Journal of Photochemistry & Photobiology, B: Biology 201 (2019) 111649

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Journal of Photochemistry & Photobiology, B: Biology

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Alpinia nigra fruits mediated synthesis of silver nanoparticles and their T

antimicrobial and photocatalytic activities
Debjani Baruaha,b, Raj Narayan Singh Yadavc, Archana Yadavd, Archana Moni Dasa,
⁎

a
Natural Products Chemistry Group, Chemical Science and Technology Division, CSIR- North East Institute of Science and Technology, Jorhat, Assam 785006, India
b
Centre for Biotechnology and Bioinformatics, Dibrugarh University, Dibrugarh, Assam 786004, India
c
Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam 786004, India
d
Biotechnology Group, Biological Science and Technology Division, CSIR- North East Institute of Science and Technology, Jorhat, Assam 785006, India

ARTICLE INFO ABSTRACT

Keywords: In the present systematic study, silver nanoparticles have been synthesized using the fruits of Alpinia nigra. Apart
Silver nanoparticles from the presence of saponins, glycosides, alkaloids, steroids, the extract of A. nigra fruits are rich in polyphenols.
Alpinia nigra The Total Flavonoid and Phenol Content of A. nigra fruits extract is 718 mgRE/g extract and 74.9 mgGAE/g
Antimicrobial extract respectively. The formation of the nanoparticles was validated through characterization techniques like
Klebsiella pneumoniae
UV–Vis spectroscopy, X- ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Energy dispersive X-
Photocatalytic
ray spectroscopy (EDX). The spherical shape of silver nanoparticles is observed in Transmission Electron
Microscopy (TEM) images. The average particle size of the silver nanoparticles is 6 nm. The biomolecules of the
fruit extract played the dual role of reducing and capping agents which is evident from Fourier Transform
Infrared (FTIR) spectrometer and Scanning Electron Microscopy (SEM) image analysis. The A. nigra capped silver
nanoparticles exhibited promising antimicrobial activity against gram negative bacteria Klebsiella pneumoniae,
gram positive bacteria Staphylococcus aureus and the pathogenic fungus, Candida albicans. Amongst the three
pathogens, Klebsiella pneumoniae is the most susceptible to silver nanoparticles. Furthermore, the nanoparticles
efficiently catalysed the degradation of the anthropogenic dyes Methyl orange, Rhodamine B and Orange G in
the presence of sunlight. The photocatalytic degradation process follows the pseudo-first order kinetics. These
results confirm that the silver nanoparticles can be efficiently synthesized via a green route using A. nigra fruits
with applications as antimicrobial and catalytic agents.

1. Introduction
Nanoparticle research has seen an increased interest in the last two
decades because of the interesting properties of particles at the nano
level and their varied applications. Noble metal nanoparticles possess
extraordinary optical, thermal, chemical properties and are being
widely used in industrial, electronics and biomedical fields [1]. As
compared to their bulk counterparts, nanoparticles have large surface
to volume ratio which significantly enhances their properties. Owing to
good conductivity, chemical stability, photonic, optoelectronic, cata-
lytic and antimicrobial properties, silver nanoparticles have attracted
an intensive research interest [2]. Silver is known for its antimicrobial
properties since ancient times. The development of resistance of pa-
thogenic organisms to antibiotics has led to the search for alternative
efficient antimicrobials. With the advent of nanotechnology this anti-
microbial potential of silver has been enhanced at the nanoscale and its
use as a bactericidal material has flourished. Besides the shape and
composition, the size and capping of nanoparticles are also important
factors affecting the properties and efficacy of the nanoparticles
[3].Chemical and physical methods of nanoparticles synthesis such as
sol-gel method, chemical vapour deposition, physical vapour deposi-
tion, laser ablation, high energy ball milling and sputter deposition
involves toxic reducing agents, high cost and high energy consumption.
Additionally, the industrial scale production is quite expensive. The use
of non-polar solvents and other toxic reagents in the synthesis process
also limit their use in clinical fields. Biological synthesis of nano-
particles, specially using plants is both environmentally benign and cost
effective. This bio-based method also facilitates easy scale-up in pro-
duction [4].
Furthermore, the small size of silver nanoparticles also enhances
their catalytic property due to the increased availability of active sites.
Industrial effluents which consist of various organic dyes contaminate

⁎
Corresponding author.
E-mail address: archanamoni@neist.res.in (A.M. Das).

https://doi.org/10.1016/j.jphotobiol.2019.111649
Received 5 June 2019; Received in revised form 11 September 2019; Accepted 9 October 2019
Available online 02 November 2019
1011-1344/ © 2019 Published by Elsevier B.V.
D. Baruah, et al.
nearby water bodies and have a toxic effect on the aquatic life [5].
Toxic dyes can be removed by various processes such as adsorption,
photo-electron degradation and microwave assisted degradation [6,7].
However, these processes consume high energy and use organic sol-
vents. Nanoparticle based dye degradation is advantageous as it offers
quick reduction of the organic dyes while being a green and simple
alternative. Recent years have seen the development of several bio-
based metal nanoparticles like Au, Ag, ZnO, CuO which have shown the
ability to degrade dyes [8–11]. Recently, Zingiber officinale [12], Ade-
nium obesum [13], Tephrosia purpurea [14], Allium ampeloprasum [15]
were used for the synthesis of silver nanoparticles.
Alpinia nigra (Gaertn.) Burtt (A. nigra) is a biennial shrub endemic to
Southeast Asia including Thailand, China, Bangladesh, Sri Lanka and
India. This plant belongs to the Zingiberaceae family. Apart from being
used traditionally for the treatment of bronchitis, gastric ulcers, in-
testinal parasitic infections [16], it is also used as a vegetable and in the
preparation of local dishes by the people of North East India. The
leaves, rhizomes and seeds have been reported to be rich in phyto-
chemicals such as terpenoids, glycosides, polyphenols and sterols
[17,18].
In our study, we have used the fruits of A. nigra for the synthesis of
silver nanoparticles. The A. nigra capped silver nanoparticles (AgNP-
AFW) were screened for their antimicrobial and photocatalytic activ-
ities.
2. Materials and Methods
The mature fruits of A. nigra were collected from CSIR-NEIST, Jorhat
campus during the month of November and December. AgNO3 and
media for antimicrobial assay and other reagents for phytochemical
tests were purchased from Merck India Ltd. Rhodamine B, Methyl or-
ange and Orange G were also purchased from Merck. The reagents used
in this research were of analytical grade and used as such without
further purification.
2.1. Plant Extract Preparation, Phytochemical Screening and HPLC
Analysis
The fruits were deseeded, dried in shade and ground into a fine
powder. Ten gram of A. nigra milled material was heated in double
distilled water at 50 °C for 45 min. After cooling, the broth was filtered
and the aqueous extract thus obtained was used for silver nanoparticle
synthesis.
The aqueous extract of A. nigra fruits (AFW) were screened for the
presence of phenols, flavonoids, saponins, steroids, glycosides, terpe-
noids and alkaloids using the standard methods of Harborne, Trease
and Evans [19,20]. Additionally, the total phenolic and flavonoid
content was determined by Folin-Ciocalteu method of Singleton [21]
and Aluminium chloride method of Chang et al. [22] with slight
modifications. The standards taken for quantitative analysis of poly-
phenolic compounds were Gallic acid and Rutin.
One milliliter of fresh AFW was filtered through a 0.22 μm filter for
HPLC analysis. AFW (20 μl) was analysed in a Dionex Ultimate 3000
HPLC, Thermo Scientific with Acclaim 120, C18 column (5 μm;
4.6 × 250 mm). The sample was eluted with 60% methanol in 1%
CH3COOH in water at a flow rate of 0.8 ml/min. Rutin was used as
standard sample.
2.2. Biosynthesis of Silver Nanoparticles Using A. nigra Fruits
To 50 ml of 2 mM AgNO3 solution, 8 ml AFW was added and stirred
vigorously at room temperature. The colour of the colloidal solution
changes to reddish-brown within 15 min. The appearance of reddish-
brown colour is an indication regarding the formation of silver nano-
particles which is further confirmed by UV–Vis spectroscopy. The stir-
ring was continued for another 2 h and monitored periodically in
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Journal of Photochemistry & Photobiology, B: Biology 201 (2019) 111649
UV–Vis spectroscopy. The aqueous extract was used for the dual pur-
pose of reduction of AgNO3 and stabilization of the synthesized silver
nanoparticles.
2.3. Characterization
The formation of silver nanoparticles was monitored by measuring
the absorption spectra in a Hitachi U3900 UV–Vis spectrophotometer.
The colloidal solution was centrifuged at 8000 rpm for 10 min and then
washed thrice with distilled water to obtain the silver nanoparticle
(AgNP-AFW) pellets. These pellets were dried and used for further
studies. The shape and size of the as-synthesized nanoparticles were
measured by transmission electron microscopy (TEM) in a JEM-2100
HRTEM. TEM samples were drop casted on a carbon coated copper grid
and allowed to dry completely and then placed at 20 kV potential. The
morphology of AgNP-AFW was studied by scanning electron micro-
scopy (SEM) in a ZEISS, SIGMA instrument. The crystalline nature of
the nanoparticles was determined in an X-ray diffractometer (ULTIMA,
Rigaku, Japan) with a Cu K α X-ray radiation (λ = 1.54Ǻ) at generator
voltage 40 kV and generator current 40 mA. The scanning was done in
the range of 5°–90° at a scanning rate of 3°/min. The possible biomo-
lecules involved in the reduction of AgNO3 and stabilization of AgNP-
AFW was determined by Fourier transform infrared spectroscopy
(FTIR). The infrared adsorption spectra were measured in a Perkin-
Elmer 2000 series FTIR spectrophotometer using KBr pellets. X-ray
photoelectron spectra (XPS) analysis was conducted on a Thermo-
Scientific ESCALAB Xi+ spectrometer with a monochromatic Al Kα X-
ray source (1486.6 eV) and a spherical energy analyser that operates in
constant analyser energy (CAE) mode using the electromagnetic lens
mode. Zeta potential and Dynamic Light Scattering values were mea-
sured in a Nano ZS Zetasizer (Malvern).
2.4. Antimicrobial Activity of A. nigra Supported Silver Nanoparticles
(AgNP-AFW)
The antimicrobial potential of AgNP-AFW was determined by the
agar well diffusion method [23]. Staphylococcus aureus (ATCC 11682),
Klebsiella pneumoniae (ATCC 13883) and Candida albicans (MTCC 3017)
were taken as test pathogens. Mueller Hinton Agar (MHA) and Potato
Dextrose Agar (PDA) were used for antibacterial and antifungal assays
respectively. Eighty microliter log culture (1 × 108 CFU/ml) of the test
pathogens were evenly spread on sterile agar plates. Using a cork borer,
6 mm wells were made in the agar plates. One hundred microliter of
AgNP-AFW (100 μg/ml) and AFW was loaded in the wells. Forty mi-
croliter of the antibacterial standard Neomycin (1 mg/ml) and anti-
fungal standard Nystatin (2 mg/ml) were taken. DMSO was used as
negative control. After loading of all the samples, the bacterial plates
were incubated at 37 °C for 16–18 h while the fungal plate was in-
cubated at 30 °C for 24–48 h. A clear zone of inhibition (ZOI) observed
after the incubation period was measured using a scale. All the tests
were done in triplicates.
The minimum inhibitory concentration (MIC) was determined by
broth dilution method [24]. Mueller Hinton Broth (MHB) and Potato
Dextrose Broth (PDB) were used for the bacterial and fungal strains
respectively. 106 CFU/ml fresh cultures were loaded onto each tube
containing sterile MHB or PDB. AgNP-AFW in varying concentrations
(10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 and 120 μg/ml) were then
loaded. The experiment included broth and inoculum as control while
broth and AgNP-AFW and only broth were kept as negative controls.
The bacteria inoculated tubes were incubated at 37 °C for 16–24 h while
the fungal tubes were incubated at 30 °C for 24–48 h. The OD was
measured at 600 nm to determine the MIC. The lowest concentration
with over 90% inhibition was the MIC. To determine the number of
viable cells at each tested concentration, 100 μl of sample was taken
from each tube and spread on MHA (bacteria) and PDA (fungal) plates.
D. Baruah, et al.
The bacterial plates were incubated at 37 °C for 24 h while the fungal
plates were incubated at 30 °C for 48 h. After incubation, the colonies
were calculated using Digital Colony Counter (Himedia). The number of
colonies counted were expressed as percentage of the number of co-
lonies grown on control plates [25]. MBC/MFC was the lowest con-
centration which reduced the viability by > 99% relative to the control
[26].
2.5. Photocatalytic Activity of AgNP-AFW
The photocatalytic activity of the green synthesized silver nano-
particles was studied for Rhodamine B (RhB), Methyl orange (MO) and
Orange G (OG) under sunlight irradiation. To each 100 mg/l solution of
the dyes, 5 mg AgNP-AFW was added and stirred in the dark for 30 min
to achieve adsorption/desorption equilibrium. Thereafter, the reaction
was shifted outside to be carried out under sunlight. The progress of the
reaction was monitored by measuring maximum absorbance of the re-
action mixtures at intervals of 15, 30, 45, 60 and 90 min after initiating
the reaction under sunlight. A control set without the catalyst was also
maintained.
3. Results and Discussion
3.1. Phytochemicals Screening and the Synthesis of Nanoparticles
The aqueous extract of A. nigra fruits (AFW) showed the presence of
a wide array of phytochemicals which is listed in Table 1. 718 mgRE/g
extract is the total flavonoid content and 74.9 mgGAE/g extract is the
total phenolic content in AFW. Studies have shown that phytochemicals
play a major role in the synthesis of nanoparticles which is accom-
panied by capping of the metallic nanoparticles to prevent agglom-
eration [27]. The presence of polyphenolic compounds also suggests
that AFW has the potential to be a bio reductant in the synthesis of
silver nanoparticles. Basically, the synthesis of nanoparticles consists of
three stages viz. reduction of ions, cluster formation and nanoparticle
growth. Here, the OH– groups of the phenols and flavonoids reduces
Ag+ to Ag0, possibly by transforming the enol form in polyphenolic
compounds to keto form. This would release a reactive hydroxyl atom
for reduction of silver ions. The silver ions may react with the poly-
phenolic compounds through the most reactive OH– group which
would reduce the Ag+ to AgNP and provide stability from agglomera-
tion [28].
The first indication of nanoparticle formation can be observed with
change in colour of the colloidal solution. Shortly after addition of AFW
to the precursor solution, the colour changes to reddish-brown in-
dicating the formation of silver nanoparticles.
3.2. UV- Vis Spectroscopy
The UV–vis spectra of silver nanoparticles synthesized using plant
extract (AFW) are shown in Fig. 1(a). The maximum absorption band at
430 nm indicates the formation of silver nanoparticles. The SPR band
was recorded at 15 mins and 60 mins. The intensity of the absorbance
band increased very slightly at 60 min and this remained constant at
even 120 mins. The peak observed for AgNP-AFW is due to surface
plasmon resonance (SPR) which is the collective excitation of the
electrons present on the surface of the nanoparticles.
Table 1
Phytochemical tests results of A. nigra fruit extract (AFW).
Phytochemical tests Phenols Flavonoids Saponins
AFW + + + +
+ indicates presence and – indicates absence.
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Journal of Photochemistry & Photobiology, B: Biology 201 (2019) 111649
The band gap of AgNPs was calculated from the tauc plot derived
from the absorption spectra obtained from UV–Vis spectroscopic stu-
dies. The tauc plot contains hʋ (energy) on the abscissa and (αhʋ)r on
the ordinate. The linear region of the plot is extrapolated to abscissa in
order to obtain the band gap value of the material under consideration.
The value was found to be 2.5 eV.
3.3. FTIR Analysis
The possible biomolecules of AFW which are involved in the re-
duction of AgNO3 and stabilization of the silver nanoparticles were
identified by FTIR spectroscopy. The FTIR spectrum in Fig. 1(b) shows
the major bands at 3413.4, 2923, 1637.3 and 1384.4 cm−1. The bands
at 3413.4 cm−1 and 2923 cm−1 correspond to OH stretching of phenol/
alcohol group and to CeH stretching respectively. The bands at
1637.3 cm−1 and 1384.4 cm−1 may be attributed to CeH bending of
aromatic and CeN stretching of amine group or OH bending of phenol
respectively. Comparison of AFW and AgNP-AFW spectra shows that
there is change in position and intensity of peaks due to reduction of
AgNO3 and its stabilization by biomolecules. A weak band appears at
1746.32 cm−1 in AgNP-AFW which corresponds to C]O bond forma-
tion. This could be as a result of oxidation of the OH group to CO group
[29]. This further indicates the role of polyphenolic compounds and
proteins in the reduction of Ag+ to Ag0. The capping of the nano-
particles by the biomolecules is also evident from the presence of the
major bands of AFW in AgNP-AFW spectra. HPLC analysis of AFW
confirmed the presence of flavonoids. A major peak for rutin was ob-
served at its characteristic retention time of 4.97 min (Fig. S1). This
further supports the probable role of the above biomolecules in the
synthesis and capping of AgNP-AFW.
3.4. XRD Analysis
Powder X-ray diffraction analysis confirmed the crystalline nature
of AgNP-AFW. The XRD diffractogram shows five distinct characteristic
peaks of silver nanoparticles at 2θ = 38.04°, 43.98°, 64.50°, 77.36° and
81.46° (Fig. 1c). These peaks can be indexed to the (111), (200), (220),
(311) and (222) planes which correspond to the face centred cubic (fcc)
crystal structure which confirms the crystalline nature of silver nano-
particles [JCPDS no. 00-001-1164]. The most intense peak which cor-
responds to the (111) plane indicates that the predominant growth of
the silver nanoparticles is in this direction.
3.5. XPS Analysis
The chemical state of as-synthesized silver nanoparticles (AgNP-
AFW) was further analysed by XPS. The Ag3d high resolution XPS
spectrum in Fig. 1(d) shows two peaks at 367.3 eV (Ag3d5/2) and
373.3 eV (Ag3d3/2). The energy spacing of 6 eV between the two peaks
suggest the metallic state of AgNP-AFW [30]. The other two peaks
which tails the Ag peaks are associated with energy loss accompanying
the surface plasmon resonance of AgNP [31,32].
3.6. Zeta Potential Analysis
Zeta potential provides information about surface charge and sta-
bility of AgNP-AFW. The zeta potential of AgNP-AFW was found to be
Glycosides Steroids Terpenoids Alkaloids
+ – +
D. Baruah, et al. Journal of Photochemistry & Photobiology, B: Biology 201 (2019) 111649

Fig. 1. (a) UV–Vis spectra of AgNP-AFW at different time intervals; inset shows αhʋ vs hʋ plot for band gap calculation of AgNP (b) FTIR spectra of AgNP-AFW and
AFW, (c) XRD spectra of AgNP-AFW, (d) High resolution Ag3d XPS spectrum.

−12.5 mV (Fig. 2). The negative zeta potential suggests repulsion and
enhanced stability. Furthermore, DLS analysis shows that the Poly-
dispersity Index (PDI) of AgNP-AFW is 0.359 and the average size was
found to be 65.9 nm. The difference in size in DLS and TEM may be due
to solvation effect in DLS analysis [33].
3.7. SEM Analysis
The morphology of the nanoparticles can be analysed by SEM
images. FE-SEM micrographs in Fig. 3 shows AgNP-AFW at different
magnifications. Monodispersed silver nanoparticles, seen here as white

Fig. 2. (a) Zeta potential of AgNP-AFW; (b) DLS of AgNP-AFW.

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Fig. 3. (a, b) SEM micrographs of AgNP-AFW at different magnifications; (c) EDX spectrum of AgNP-AFW showing the presence of Ag, C, O and Ca.

dots, were formed on the surface of the AFW derived biological mate- plane of fcc crystal structure of silver nanoparticles. The SAED pattern
rials as indicated in Fig. 3(a). The silver nanoparticles are spherical in which shows bright spots corresponding to the (111) and (200) planes
shape. The EDX data provides definite information about the elements of silver nanoparticles further confirms the crystalline nature of AgNP-
present in the sample. Fig. 3c shows the presence of Ag at 3 keV. AFW.

3.8. TEM Analysis 3.9. Antimicrobial Activity of AgNP-AFW

The distribution, shape and size of the silver nanoparticles were
analysed by TEM analysis. The TEM micrographs of AgNP-AFW at
different magnifications along with the SAED pattern and average
particle size distribution is shown in Fig. 4. It is seen that the silver
nanoparticles are uniformly distributed and spherical in shape. The
average particle size of as-synthesized silver nanoparticles is 6 nm with
a standard deviation of 2 nm. The HR-TEM image in Fig. 4(d) shows
that the lattice fringe spacing is 0.24 nm which corresponds to the (111)
The antimicrobial activity of the biomolecules supported silver na-
noparticles (AgNP-AFW) was determined against gram positive bac-
teria, gram negative bacteria and a fungus. The agar well diffusion
assay showed high antimicrobial activity of AgNP-AFW against all test
pathogens. The mean diameter of ZOI for the three pathogens are
presented in Table 2 along with MIC and MBC. The negative control,
DMSO, showed no zone of inhibition. The ZOI indicated that the Gram-
negative bacteria has the highest antimicrobial activity. The crude plant

Fig. 4. TEM micrographs of AgNP-AFW at different magnification of (a) 200 nm (b) 50 nm (c) 20 nm (d) HR-TEM image showing clear lattice fringe spacing (e) SAED
pattern of AgNP-AFW and (f) average particle size distribution of AgNP-AFW.

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Table 2 Table 3
Zone of Inhibition (mm) and MIC(μg/ml) of AgNP-AFW along with their MBC/ Comparison of antimicrobial activity of silver nanoparticles synthesized using
MFC (μg/ml) for two bacteria and one fungus. different plants.
Test pathogens AgNP-AFW Standard Negative MIC MBC/ Plant and its part Test pathogen ZOI (mm) Reference
(100 μg/ml) (neomycin/ control (μg/ MFC
nystatin) (DMSO) ml) (μg/ Zingiber officinale rhizome S. aureus 6.5 [34]
ml) Iresine herbstii leaves S. aureus 9.7 [36]
Zone of inhibition (diameter in K. pneumoniae 8
mm) Cleome viscosa fruit S. aureus 17 [37]
K. pneumoniae 14
S. aureus 17.67 ± 0.57 25 NI 30 30 Caesalpinia coriaria leaves S. aureus 10.3 [38]
K. pneumoniae 19.67 ± 0.57 24 NI 20 20 K. pneumoniae 14.6
C. albicans 12.33 ± 0.57 18 NI 50 100 Rosa brunonii Lindl leaves K. pneumoniae 12 [39]
Withania somnifera leaves S. aureus 11 [40]
The ZOI results are in Mean ± SD. C. albicans No Inhibition
Banana peel C. albicans 10 [41]
Dioscorea batatas rhizome C. albicans 10 [42]
extract, AFW, did not exhibit any inhibitory activity against the tested
pathogens. The minimum inhibitory concentration is the lowest con-
centration of the drug that prevents visible growth of microbes. The antimicrobial activity, thus eliminating the concept of electrostatic in-
determination of MIC through broth dilution method demonstrates that teractions causing cell membrane communications [39,40,44,45]. The
the inhibitory activity of AgNP-AFW is strongest against K. pneumoniae size and shape of nanoparticles are considered important parameters of
followed by S. aureus and C. albicans. AgNP-AFW inhibits the growth of antimicrobial activity. Silver nanoparticles accumulate at different sites
both the bacteria and fungus which is evident from the ZOI and MIC. on the cell and cause destabilization of membrane of fungi, gram-po-
Fig. 5a shows the number of colonies (expressed as percentage) grown sitive and gram-negative bacteria. This leads to cell lysis via formation
on the agar plates as a function of the concentration of silver nano- of pits and accumulation of nanoparticles in the cytoplasm. Further-
particles. It was observed that upon treatment with AgNP-AFW, the more, silver nanoparticles of size 10 nm or less has more cell-particle
number of colonies reduced significantly as compared to the control. contact than larger nanoparticles which leads to internalization of na-
The low susceptibility of S. aureus may be attributed to large pepti- noparticles into cells and then release of toxic ions [43,46]. Thus, the
doglycan layer in the cell wall of S. aureus. size of the nanoparticles is an important parameter for its antimicrobial
A comparison of the antimicrobial activity of the silver nano- activities and the better antimicrobial activity of AgNP-AFW could be
particles synthesized using A. nigra fruits (AgNP-AFW) with previously attributed to their small size.
reported studies are presented in Table 3. The MIC and MBC of silver
nanoparticles synthesized using Z. officinale is less than that observed
3.10. Photocatalytic Activity of AgNP-AFW
for AgNP-AFW while the MIC and MBC of Enteromorpha flexuosa is si-
milar to our results [34,35]. This establishes the fact that AgNP-AFW
Rhodamine B, Methyl Orange and Orange G are popular water-so-
exhibits better antibacterial as well as antifungal activity.
luble dyes which are extensively used in industrial sectors such as
The antimicrobial activity of AgNP-AFW and the antibiotics show
textile, leather, printing, paper and pharmaceuticals. Degradation of
mild variation in efficacy. Standard antibiotics have specific targets but
these dyes in the presence of AgNP-AFW was monitored with a UV–Vis
their excessive use is leading to development of resistance.
spectrophotometer. The absorption maxima of Rhodamine B, Methyl
Nanoparticles exhibiting antimicrobial activity are gaining attention as
Orange and Orange G were centred around 553, 464 and 482 nm re-
they act via multiple mechanisms and hence to elicit any form of re-
spectively. The silver nanoparticles catalysed degradation of the or-
sistance to nanoparticles, complex multiple gene mutations are required
ganic dyes under sunlight irradiation is apparent from the gradual de-
in the same cell [43]. In the present work, the silver nanoparticles with
crease in the main absorption peak of the dye solutions. It is clearly
a negative surface charge exhibited antimicrobial activity which is in
demonstrated in Fig. 4 that with time there is decrease in the absorption
contrast to the assumption that only positively charged nanoparticles
band of Rhodamine B, Methyl Orange and Orange G centred around
can interact with the negatively charged bacterial membrane. Several
553, 464 and 482 nm respectively which finally approached the base
studies have reported that AgNPs with a negative surface charge exhibit
line indicating the successful degradation of the dyes in the presence of

Fig. 5. (a) Number of colonies as a function of the concentration of AgNP-AFW expressed as a percentage of the number of colonies grown on control plates, (b) MIC
of AgNP-AFW against the three test pathogens.

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Fig. 6. (a) UV–vis spectra of Rhodamine B in the presence of AgNP-AFW, (b) Plot of ln (A/A0) vs time (min) for catalytic degradation of Rhodamine B, (c) UV–vis
spectra of Methyl orange in the presence of AgNP-AFW, (d) Plot of ln (A/A0) vs time (min) for catalytic degradation of Methyl orange, (e) UV–vis spectra of Orange G
in the presence of AgNP-AFW, (f) Plot of ln (A/A0) vs time (min) for catalytic degradation of Orange G.

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the nanoparticles. Fig. 6(b, d, f) depicts the Ln (A/A0) vs time plot for
Rhodamine B, Methyl Orange and Orange G. The degradation rate
constant calculated for Rhodamine B is 1.734 × 10−2/min, Methyl
Orange is 1.607 × 10−2/min and for OG is 1.413 × 10−2/min. De-
crease in Ln (A/A0) value with time indicates that the reaction process
followed pseudo first order kinetics.
In addition, a comparison of the photocatalytic activity of the AgNP-
AFW with other similar nanoparticles clearly demonstrates the effi-
ciency of the former against a number of toxic dyes. Previous studies
have reported that 40% RhB degradation was achieved by silver na-
noparticles synthesized via solid state thermal decomposition method
after 60 min [47]. In another report, 75% degradation of MO was
achieved in 480 min by silver nanoparticles synthesized using Solanum
tuberosum infusion [48]. A report by Liang et al. revealed that there was
no significant activity observed for degradation of OG with the use of
silver nanoparticles synthesized using Citrus lemon fruit extract [49]. Fig. 8. Chemical structure of the organic dyes.
On the other hand, the percent dye degradation calculated in our study
from the absorbance spectra is 85.9%, 83.4% and 79.9% for RhB, MO
and OG respectively. This data clearly substantiates the fact that the
AgNP-AFW synthesized in the present work show better photocatalytic
activity when compared to silver nanoparticles synthesized from other
sources.
The degradation process takes place on the surface of the catalyst.
The average particle size as obtained from TEM analysis is 6 nm and the
XRD studies establish that AgNP-AFW have FCC crystal structure. With
increased surface area of nanoparticles, there is an increased number of
available active sites where the degradation of the complex dye mole-
cules can take place through electron transfer. Upon irradiation with
light, the electrons get excited and move from the valence band of Ag to
the empty conduction band. This leads to the formation of electron-hole
pair which are good oxidizing and reducing agents. On reacting with
the oxygen in the reaction mixture, these excited electrons generate free
radicals like HO•, O•22− and HOO• at the surface of the catalyst i.e. at the
“active sites” [29,50]. The entire process is depicted pictorially in
Fig. 7. The structure of the dyes also plays a vital role in the rate of
degradation. The dyes used in this study are polycyclic and their
structures are presented in Fig. 8. The N]N bond in azo dyes are
weakened by the hydroxyl and amino groups through resonance effect,
Fig. 9. Dye degradation activity of AgNP-AFW upto 5 runs.
thereby assisting in the rapid degradation of these dye molecules.
However, the presence of sulphonyl and nitro groups slows down the
degradation process. Rhodamine B undergoes faster degradation be- such, the AgNP-AFW were recovered after each run and reused 5 times
cause of the absence of a sulphonyl group. Amongst the two azo dyes, to observe its efficiency after each run. The catalyst was effectively used
the degradation of Orange G is slower due to the likelihood of in- three times and thereafter we recorded a decline in activity which is
tramolecular hydrogen bonding between the substituents and the azo shown in Fig. 9. This demonstrates that AgNP-AFW can be recovered
group which leads to strengthening of the N]N bonding [29]. Thus, the and reused successfully thus making it suitable for waste water re-
para substituted Methyl Orange undergoes faster degradation as com- mediation.
pared to the ortho substituted Orange G.
In a practical situation it is vital for the catalyst to be reusable. As 4. Conclusion

Fig. 7. A mechanistic view of dye degradation in the presence of AgNP-AFW.
This work reports a simple and efficient method for the synthesis of
silver nanoparticles using the aqueous extract of A. nigra fruits as re-
ducing and stabilizing agents. The capping of the biomolecules is evi-
dent from the FTIR spectra. The SEM micrograph clearly shows both
silver nanoparticles and biomaterials indicating the formation of silver-
phytonanocomposite. The present work is significant as it intergrates
the tenets of green synthesis by using only eco-friendly solvents. These
phytonanocomposites with an average particle size of 6 nm showed
potent antimicrobial activity against K. pneumoniae, S. aureus and
C. albicans. The antimicrobial potential of these nanoparticles suggest
that it can be used to avert and combat nosocomial infections. The
nanoparticles also demonstrated its potential as a catalyst in the de-
gradation of organic dyes such as Rhodamine B, Methyl orange and
Orange G which are found in industrial effluents. On the whole, this
work presents a green method for the rapid synthesis of silver nano-
particles which can be used as antimicrobial agents and can also be
used for treatment of waste water.

8
D. Baruah, et al.
Declaration of Competing Interest
All the authors declare that they have no conflict of interest.
Acknowledgements
The authors thank the Director, CSIR-NEIST, Jorhat for providing la-
boratory facilities. The authors express their sincere gratitude to SAIF,
NEHU, Shillong for TEM analysis of the samples. Monmi Goswami is
gratefully acknowledged for her conceptual support. Debjani Baruah
would like to thank University Grants Commission, New Delhi for fel-
lowship. The IDs of the research is UGC Ref. No.: 4992/(NET-JUNE 2012).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jphotobiol.2019.111649.
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