Biogenic synthesis of palladium nanoparticles and their applications as catalyst and antimicrobial agent

Article abstract of DOI:10.1371/journal.pone.0184936

This paper describes a simple in-situ process of synthesizing highly dispersed palladium nanoparticles (PdNPs) using aqueous leaf extract of GarciniapedunculataRoxb as bio-reductant and starch (0.3%) as bio-stabilizer. The PdNPs are characterized by techniques like FTIR, TEM, SEM-EDX, XRD and XPS analysis. It is worthnoting thatwhen the synthesis of nanoparticles was carried out in absence of starch, agglomeration of particles has been noticed.The starch-assisted PdNPs showed excellent aqueous-phase catalytic activities for three important reactions: the Suzuki-Miyaura cross-coupling reactions of aryl halides (aryl bromides and iodides) with arylboronic acids; selective oxidations of alcohols to corresponding carbonyl compounds; and reduction of toxic Cr(VI) to nontoxic Cr(III). Our catalyst could be reused up to four cycles without much compromising with its activity. Furthermore, the material also demonstrated excellent antimicrobial and anti-biofilm activities against a novel multidrug resistant clinical bacterial isolate Cronobactersakazakii strain AMD04. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PdNPswere found to be 0.06 and 0.12 mM respectively.

Full text of DOI:10.1371/journal.pone.0184936

RESEARCH ARTICLE
Biogenic synthesis of palladium nanoparticles
and their applications as catalyst and
antimicrobial agent
Munmi Hazarika1☯, Debajit Borah2☯, Popymita Bora2, Ana R. Silva3, Pankaj Das1*
1 Department of Chemistry, Dibrugarh University, Dibrugarh, India, 2 Centre for Biotechnology and
Bioinformatics, Dibrugarh University,Dibrugarh, India, 3 CICECO-Aveiro Institute of Materials, University of
Aveiro, Aveiro, Portugal
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☯ These authors contributed equally to this work.
* pankajd29@yahoo.com
Abstract
This paper describes a simple in-situ process of synthesizing highly dispersed palladium
nanoparticles (PdNPs) using aqueous leaf extract of GarciniapedunculataRoxb as bio-
reductant and starch (0.3%) as bio-stabilizer. The PdNPs are characterized by techniques
like FTIR, TEM, SEM-EDX, XRD and XPS analysis. It is worthnoting thatwhen the synthesis
of nanoparticles was carried out in absence of starch, agglomeration of particles has been
noticed.The starch-assisted PdNPs showed excellent aqueous-phase catalytic activities for
three important reactions: the Suzuki-Miyaura cross-coupling reactions of aryl halides (aryl
bromides and iodides) with arylboronic acids; selective oxidations of alcohols to correspond-
ing carbonyl compounds; and reduction of toxic Cr(VI) to nontoxic Cr(III). Our catalyst could
be reused up to four cycles without much compromising with its activity. Furthermore, the
material also demonstrated excellent antimicrobial and anti-biofilm activities against a novel
multidrug resistant clinical bacterial isolate Cronobactersakazakii strain AMD04. The mini-
mum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of
PdNPswere found to be 0.06 and 0.12 mM respectively.
OPEN ACCESS
Citation: Hazarika M, Borah D, Bora P, Silva AR,
Das P (2017) Biogenic synthesis of palladium
nanoparticles and their applications as catalyst and
antimicrobial agent. PLoS ONE 12(9): e0184936.
https://doi.org/10.1371/journal.pone.0184936
Editor: Yogendra Kumar Mishra, Institute of
Materials Science, GERMANY
Received: March 29, 2017
Accepted: September 1, 2017
Published: September 28, 2017
Copyright: © 2017 Hazarika et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. The GenBank accession number is KJ812198.
Over the past few decades, delving into the synthesis of metalnanoparticlesviz. gold, silver,
Zinc, platinum, palladium, etc.have fetched considerable attention in the fields of material and
biological sciences [1–8]. Amid other metal NPs, Pd is gaining special mention because of its
profound applications as catalyst in large number of organic transformations which include-
various types of carbon-carbon cross-coupling [9,10], oxidation [11,12]and reduction reac-
tions. [13,14]. Traditionally, Pd NPs are synthesized via various physical or chemical methods
with the aid of toxic and hazardous reducing and stabilizing agents, however, over the years, in
the quest of ‘going green’, there has been a paradigm shift towards bio-inspired strategies for
the synthesis of metal NPs [15–17]. The biocompatibility and environmentally benign proper-
ties attributes to these biological techniques to supersede the conventional physical and wet-
Funding: The Department of Biotechnology is
gratefully acknowledged for financial support
(Grant no:MED/2013/23) under DBT-Twinning
programme and also for providing Delcon as well
as IBT hub facility to the Centre for Biotechnology
and Bioinformatics, Dibrugarh University. The
UGC, New Delhi has also been acknowledged for
the SAP-DRS-I grant awarded to the Department of
Chemistry.
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Green synthesis of palladium nanoparticles and their multifunctional activity
Competing interests: The authors have declared
chemical methods. The field of biological synthesis of Pd NPs encompasses the use of plant
extracts, microorganisms, marine organisms, etc. as green-reductants. Amongst those meth-
ods, the tapping of bio-resources, particularly the plant extracts for the synthesis of the NPs
seems promising owing to their ready availability, rapid process, better cost-effectiveness, and
the ability to use in large-scale biosynthesis [18]. Hence, plant mediated syntheses of PdNPs,
has gained many recent attentions[19,20]. Examples include use of peel-extract of banana [21],
leaf-extracts of Pulicaria glutinosa[16],Perilla frutescens[17],Euphorbia granulate[22],Ocimum
Sanctum[23], Origanum vulgare [24],etc. and some of the systems have been successfully
applied as catalysts for organic reactions like Suzuki-Miyaura reactions[16,21,25–28], oxida-
tion of alcohols [24], hydrodechlorination of p-chlorophenol [29], etc. Unfortunately, baring a
couple of examples [22,25,28], majority of these reported plant-based catalysts often suffer
from limitations like high reaction temperatures (up to 120˚C), high catalyst loadings (up to
12 mol%), limited substrate scopes and / or use of undesirable organic solvents as reaction, etc.
Moreover, the reported plant-based Pd NPs mostly tested for a single catalytic system, while
their multifunctional potentiality remains largely under-explored. Thus, the appropriate selec-
tion of plant extract which can bestow multifunctional roles viz., bio-reduction of Pd salts, aid
in multiple catalytic reactions and afford biological activities is highly desirable. In this context,
we would like to exploit a bio-resource namely Garcinia pedunculata Roxb aqueous leaf extract
for the synthesis of Pd NPs. This leaf extract has been traditionally known to possess multiple
medical uses, validated by ethno-pharmaceutical research [30]. Literature reveals the presence
of phytochemical constituents like polyphenol, flavonoid, hydroxycitric acid in the leaf extract,
which functions as active components for bio-reduction [31]. To the best of our knowledge,
the potentiality of this plant has not been explored for synthesizing and functionalizing metal
NPs till date. Herein, along with the synthesis of PdNPs, we have also explored the versatility
of these NPs as multifunctional catalyst for Suzuki-Miyaura cross-coupling reactions of aryl
halides, alcohol oxidation and Cr(VI) to Cr(III) reduction reactions using water as a reaction
media. The antibacterial activity of the Pd NPs was also was investigated against a novel multi-
drug-resistant clinical bacterial isolate Cronobacter sakazakii strain AMD04. It is worth men-
tioning that the novelty of this reported synthesis of Pd NPs lies in appropriate selection of the
bio-resource, which not only aids in the synthesis, but unlike other biogenic process plays a
crucial role in bestowing multiple catalytic and antibacterial properties. Of note, the multifunc-
tional catalytic aspects of the synthesized PdNPs viz., C-C cross-coupling, oxidation and
reduction reactions constitute an added asset.
that no competing interests exist.
Experimental materials and methods
Fresh green leaves of G. pedunculata Roxb (ESI: S1 Fig) were collected from Dergaon area
(26.6969˚ N, 93.9853˚ E) of Golaghat District, Assam, India. Palladium acetate [Pd(OAc)2]
was purchased from Merck India Pvt. Ltd. All the bacteriological media used in the present
study were procured from HiMedia Laboratories Pvt. Ltd. The other common laboratory
chemicals are of analytical grade and were purchased from different Indian firms. FTIR spectra
(400–4000 cm-1) were recorded in KBr using Shimadzu (Prestige-21) spectrophotometer.
The X-ray diffraction (XRD) study was performed in a Rigaku X-ray diffractometer (model:
ULTIMA IV, Rigaku, Japan) with a Cu Kα X-ray source (λ = 1.54056 Å) at a generator voltage
of 40 kV and a generator current of 40 mA. The Transmission electron microscopy (TEM)
and high were carried out on a JEOL JEM-2011 electron microscope operated at an accele-
rating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) study was performed at
‘‘Centro de Materiais da Universidade do Porto” (Portugal), using a Kratos Axis Ultra HSA
spectrometer with a non-monochromatized Mg Ka radiation (1253.6 eV). The scanning
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Green synthesis of palladium nanoparticles and their multifunctional activity
electron microscope (SEM) spectra were recorded using JEOL Model JSM - 6390LV and
energy dispersive X-ray (EDX) peaks were recorded in JEOL Model JED– 2300. Bacterial
growth was measured with the help of UV-VIS spectrophotometer (Shimadzu UV-1800,
Japan).HPLC analysiswas done with Agilent HPLC System (Infinity 1200) equipped with
Multi-Wavelength Detector. Thermogravimetric analysis (TGA) was performed using Perkin
Elmer STA-8000 at the heating rate of 10˚C per min under nitrogen atmosphere. Dynamic
Light Scattering (DLS) study was done by using Zetasizer analyser (Model: Nano ZS, Malvern,
UK) instrument.
Preparation of plant leaf extract
100 gm fresh leaves of G. pedunculataRoxbwere chopped nicely and then added slowly in 500
mL deionized water with occasional shaking. The aqueous mixture was boiled for 30 min with
continuous stirring and then allowed to cool to room temperature. The leaf extract was then
filtered using Whatman no.1 filter paper and the filtrate was stored at 4˚C for further use.
Synthesis of palladium nanoparticles
500 ml aqueous leaf extract of the plant G. pedunculataRoxb was mixed with 1000 ml of palla-
dium acetate (1mM) solution containing 0.3% of starch. The reaction mixture was allowed to
stir continuously for overnight and then autoclaved at 121˚C for 15 min. The color of the solu-
tion changed from orange to dark brown, consistent with the formation of PdNPs. The NPs
were then separated from the aqueous suspension by centrifugation at low temperature at
30,000 g for 30 min and then washed twice by deionized water and finally dried in an oven at
80o C.
General procedure for the Suzuki-Miyaura reaction
Forthe Suzuki-Miyaura reaction, a 50 mL round-bottomed flask was charged with aryl halide
(0.5 mmol), arylboronic acid (0.65 mmol), K2CO3 (1 mmol), catalyst (0.002 g; 0.0005 mmol of
Pd) and water (4 mL) and stirred at appropriate temperature. The progress of the reaction was
monitored by thin layer chromatography using aluminum coated TLC plates (Merck) under
UV light. At the end of reaction, the mixture was cooled down to room temperature and the
product diluted with water (10 mL) and extracted with ether (3 x 15 mL). The combined
extract was washed with brine (3 x 15 mL) and dried over Na2SO4. After evaporation of the sol-
vent under reduced pressure, the residue was subjected to column chromatography with ethyl
acetate/hexane (1:9) as eluent to get the desired product.
Reusability experiment
A 100 mL round-bottomed flask was charged with p-bromotoluene (2.5.mmol), phenylboro-
nic (3.25 mmol), K2CO3 (7.5 mol), H2O (20 mL), catalyst (10 mg; 0.0025 mmol) and stirred at
50˚C for 3 h. After the reaction was over, the reaction mixture was cooled to room temperature
and then 20 mL of EtOH was added and stirred for 10 min. The catalyst was then separated by
centrifugation (14,000 rpm for 15 min), washed thoroughly with EtOH-H2O (1:1) followed by
ethyl acetate, dried under vacuum and then used for subsequent runs.
General procedure for alcohol oxidation reaction
A 50ml round bottomed flask was charged with alcohol(1.2mmol),TBHP(2.4mmol),
K2CO3(0.0075 mmol), catalyst (3mg, 0.0007 mmol) and H2O (10mL). The reaction was carried
out at 80˚C with continuous stirring and monitored by using TLC.On completion of the
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Green synthesis of palladium nanoparticles and their multifunctional activity
reaction, the mixture was extracted with ether and dried over Na2SO4.The solvent was evapo-
rated under reduced pressure to obtain the concentrated organic product which was extracted
with ethyl acetate and analyzed by gas chromatography (Agilent 7820A) with mass detector
(Agilent 5975 series).
Procedure for Cr (VI) reduction
Formic acid (0.3 ml) was added to 50ml of aqueous K2Cr2O7(0.001M) to prepare an acidified
solution of Cr(VI). To this solution, 10ml of aqueous sodium acetate trihydrate(0.5M) was
added dropwise to maintain a pH of 2.The reaction mixture was stirredat room temperature in
presence of 3mg(0.0007 mmol) of the PdNP catalyst and then monitored with UV-vis
spectrophotometer.
Isolation, screening and identification of novel MDR isolates
MDR bacterial isolates were screened from clinical samples collected from Assam Medical
College Hospital, Dibrugarh by observing their resistance patterns against at least four antibio-
ticsout of the following: Metropenem (10mcg), Polymixin B (10mcg), Metronidazole (5mcg),
Novobiocin (30mcg), Cefixime (5mcg), Ciprofloxacin (5mcg), Tetracycline (30mcg), Genta-
mycin (30mcg), Amikacin (30mcg), Rifampicin (5mcg), Erythromycin (15mcg), Ampicillin
(10mcg), and Imipenem (10mcg). The isolate showing resistanceagainst maximum number of
antibiotics was then identified by 16S rDNA sequencing technique using 704F forward primer
(5’-GTAGCGGTGAAATGCGTAGA-3’) and 907R reverse primer (5’-CCGTCAATTCMTTT
GAGTTT-3’).
Evaluation of antimicrobial activity
Antimicrobial activity of Pd NPs was evaluated by agar plate well diffusion method by loading
10 μL of the plant extract, 1 mMPd(OAc)2 solution, and aqueous suspension of PdNPs in
respective wells. The plates were then incubated overnight at 37˚C and zone of inhibition was
observed around the wells. The antimicrobial activity in terms of minimum inhibitory concen-
tration (MIC) and minimum bactericidal concentration (MBC) of Pd NPs was examined by
the standard broth dilution method[32]. The lowest concentration of Pd NPs causing signifi-
cant decline in the bacterial growth as compared to that of control was considered as the MIC,
while the minimum concentration of PdNPs which completely inhibits the bacterial growth
was considered as MBC. Bacterial growth was measured as an increase in the absorbance of
the 600 nm peak determined by a spectrophotometer at an interval of 1 h. Sterile test tubes,
each containing 1 mL of LB (Luria Bertani) broth were inoculated with 100μL of freshly pre-
pared bacterial suspension in order to maintain initial bacterial concentration (103−104 CFU/
mL). 1 mM of PdNPs solution was prepared and diluted to 0.02, 0.04, 0.06, 0.08, 0.1, 0.12 mM
in 1mL culture of above mentioned Cronobactersakazakiistrain AMD04 and then incubated in
an orbital shaker at 200 rpm and 37˚C (Sartorius StedimCertomat BS-1 shaker incubator, Ger-
many Ltd.). The control was devoid of NPs containing inoculums and LB broth.
Evaluation of anti biofilm activity
Inhibition of biofilm formation in presence of PdNPs was determined and quantified by crys-
tal violet staining method [33]. Overnight culture of novel MDR clinical isolate Cronobacter
sakazakii strain AMD04 was diluted (1:100) and inoculated into a micro titre plate containing
increasing concentrations of PdNPs in dilute (1:100) Luria Bertani (LB) Broth. After 24 h of
incubation, the micro titre plate was washed with phosphate buffer saline (PBS) to remove the
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Green synthesis of palladium nanoparticles and their multifunctional activity
unbound cells and left to air dry. The plate was stained with 0.1% crystal violet for 10–15 min-
utesand then washed thoroughly in phosphate buffer saline (PBS) followed by drying at 60˚C.
Crystal violet was extracted using 30% acetic acid for 10–15 min and absorbance was recorded
at 595nm.
Statistical analysis
All the biological experiments related to antimicrobial study were carried out in triplicate and
results are expressed in mean S.D. Significance of the results were checked by performing
student’s t-test (p<0.05) using online statistical tool graphpad (https://www.graphpad.com/).
Results and discussion
Synthesis and characterization of palladium nanoparticles
The PdNPs are synthesized by a simple in-situ method treating a solution of [Pd(OAc)2] with
aqueous leaf extract of the plant G. pedunculataRoxb in presence of 0.3 mol% of starch. Since,
we have not used any external reducing agent, it is logical to assume that the phytochemical
constituents performed the reduction Pd+2!Pd0. In order to investigate the phyto-constitu-
ents responsible for this reduction, we have performed HPLC analysis of the leaf extract and
gallic acid was found to be the major constituent present in the bio-extract. The presence of
gallic acid was further confirmed by HRMS analysis which shows a molecular ion peak [M-
2H]+atm/e 168 [ESI:S2 Fig].It is worthy to note that the reducing potential of gallic acidfor syn-
thesizing metal NPs has been reported in literature [8,34]. Hence, we believe that in our case
also the gallic acid has been acted as bio-reducing agent, although the possibility of other
minor constituents cannot be ruled out.
The morphology of the starch-assisted Pd NPs was studied by SEM and TEM analysis. The
TEM images reveal that NPs are well dispersed and are of both spherical and non-spherical
morphologies with sizes ~2–4 nm (Fig 1A and 1B). Interestingly, when the same synthesis was
carried out in absence of starch, the nanoparticles are found to be in the agglomerated form
[ESI: S3 Fig].The high magnification TEM image of the Pd NPs along with the lattice fringes
with an inter fringe distance of 0.21 nm corresponding to the (111) plane of PdNPs is pre-
sented in Fig 1C. The selected area electron diffraction (SAED) pattern (Fig 1D) is consistent
with the face centre cubic (fcc) arrangement of the Pd nanocrystals [35].Unfortunately, the Pd
particles are not clearly visible in the SEM image, although the EDX analysis shows the pres-
ence of Pd along with other elements such as N, O, and S (ESI: S4 Fig). The FTIR spectra of the
Pd-based material (ESI: S5A Fig) shows three very prominent peaks at 3338(br), 1616 and
1750 cm-1. The first two peaks could be attributed to theυOHstretching andυOHbending of the
phenolic hydroxyl groups, while the 3rd peak could be due to the carbonyl group of gallic acid.
For comparison, an FTIR spectrum of the powdered dry leaf sample was also recorded (ESI:
S5B Fig). In fact, those three peaks of the Pd NPs were also present in the FTIR spectra of dry
leaf sample. However, significant shifts of the υOH stretching (from 3338 cm-1 to 3269) and
bending (from 1616 to 1652 cm-1) were noticed suggesting involvement of a coordinative
interaction between the Pd and the galic acid.In addition, some minor changes have also been
observed in the far-infrared region. For instance, a band at 384 cm-1 in the dry leaf sample
shifts towards lower wavenumber upon interaction with Pd.
The formation of PdNP was further evidenced from the XRD investigation. The XRD pat-
tern of the Pd NPs is shown in Fig 2. The distinct peaks of the Pd NPs were observed at 2θ of
39.96˚, 46.50˚, 68.54˚ and 82.16˚ diffracted from the (111), (200), (220) and (222) planes with
corresponding d-spacing values of 2.254, 1.951, 1.37 and 1.172 Å respectively (JCPDS card No.
001–1201). In addition, three other peaks were also observed at 2θ of 34.22˚, 55.72˚, 86.38˚
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 1. TEM images; (a) & (b) Highly dispersed spherical PdNPs synthesized by using G. pedunculataRoxb. leaf extract; (c) magnified TEM image of a single
metal nanoparticle along with lattice fringes for (111) fcc plane; (d)selected-area electron diffraction (SAED) pattern.
https://doi.org/10.1371/journal.pone.0184936.g001
with corresponding d-spacing values of 2.62, 1.65, and 1.12 Å indicated the presence of PdO
phases along with the Pd NPs (JCPDS card No. 002–1432). It may be noted that often wide-
angle XRD issued to investigate size and shape of nanoparticles[36].UsingScherrer equation,
the average size of the Pd nanoparticles are found to be about 1.5 nm which is close to that
obtained from the TEM spectra. However, when the particle size distribution was measured
through DLS, an average hydrodynamic diameter of 322 nm was found; possibly imply
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 2. Wide-angle powder XRD pattern of biosynthesized PdNPsin presence of starch (0.3%).
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aggregation of nanoparticles occurs in the aqueous suspension. Similar types of larger particle
sizes determined by DLS study(ESI. S6 Fig) compared to those determined by TEM are also
found in literature with other Pd-based materials [37].
The presence of two different oxidation states of Pd was further corroborated by the charac-
teristics 3d3/2 and 3d5/2 peaks in the XPS spectra. The high resolution peak of Pd 3d5/2 (Fig
3) can be deconvoluted into two peaks: a larger one is at 337.8 eV and a smaller one is at 335.7
eV. The first peak is typical for a Pd(II) species, while the second one is for Pd(0). The XPS sur-
vey spectrum of the material shows in addition to palladium, the material also contain carbo-
nand oxygen From their atomic percentages, the surface amount of Pd was calculated as 1.20
mM/g. The O1s peak (ESI. S7 Fig) appears at 533.1 eV which is very near to that reported for
PdO [38,39].The thermal decomposition study of the bio-synthesized PdNPs indicated a
three-step degradation process (ESI: S8 Fig). The initial minor decay of about 1.5% at around
100˚C was attributed to phsyi-adsorbed water associated with the nanomaterials, while the sec-
ond and third decay at 235–345˚C and 345–682˚C with corresponding weight lossesof 69%
and 23% could be attributed to the phenolic–OH and the bio-organic molecules respectively.
Residual weight of14% could be ascribed to metallic Pd or PdO.
Suzuki-Miyaura activity
The palladium catalyzed Suzuki-miyaura cross-coupling reactions between aryl halides and
aryboronic acids is one of the most extensively studied reactions in organic synthesis [3,40]. A
wide range of Pd-catalysts including Pd NPs are known that could perform this reaction under
mild conditions. It is worth to note that PdNPs of less than 5 nm often show very good cata-
lytic activities in the Suzuki-Miyaura cross-coupling reaction[41–43]. To check the catalytic
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 3. The high resolution spectrum of the starch-assisted PdNPs (Pd 3d region); inset: curve fitted 3d5/2 peak.
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activity of the synthesized PdNPs, we have preformed the Suzuki-Miyaura reaction using p-
bromotolouene and phenylboronic acid as model substrates. Initially, the model reaction was
performed at room temperature in water with 2 mg of catalyst using K2CO3 as base and 72%
(Fig 4, entry 1) product formation was achieved after a span of 3h. However, increasing the
temperature to 50˚C, almost quantitative formation of biphenyl was obtained within 2h (entry
2). Based on our initial success; we have tested the scope of our catalytic system for other re-
presentative aryl bromides or iodides including heteroaryl halides that are usually difficult to
activate in the Suzuki reaction. Our study revealed that aryl bromides and iodides bearing elec-
tron-neutral (entry 4 & 5), electron-withdrawing (entry 3 & 6) and electron-donating groups
(entry 2) underwent smooth coupling with phenylboronic acid and almost quantitative con-
version was obtained in all the cases. Interestingly, this high yields were also maintained when
phenylboronic acid was replaced with p-tolyl- or p-chloroboronic acid (Entry 7 & 8). More-
over, at an elevated temperature (80˚C) and with an extended reaction time our system can
also tolerates difficult substrates like sterically demanding 2-iodotoluene (entry 10) and
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 4. Suzuki-Miyaura reactionsa of various aryl/heteroaryl halides with arylboronic acids using PdNPs as catalyst.
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1-bromo-2,5-dimethoxybenzene (entry 11), or heteroaryl halides like 3-iodopyridine (entry
12) or 5-bromopyrimidine (entry 13) and provided the cross-coupling products in good-to-
excellent yields.It needs to mention that aturnover number (TON) close to 1000 was obtained-
with our catalyst for cross-coupling with a few aryl bromides in water,and this value is either
comparable[44] or superior [45]with some of the reported PdNPs-based catalysts that are
chemically synthesized. Unfortunately, despite good results with aryl bromides or aryl iodides,
our catalyst is not suitable for activating aryl chlorides as substrate (Entry 15 & 16).Noteworthy
to mention that usually aryl chlorides are difficult substrates to be activated in Suzuki reaction
because of stronger C-Cl bond compared to C-Br or C-I bond [9].
It may be important to note that one of the key advantages of NPs based catalytic system is
their potential recyclability, and to check this property, we have performed the reusability
experiment with our model system taking p-bromotoluene and phenylboronic acid as model
substrate Table 1. For handling convenience, we have performed the reaction with 10 mg cata-
lyst using proportionate amount of substrates, base and water. Our study revealed (Fig 4) the
nanocatalyst could be reused at least for four cycles; however a gradual decrease in yield was
observed which might be due to the handling loss of the catalyst during centrifugation. The
TEM pictures (Fig 5) of the reused catalyst showed almost similar morphology with no sign of
aggregations suggesting that the catalyst could be reused for further runs.
Table 1. Recycling of the catalyst for the reaction between 4-bromotoluene and phenylboronicacida.
Cycle
Yieldb
95
1
2
3
4
92
91
89
aReaction condition: 4-bromotoluene (2.5mmol), phenylboronic acid (3.25 mmol), K2CO3 (7.5 mmol), H2O
(20 mL), Pd catalyst (10 mg); 50˚C
bIsolated yield.
https://doi.org/10.1371/journal.pone.0184936.t001
Fig 5. TEM images of the recycled catalyst showing lattice fringes corresponds to (111) plane of Pd NP.
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 6. Oxidation of selective alcohols in aqueous media catalyzed by PdNPs as catalyst.
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Alcohol oxidation reaction
Oxidation of alcohols to carbonyl compounds is one of the most important reactions in
organic synthesis. Palladium-based catalysts, including Pd NPs,are particularly known to
promote this reaction under mild conditions using environmentally benign oxidants such as
O2, H2O2, tert-butyl hydrogen peroxide (TBHP), etc [11,46]. However, in the majority of the
cases, the reactions are usually conducted in organic solventsthat are not only toxic but also
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 7. Cr(VI) reduction: (a) in absence of catalyst; (b) in presence of Pd(OAc)2 salt; (c) in presence of starch-assisted Pd NPs as catalyst.
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expensive. With the bio-synthesizedPd NPs at hand, we have expanded the scope of the cata-
lyst for alcohol oxidation reactions using water as solvent. Initial study was performed at room
temperature using p-chlorobenzylalcohol as model substrate withTBHP as oxidant in presence
of K2CO3 as base using 3 mg of the Pd NPs. After a reaction time of 4h, only 19% p-chloroben-
zaldehyde was formed. However, on increasing the temperature to 80˚C, nearly quantitative
yield of product was obtained in 4h. Encouraged by this result, we have tested some more rep-
resentative alcohols for the oxidation reaction. Alcohols bearing electron-withdrawing groups
like p-nitrobenzylalcohol (Fig 6, Entry 5) or electron-donating groups like p-methybenzylalco-
hol (Fig 6: Entry 4) could be converted to corresponding aldehydes with moderate to high
yields. Our results showed that the position of substituent also have certain impacts on the per-
formance of the catalyst. For instance, oxidation of o-chlorobenzylalcohol gave 75% yield,
while under similar condition p-chlorobenzylalcohol gave 94% yield.
Reduction of Chromium(VI) to Chromium(III)
Chromium(VI) compound is considered to be one of the major pollutantscommonly found in
ground water at the industrial sites [47]. At present, various remediation strategies are avail-
able that can detoxify chromium, and one such method is the catalytic reduction of Cr(VI)
to Cr(III). Among various catalysts, NP-based systemsincluding Pd NPs are found to be par-
ticularly promising [48]. To investigate the effectiveness of our catalyst for this reaction, an
aqueous solution of K2Cr2O7 was treated with bio-synthesized Pd NPs in presence of formic
acid as reducing agent and the degradation of Cr(VI) to Cr(III) was monitored with UV-
Vis spectrophotometer. The aqueous solution of K2Cr2O7 exhibits a strong peak at 350 nm
which gradually decreases with time (Fig 7). Upon stirring the reaction for 40 min. this
peak finally vanishes indicating complete reduction of Cr(VI) to Cr(III) and the light yellow
colour of K2Cr2O7 solution changed to colourless. It is worth noting that under the same set of
experimental conditions, K2Cr2O7 and Pd(OAc)2 showed only 17 and 57% degradations
respectively.
Evaluation of anti-microbial activity against MDR isolate
The most potential MDR bacterial isolate (i.e. showing resistance against maximum number
of antibiotics viz., ampicillin, imipenem, tetracycline, and rifampicin) was identified as Crono-
bactersakazakiistrain AMD04 by 16S rDNA sequencing technique. The NCBI Gene Bank
accession no. KJ812198.1 was received for the novel isolate (ESI.S9 Fig). Antimicrobial activity
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 8. Figure showing clear zone of inhibition around the well containing Pd NPs confirm its antimicrobial
activity against MDR Cronobactersakazaki AMD04 strain; whereas the aqueous plant extract (PE) shows no
effect and Pd(OAc)2 shows minor effect against the isolate.
https://doi.org/10.1371/journal.pone.0184936.g008
of PdNPs was carried out by agar well diffusion method. Clear zone of inhibition with a diame-
ter of 31.67 1.53 mm around the well containing PdNPs in bacterial culture plate clearly con-
firm its bactericidal effect against the isolates (Fig 8). Whereas, aqueous plant extract and Pd
(OAc)2 solution show no effect against the isolate. The PdNPs exerted an MIC and MBC
of0.06mMand0.12mMrespectively against Cronobactersakazakii AMD04 (Fig 9) which was
measured spectrophotometricallyat an absorbance of 600 nm. It may be noted that although
bacterial growth can be measured at any of the visible light range, measuring the 600 nm peak
is a standard method of monitoring growth, as this wavelength corresponds to orange colour
and most bacterial cultures tend to grow orange as their culture grows more dense[49].These
MIC and MBC values aremuch lower than FDAoral tolerable daily intake for dosage forms
and components (i.e. 100μg/day) [50]. This finding makes PdNP a potential candidate for
using as a drug component to fight against MDR bacteria.
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 9. Graph showing MIC and MBC of PdNPs against Cronobactersakazakii strain AMD04 at different time intervals.
https://doi.org/10.1371/journal.pone.0184936.g009
Biofilm inhibition activity by PdNPs
Antibacterial activity with chemically synthesized Pd NPs has been previously reported[35],
although reports on biofilm inhibition activity by Pd NPs have not been seen so far.We are
intrigued to see whether our biosynthesized PdNPs are able to eradicate biofilms which are
known to display high resistance to toxic doses of antimicrobial agents that usually eradicate
planktonic cells. It may be noted that inhibitory effects of metal nanoparticles (e.g. AuNPs,
[51] ZnO-NP[52], etc) on pathogenic biofilm formation is an active area of research that has
got many recent attentions. When tested the PdNPs against novel MDR clinical isolate Crono-
bactersakazakiistrain AMD04, it displayed significant decrease of biofilm biomass. The experi-
ment was done in triplicate and results are expressed in terms of mean S.D. Maximum
inhibition of biofilm was recorded at low concentration (0.26 0.02 mM) of PdNPs (Fig 10).
Comparable decrease in biomass formation was also observed at 0.39mM and 0.52mM con-
centrations. At concentrations below 0.26mM and above 0.65mMPdNP, Cronobactersakazakii
exhibited slight increase in absorbance. This increase in absorbance is due to the interference
caused by increased concentration of PdNPs. Most unremitting and relentless bacterial infec-
tions are associated with biofilm growth, a strategy that has accelerated the emergence and
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Green synthesis of palladium nanoparticles and their multifunctional activity
Fig 10. Graph showing anti-biofilm effect of PdNPs against MDR clinical isolate Cronobactersakazakii strain AMD04.
https://doi.org/10.1371/journal.pone.0184936.g010
hasty spread of multidrug resistant bacteria. It is well known that biofilm associated bacteria
are much more difficult to be eradicated by traditional bactericidal antimicrobials than plank-
tonic cells. Moreover the PdNPs exhibited their potential as biofilm inhibitor against novel
MDR clinical isolate CronobactersakazakiiAMD04.On the basis of literature report [51], a pro-
posed mechanistic pathway of biofilm inhibition by Pd NPs has been shown in (ESI.S10 Fig).
Planktonic bacterial cells adsorb on the substrate and attaches with each other by cell signaling
and subsequent release of exopolysaccharide (EPS) forming a protective matrix. Similar studies
carried out using AuNPs suggested that the increased stress exerted by nanoparticles on
microbial cells resulted in increased planktonic growth and decreased adhesion of microbes
on substrate.
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Green synthesis of palladium nanoparticles and their multifunctional activity
Conclusion
In summary, we presented a green protocol of synthesizing PdNPs by using aqueous leaf
extract of GarciniapedunculataRoxb as bio-reductant. The PdNPs showed excellent activity as
catalyst for three important reactions namely the Suzuki-Miyaura cross-coupling reaction,
alcohol oxidation and reduction of Cr(VI) to nontoxic Cr(III). The Pd NPs also showed excel-
lent antimicrobial and anti-biofilm activities against a novel MDR clinical isolate Cronobacter-
sakazakii AMD04. The MIC and MBC values of Pd NPs are much lower than that of USP
administration limit for oral tolerable daily intake, which make these PdNPs potential drug
candidate to fight against MDR bacteria. The multifunctional applications along with ‘in
water’ reactions for both synthesis and catalysis are the main features of our catalyst.
Supporting information
S1 Fig. Garcinia pedunculata roxbleaves used for nanoparticle synthesis.
(TIF)
S2 Fig. HRMS spectrum of bio-extract of Garcinia pedunculata Roxb.
(TIF)
S3 Fig. TEM images of PdNPs in absence of starch.
(TIF)
S4 Fig. SEM-EDX images of the PdNPs.
(TIF)
S5 Fig. FTIR spectra of PdNPs andpowdered dry leaf of Garcinia pedunculata Roxb.
(TIF)
S6 Fig. DLS spectra of PdNPs.
(TIF)
S7 Fig. XPS survey spectrum of the PdNPs.
(TIF)
S8 Fig. TG thermogram of biosynthesized PdNPs.
(TIF)
S9 Fig. Phylogenetic relationship of the MDR isolate Cronobacter sakazaki strain AMD04
(highlighted in yellow colour) with ten most closely related strains based on BLAST results
taking Erwinia amylovora ATCC 15580 as out group.
(TIF)
S10 Fig. 1H NMR spectra of the products for the Suzuki-Miyaura reaction.
(PDF)
S11 Fig. Mass spectra of selected products for the Suzuki-Miyaura reaction.
(PDF)
S12 Fig. Mass spectra of products for the alcohol-oxidation reaction.
(PDF)
Acknowledgments
The Department of Biotechnology is gratefully acknowledged for financial support (Grant no:
MED/2013/23) under DBT-Twinning programme and also for providing Delcon as well as
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Green synthesis of palladium nanoparticles and their multifunctional activity
IBT hub facility to the Centre for Biotechnology and Bioinformatics, Dibrugarh University.
The UGC, New Delhi has also been acknowledged for the SAP-DRS-I grant awarded to the
Department of Chemistry. The service of SAIF, NEHU, Shillong was gratefully acknowledged
for TEM analysis. Dr. S. Pramanik was acknowledged for English editing in the revised
manuscript.
Author Contributions
Conceptualization: Pankaj Das.
Data curation: Munmi Hazarika, Debajit Borah, Popymita Bora.
Formal analysis: Munmi Hazarika, Ana R. Silva.
Investigation: Debajit Borah.
Methodology: Munmi Hazarika, Debajit Borah, Popymita Bora.
Project administration: Pankaj Das.
Supervision: Pankaj Das.
Writing – original draft: Debajit Borah.
Writing – review & editing: Munmi Hazarika, Ana R. Silva, Pankaj Das.
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