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Plant mediated green synthesis: modied approaches

Cite this: Nanoscale, 2013, 5, 10155

Ratul Kumar Das and Satinder Kaur Brar*

Plant mediated green synthesis of dierent metallic nanoparticles has emerged as one of the options for
implementation of green chemistry principles, and successfully made an important contribution towards
green nanotechnology. However, beyond the synthesis and application aspects, the science of green
synthesis has carried some wrong perceptions in an unforeseen fashion. In this review, some of the key
issues related to the green synthesis of metallic nanoparticles employing plants as reducing/capping
agents have been addressed. Random selection of plants and its overall impact on the dierent aspects
of green synthesis have been discussed. Emphasis is given to the setting of some standard selection
criteria to be adopted for selecting a plant for use in green synthesis. How selection of a plant can
positively or negatively inuence both procedure and products of a green synthesis process is the prime
concern of this article. In addition to selection, the key issue of biocompatibility associated with green

Received 17th May 2013

Accepted 22nd August 2013
DOI: 10.1039/c3nr02548a
www.rsc.org/nanoscale

synthesized metallic nanoparticles has been considered. Both selection of plant and biocompatibility
were reconsidered for their minute details in terms of synthesis, analysis and data interpretation in the
green synthesis approach. The key factors capable of ne tuning the core meaning of green in the
synthesis of any metallic nanoparticles were taken into consideration. This article is an eort towards
keeping the core meaning of green synthesis.

1.
INRS-ETE, Universite du Quebec, 490 Rue de la Couronne, Quebec (QC) G1K 9A9,
Canada. E-mail: satinder.brar@ete.inrs.ca; Fax: +1 418 654 2600; Tel: +1 418 654
3116

Ratul Kumar Das graduated

from Tezpur University, India,
with a MSc. degree in the eld of
Molecular Biology and Biotechnology in 2007. He worked as a
Junior Research Fellow and
Senior Research Fellow in the
Dept. of Biotechnology and
Centre for the Environment,
Indian Institute of Technology
Guwahati (IITG) from 2008 to
2012 and published many
research articles on nano drug
formulations and plant mediated green synthesis of gold nanoparticles. He is currently a Ph.D. candidate at Institut National de
la Recherche Scientique (Eau, Terre et Environnement, INRS-ETE,
Universite du Quebec, Quebec, Canada) under the guidance of
Prof. Satinder Kaur Brar. His research interests include wastewater management and green nanotechnology.

This journal is The Royal Society of Chemistry 2013

Introduction

The plant world has been revisited with the renewed interest
since the perception of nature's ability to work as a nanofactory. As far as synthesis mechanisms are concerned, the

Satinder Kaur Brar has been an

Associate Professor at Institut
National de la Recherche Scientique (Eau, Terre et Environnement, INRS-ETE) since June,
2007. She graduated with a
Master's in Organic Chemistry
from National Chemical Laboratory, Pune, India with Master's
in
Technology
in
Environmental Sciences and
Engineering from Indian Institute of Technology, Bombay,
Mumbai, India and a Ph.D. in biochemical engineering from INRS,
Quebec, Canada. Her research interests lie in the development of
wastewater and wastewater sludge based value-added bioproducts, such as enzymes, organic acids, platform chemicals,
biocontrol agents, biopesticides, butanol and biohydrogen, and in
the fate of endocrine disrupter compounds, pharmaceuticals,
nanoparticles and other toxic organic compounds during valueaddition of wastewater and wastewater sludge.

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Nanoscale
green synthesis (GS) concept is still in its infancy, but the
success rate of synthesis is very high and has exhibited great
diversication in the taxonomic groups of explored plants.15
The approach was adopted to make the nanoproducts safely
applicable for human use. However, one has to be very selective
in the case of plants, as a common repertoire of phytochemicals
or oxidationreduction systems imparts the same reducing
power in taxonomically distant plant groups. For instance,
bioactive components belonging to the phenolic, avonoid,
alkaloid and terpenoid families are common to angiosperms,
gymnosperms, pteridophytes and bryophytes, which can play
important role in green synthesis mechanisms.612 In this
regard, the present plant selection process seems to be suering
from a lack of any authentic guidelines, except for a few real
concerns.1,13 Selection of a plant with a proper scientic background of broader applications and full of qualities for recommendation as a representative for a particular taxonomic group
is very important. However, this can be quite challenging as it
needs extensive study on the phytochemical evolutionary
aspects of plants and to relate the same with GS eciency.
Further to the selection of a plant, another prime issue of GS
is the biocompatibility of the green synthesized products. In
general, prior to any bio-medical applications of engineered
nanoparticles (ENPs), testing for biocompatibility by carrying
out quantitative and/or qualitative in vitro cytotoxicity experiments on mammalian cell lines is a very important step.1420
This ensures that ENPs intended for biological applications do
not cause any adverse eects. For green synthesized MNPs,
reports available on the cytotoxicity studies at in vitro and in vivo
levels are very informative and conclusive.2125 Such basic
studies conrm the biocompatibility of MNPs on dierent
mammalian cells lines. However, in terms of the procedure
followed and the product obtained, the scientic ground of
claims over the biocompatibility of conventionally or green
synthesized MNPs can not be considered to be the same. It is
now a well-established fact that toxicities associated with
conventionally synthesized MNPs are due to the le-over or
unused amounts of toxic and hazardous reducing and stabilizing agents carried by MNPs during their synthesis.2628 This
renders MNPs unsafe for bio-medical applications. This fact is
also applicable to green synthesized MNPs as they follow the
same steps (formation of embryos, nucleation, primary nanoparticles and nanoparticles) of MNPs synthesis in a bottom-up
process.28 However, due to the inherent biocompatible nature of
phytochemicals carried over by MNPs in GS, they are much safer
and well tolerated by mammalian cells. Meanwhile, chemical
inertness renders gold useful for internal applications in
humans such as teeth implants and radioactive gold used in
cancer treatment.29,30 Thus, human interference in the form of
laboratory chemicals (reductants and surfactants) have made
otherwise highly biocompatible gold hazardous. However, in a
real sense, any claim over the biocompatibility of nanoproducts
needs the incorporation of sucient scientic support and to
achieve that, setting of some standard parameters of biocompatibility is a must. To be more specic with GS, in a broader
sense, it seems to be highly feasible that every MNP of GS origin
passes through the cytotoxicity barrier provided that applied

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MNPs concentration is within the range of cell tolerance.
However, for dierent plants, biocompatibility levels showed
great variation in terms of cell viabilities.22,25 In addition, the
unused phytochemicals carried over by MNPs during their GS,
might exert their own impact on the overall assessment of
biocompatibility. These issues are very important to dene the
core zone of GS and also to make it more generalized in terms of
the designing of its workable principles.
In this article, pros and cons of random selection of a plant
candidate for GS have been reviewed followed by emphasis on
ne tuning of biocompatibility of green synthesized MNPs.
Proposals for new approaches have been put forward for constructing a strong platform for the better execution of GS
principles.

2.

Plant aspects in green synthesis

2.1. Plant groups vs. green synthesis

From a taxonomic point of view, reports on GS have covered
from the less evolved non-vascular (algae, bryophytes and
pteridophytes) to highly evolved vascular plant groups
(gymnosperms and angiosperms). Among all the explored
taxonomic plant groups explored for GS, angiosperms outnumbered the others (Fig. 1). For lower groups of plants, reports
on algae are piling up, while reports on GS employing pteridophytes, bryophytes and gymnosperms are very scarce (Table 1).
One quite obvious reason for the very high frequency of
involvement of angiosperms in GS is the cosmopolitan nature
in their habitats as compared to other plant groups. Being the
most highly evolved taxonomic plant group, angiosperms are
expected to perform better in GS irrespective of any medicinal
background history. However, as mentioned before, a common
repertoire of phytochemicals are responsible for imparting the
reducing and capping properties to both higher and lower plant
groups. So, the valid reason that stands for selecting angiosperms as the frontline candidates for GS is not based on the
eciency level but probably the worldwide ease of access to
them and most explored plant groups with dierent interests.
On the other hand, while taking into consideration both organic
and phytochemicals evolutionary aspects for GS, it is dicult to
organize the plant groups into a hierarchical trendline. No
evidence exists for arranging the plant groups in an ascending
or descending order of GS eciency. Approaches can be made
towards the quantitative or qualitative evaluation of phytochemicals for GS and then to correlate it with GS eciency.
2.2. Mechanisms of green synthesis
Mechanisms behind GS and causative agents for reduction and
capping exhibited variation within same and among dierent
plant groups.1,31,32 However, compared to the number of
explored plants, investigation proceedings on reduction and
capping mechanisms are exceptionally rare (Table 1). Most of
the reports on GS have claimed common phytochemicals as
causative agents for the reducing properties.4045 If these
phytochemicals are held responsible for GS, there should not be
many variations in the GS mechanisms for dierent taxonomic

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Fig. 1

Nanoscale

The green synthesis aspects of the plant kingdom.

plant groups. This assumption is quite rudimentary as each

class of phytochemicals is actually a library of functionally
dierent molecules. For example, alkaloids alone represents
over 12 000 cyclic nitrogen-containing compounds that are
found in over 20% of plant species.46 The scenario becomes
even more complex when the fact that more than 5000 phytochemicals have been identied is considered.47 The reduction
property displayed during GS might be a coordinated function
of all those phytochemical molecules. Moreover, phytochemical
evolution might have its own inuence on the reduction and
capping mechanisms for each plant group. A better comprehension of the plant biochemistry, plant physiology and plant
molecular biology involved in GS can lead to a general conclusion on the mechanism domain.

2.3. Setting standards in plant selection

Undoubtedly, nature has gied the plant kingdom with excellent powers of reduction. However, only ethanobotanical based
ndings are not enough for selecting a plant. If bio-reduction of
metallic cations is considered as a part of a plants defense

This journal is The Royal Society of Chemistry 2013

mechanism against ionic stress, then there is every possibility

that many plants should be exploitable for GS. In this regard,
the bio-reduction process must be reconsidered in terms of
chemical evolution of phytochemicals in dierent plant groups.
In general, the whole process of GS has been given a haphazard
look. Instead of nding the exact causative agents of bioreduction for individual plans, eorts should have been made
to pick up some representatives for each plant group and
standardize a protocol. An ascending or descending pattern of
eciency for GS within the same plant group has to be followed.
At the same time, one must be aware of the fact that too many
process parameters (reaction time and temperature, concentrations of metallic salt and plant extract, pH of reaction
medium, size and shape manipulations of MNPs etc.) are
generally xed while standardizing protocols in GS. Such
approaches are of little importance if not done for taxonomically close plant members. Moreover, a requirement with the
motivation of scaling up GS from the lab scale to a commercial
level must be xed. A plant with all the desired qualities for GS
may not be failproof while translating the technology onto a
large scale level. It's worth mentioning that some of the plants

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Table 1

An overview of the plant mediated green synthesis approaches of metallic nanoparticle synthesis

Plant group

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Type of metallic Mechanism of

nanoparticle
synthesis

Plant name

Algae (examples Chlorella vulgaris

with known
mechanisms)
Chlorella pyrenoidusa
Bryophytes
Anthoceros
Riccia
Fissidens minutus
Pteridophytes
Adiantum capillus verenis
Adiantum caudatum
Adiantum philippense
Gymnosperms
Cycas
Ginkgo biloba
Pinus thunbergii
Angiosperms
Camellia sinensis
(example
with known
mechanism)

Au

Ag

Protein (28 kDa)

mediated
Reductase enzyme
Not explored

Ag

Not explored

Au, Ag
Au

Not explored

Ag
Au

Reference

For other algal members

applied for GS, mechanisms
are yet to be explored
From a phytochemical
evolutionary point of view,
exploration of the mechanisms
is very important

31 and 32

Catechins, Theaavins and For other reported members,

Thearubigins played
mechanisms and causative
a major role
agents are not specied/explored

exploited for GS have huge potential for bio-medical applications and can be considered for scaling up.1,6,48 To screen out a
plant as the best candidate, one has to x some standard criteria
with exclusion or inclusion principles. If a random mode of
plant selection for GS is employed, advantages and disadvantages arising out of such procedures can be helpful to scrutinize
a plant for designing a greener technology. As shown in Table 2,
the plant selection parameters presently followed in GS leads
nowhere in making conclusive remarks. None of these parameters are based on standardized protocols or authentically
guided selection criteria. This trend is being followed just to
orient the ndings with a new plant towards the basic working

Table 2

Important remarks

2, 3, 3339

principles of GS. The outcome of the comparative analysis of the

advantages and disadvantages of these parameters have been
presented as the important remarks in Table 2. For each
parameter, the disadvantages seem to be more practical problems or queries that need to be addressed in future. Another
important aspect is the lack of scientically evaluated data on
those plants not capable of GS under any reaction conditions.
This can be of major concern towards the comparative analysis
of plants with and without the capabilities of GS.
Contrarily, an attempt can be made to make GS more organized and generalized so that every new report on GS truly
contributes in constructing a much needed plant repository to

The pros and cons of plant selection for green synthesis of metallic nanoparticles

Plant selection parameters

Advantages

Disadvantages

Important remarks

First reporting

An unexplored plant is rst time

evaluated for GS, thus enriching the
repository of natural reductants
Less time consuming, high process
yield, MNPs of desired size and
shapes
Acts both as reducing and capping
agents

Many reports lack proper scientic

grounds of selection of the used
plant
No standard is set to measure
eciency level

Ratio should be maintained

between the importance of the plant
and its exploration for GS
Precision is required

Need more sophistication of data

supporting the capping

Ecient under dierent sets of

reaction parameters
Can be reported individually

Should not be generalized, capping

is a complicated process, not always
subsequent to reduction
May look virtually dierent, but
governed by same principles
Leads to redundancy

Broad application

Covers synthesis of dierent MNPs

Number of reports appears scarce

Medicinal values

Pre-requirement before exploitation

for GS

Availability

Easy access of the plant round the

year, thus no time barrier for
sample collection

Majority of reports are based on

ethanobotanical ndings, not
executed even at in vitro level
Plant physiology is highly
susceptible to soil parameters,
which are in turn weather
dependent

Eciency

Dual functions

Propensity
Use of dierent body
parts of the same plant

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Not all plants exhibit propensity

Should always be compared to the
results obtained using the whole
plant
Should be addressed in terms of the
redox potentials of dierent
metallic ions
Plants of clinical relevance should
only be preferred
Plant tissue culture can be vital to
better address this problem

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be referred as shown in Table 3. This is a very preliminary step

towards bridging the gap of knowledge between exploitation of
natures ability and the scope of feasibility to make GS more
scalable and predictable. The proposal for the new approaches
in plant selection criterion is quite feasible and applicable to all
groups of plants. For example, genetic manipulation and plant
tissue culture techniques can be very eective for making GS
more sustainable. Similarly, eorts to construct a reference
plant for more closely related plant members will make the GS
approach more convergent in terms of the number of such
plants studied and reference data for the other unexplored
members.

3.

Biocompatibility and green synthesis

The green nanotechnology of MNPs synthesis is preferred over

conventional synthesis methods provided that the products of
GS are biocompatible. However, the core meaning of the term
biocompatibility lies in many important deciding factors
associated with GS. It is important to delve into those overlooked or underestimated factors that might have the controlling key of ne tuning of biocompatibility and try to extract out
the best resultant part of all such factors.
3.1. Plants have big role in conferring biocompatibility to
metallic nanoparticles
Plants have control over the morphology and biocompatibility
of MNPs.19,49 In addition, as mentioned before, in MNP
synthesis by conventional methods the le-over amount of
toxic chemicals carried by the MNPs on their surfaces can cause

Table 3

toxicity in the human body. It is obvious that the le-over

amount has a big role to play in the determination of biocompatibility whether it is of chemical or phytochemical origin. In
fact, random selection of plants makes the le-over amount
worth worrying. In GS, the le-over amount of phytochemicals carried by MNPs virtually should not make any sense of
toxicity as the original chemical has been replaced by natural
ones. However, while doing cytotoxicity studies with MNPs
carrying unused phytochemicals, apart from the MNPs alone,
this repertoire of plant molecules may exert its own positive or
negative eects on the cells being used. Hence, the resultant
eects of the MNPs are actually a combined one. The viabilities
shown by various cell lines are also an indication of tolerance
level for the phytochemicals of a particular plant origin. Thus,
even if the sizes and shapes of MNPs are kept favorable for a
particular cell line, biocompatibility might be a sole response to
the surface bound plant molecules coming in contact with the
cells. The threshold limit of concentrations of MNPs for toxicity
is actually the borderline response of cells. Practically, it will be
very dicult to quantify the phytochemicals carried by MNPs;
otherwise the biocompatibility could have been better judged
separately for both MNPs and the phytochemicals they carry.
Moreover, it is dicult to recognize all those plants with same
repertoire of phytochemicals for GS, as there is great variation
in the structure and function of plant molecules at the molecular level which will ultimately inuence the biocompatibility to
a great extent. This will be a huge challenge for researchers to
nd out the the impacts of individually unique plant molecules
on the determination of overall biocompatibility. Thus, it is
advisable that preference should be given to plants that we are

The thrust areas of research of plant mediated green synthesis

Standard parameters

Criterion of selection

Present status

Important remarks

The selected plant should have

clinical relevance

Previously well documented

scientic reports on applications
for human health
Representative from a monophylogenetic line within a group
and extensive experimentation to
set it as reference plant
In vitro level study on normal
human cell lines including
cellular damage

Badly suering from random

selection

Scaling up the process for

commercialization. To replace
MNPs with green synthesized ones
Can be referred to the
representative while reporting a new
plant

Phylogeny

In vitro study

Precision

Identication of the fraction

causing the synthesis and the
associated mechanisms

Geographical distribution

Though not cosmopolitan, at

least should not be very narrow
in its distribution. Alternatively,
plant tissue culture can be
a good approach
Selection can also be made at a
molecular level. Control over
genes regulating the reducing
actions of plants can be very
useful for designing a laboratory
model for GS

Genetic Aspects

This journal is The Royal Society of Chemistry 2013

No such parameter has

been xed

Mostly reported without in vitro

study or reported with cell
deaths or viability (extreme
ends of cell treatment)
Crude forms of plant extracts
mostly exploited, except
a few with fractionated ones
Some reported plants are very
specic to a particular zone of
inhabitation which is a barrier in
accessing the plant sample other
than its native place
Unexplored area

In vitro studies lead to in vivo and

clinical trials

Isolation, purication and

identication of a particular
fraction will denitely promote a
plant for commercialization
Plants with huge potentials in GS,
can be considered for better
protection and their cultures can
help in accessing them worldwide
Genetic manipulation can lead to
transgenic plant with enhanced
quality for GS

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more familiar to and to be more specic, edible ones should be
the prime choice as they have the added advantage of naturally
proven body tolerance.

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3.2. The purity of MNPs inuence the biocompatibility

In GS, as a part of purication process, MNPs are subjected to
high speed centrifugation (10 00020 000 rpm) to remove all the
uncoordinated plant molecules from the colloidal solution.12,50,51 The pellet of MNPs obtained is considered to be free
of any unused chemicals or plant extract. However, if a crude
plant extract is considered, it has arrays of compounds of
dierent molecular weights (MW). Hence, while putting the
colloidal solution into centrifugation, there is a chance that
apart from those phytochemicals tightly coordinated to MNPs
surfaces, plant compounds of high MW also form pellets and
settle (Fig. 2). This can only be avoided if the purication is
done in multiple steps of centrifugation with an increasing
speed. However, these steps are not followed in the common
practices of GS. Thus the extra spell of phytochemicals carried
by MNPs can manipulate the analysis of their biocompatibility.
3.3. Instrumental analysis
Most of the reports on GS incorporate Fourier transform
infrared (FTIR) and energy-dispersive X-ray analysis (EDX) data
to show the presence of phytochemicals on the surfaces of
MNPs (capping behavior). Except only a few, others lack the
more sophisticated instrumental investigations, such as X-ray
photoelectron spectroscopic (XPS) and circular dichroism (CD)
spectral analysis of the capped MNPs.52,53 Merely showing the
presence of compounds or elements is not enough to conclude
about the close interaction between MNPs and phytochemicals.
In XPS, detailed elemental analysis and information on oxidation states of metallic ions or surrounding elements of plant

Fig. 2

Minireview
origin can predict the chemical environment by measuring the
binding energy levels of dierent elements and ions, while CD
analysis can strongly support the existence and conformation of
biological moieties (e.g. peptide capping) on MNP surfaces.52
These analyses are important as cells might behave dierently
to the dierent ionic states of the same element and the surface
elemental composition of MNPs plays an important role in the
biocompatibility behavior of MNPs.54,55
3.4. Cytotoxicity studies
The cytotoxicity studies of green synthesized MNPs on dierent
mammalian cell lines are generally accompanied with quantitative cell viability data based on dye conversion assays like MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide); while few investigations have included
qualitative ndings (acridine orange and ethidium bromide
live/death double staining assays).2125 What we have learned
from all those contemporary research outcomes is that the
background of biocompatibility assessments is based on the
two extreme fates of treated cell lines, i.e. (a) viability and (b)
death. The rst one recommends MNPs to be biocompatible,
while the second one rules out the same. However, in between
these two extreme consequences of life and death, cellular
damage may be possible. Live cells can still be considered for
structural (cell membrane, cytoskeleton, DNA etc.) or functional
(cellular transportation, protein synthesis) abnormalities when
exposed to MNPs. It is also supported from the facts that MNPs
are highly explored for use in various biological molecular level
applications due to their high anity with nucleic acids and
proteins.5658 Thus, nanoscale analysis of MNPbiomolecule
interactions is vital in determining the biocompatibility of
MNPs. However, the number of reports in this direction are

The role of plant molecules in conferring biocompatibility to MNPs.

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scarce. In addition, while designing a cell line specic in vitro
experiment for any green synthesized MNPs, one must carry out
the control experiment with chemically synthesized MNPs
against the same particle concentration and incubation time
used for the green synthesized one. For a constant particle dose,
comparative analysis of cell viabilities would provide more
precise data on biocompatibility for green synthesized MNPs.

4.

Concluding remarks

An attempt to address some of the key issues of newly emerging

green nanotechnology for the synthesis of MNPs was made in
this review article. A better comprehension of the roles played
by the two important parameters namely selection of the
plant and biocompatibility in sticking to the original principles of GS has been highlighted. The modied approaches
proposed in the dierent context of common practices of GS are
important to make this green science more precise and practical
in its execution. Selection of a plant with strong scientic
background and socio-economic importance has been foreseen
as the common adoptable approach for GS. Taxonomic and
evolutionary aspects of plants were projected for setting the
guidelines of plant selection for dierent plant groups. Further,
instead of optimizing parameters set up for each plant,
designing of basic protocols applicable to taxonomically related
plants was emphasized. A novel concept of a reference plant has
been sought for making GS more convergent. Referral standards for measuring the various qualitative and quantitative
parameters of a plant used in GS were suggested for the rst
time. A critical analysis on the biocompatibility of conventionally or green synthesized MNPs lead us to the conclusion that
current methodologies followed in GS have imparted some
elements of imperfections in an unforeseen fashion. These were
highlighted for ne tuning of biocompatibility and to have
more precision in its assessment. The present article is an
attempt to technically improve GS at its grass roots level without
any intention of highlighting the dark side of GS. Discussions
made on the deviations of common practices of GS and their
rectications are merely an outcome of in depth studies on the
present scenario of plant mediated GS of MNPs.

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