, at the time points of 24-hour, 48-hour, 72-hour and 96-hour incubation, A. niger diame-
ters were 24.8, 44.3, 64.8 and 88.6 mm; A. flavus diameters were 20.3, 31.0, 56.0 and 88.3 mm;
F. oxysporum diameters were 11.0, 23.5, 50.1 and 71.3 mm. On PG.broth, with these respec-
tively above time intervals, A. niger diameters were 24.5, 44.8, 64.2 and 89.5 mm; A. flavus
diameters were 20.7, 32.3, 54.7 and 88.8 mm; F. oxysporum diameters were 10.9, 24.5, 52.0 and
72.8 mm. Making the comparison between two extracts with PDA dishes, the radial growth of
three fungal strains were similar together at each time interval (P > 0.05). MC.broth and PG.
broth didn’t exhibit the antifungal effect against A. niger, A. flavus and F. oxysporum at the
using concentration for synthesis of AgNPs. Being similar to other studies [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. fla-
vus 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 concentra-
tion 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 exhib-
ited 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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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

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, sur-
face coating of nanoparticles has been become the strategy for controlling the biodegradation
of metallic nanoparticles in physiological conditions. Polysaccharides and poly(ethylene gly-
col) 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 situ-
ation also occurred for other synthetic polymers [30], so they should be not considered in sur-
face 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 emerg-
ing 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 compli-
cated 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 mor-
phology 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 stabi-
lized 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. oxy-
sporum. So these green AgNPs were synthesized by ecofriendly method overcoming the disad-
vantages 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.

Author Contributions
Conceptualization: Dai Hai Nguyen.
Data curation: Dai Hai Nguyen, Thanh Nguyet Nguyen Vo, Thai Thanh Hoang Thi.

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PLOS ONE Biogenic silver nanoparticles formed by M. charantia and P. guajava leaf extract

Formal analysis: Thanh Nguyet Nguyen Vo, Thai Thanh Hoang Thi.
Funding acquisition: Dai Hai Nguyen.
Investigation: Yern Chee Ching.
Methodology: Dai Hai Nguyen, Ngoc Tung Nguyen, Yern Chee Ching, Thai Thanh Hoang
Thi.
Project administration: Dai Hai Nguyen, Yern Chee Ching.
Supervision: Dai Hai Nguyen, Ngoc Tung Nguyen, Yern Chee Ching.
Validation: Thai Thanh Hoang Thi.
Visualization: Yern Chee Ching.
Writing – original draft: Thai Thanh Hoang Thi.
Writing – review & editing: Thai Thanh Hoang Thi.

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