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Sustainable production of biomass and industrially important secondary
metabolites in cell cultures of selfheal (Prunella vulgaris L.) elicited by silver and
gold nanoparticles
Hina Fazal
a
, Bilal Haider Abbasi
b
, Nisar Ahmad
c
, Mohammad Ali
c
, Syed Shujait Ali
c
, Abbas Khan
d
and
Dong-Qing Wei
d
a
Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex, Peshawar, Pakistan;
b
Department of Biotechnology,
Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan;
c
Center for Biotechnology and Microbiology, University of Swat,
Swat, Pakistan;
d
Department of Bioinformatics and Biostatistics College of Life Sciences and Biotechnology, The State Key Laboratory of
Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China
ABSTRACT
Elicited artificial in vitro cultures are gaining more interest due to their uniform biosynthesis of industri-
ally valuable secondary metabolites. In this study, a unique methodology was applied, in which differ-
ent ratios of gold (Au) and silver (Ag) nanoparticles (NPs) were supplemented to submerge cultures to
investigate sustainable production of biomass and antioxidant secondary metabolites. Cell suspension
cultures were exposed to Ag and AuNPs alone or different ratios of AgAuNPs (1:2; 1:3; 2:1; 3:1) in com-
bination with NAA. The combination of AgAuNPs (3:1) with NAA enhanced fresh (9.25 g/100 ml) and
dry biomass (0.64g/100ml) of suspended cells than control (6.67; 0.233 g/100 ml). AuNPs with NAA-
augmented media enhanced biomass accumulation in lag, log and stationary phases in a period of
49 days. Furthermore, AgAu (3:1) and AgAuNPs (2:1; 1:2) with NAA enhanced protein contents, peroxid-
ase and superoxide dismutase enzymes. However, maximum phenolics (TPC; 10.61 mg/g-DW) and flavo-
noids (7.62 mg/g-DW) were observed in cell cultures exposed to a combination of AgAuNPs (1:3) and
NAA than control (6.27, 5.49mg/g-DW). The combination of AgAuNPs (2:1) with NAA enhanced antioxi-
dant activity (87.85%) in cell cultures. This study will help in illuminating the impact of NPs on cultures
development and production of natural antioxidants.
ARTICLE HISTORY
Received 18 April 2019
Revised 29 April 2019
Accepted 29 April 2019
KEYWORDS
Prunella vulgaris; cell
culture; secondary
metabolites; stress enzymes;
nanoparticles
Introduction
Nanotechnology is a multipurpose field that has got ever
escalating applications in nearly every field of science. It is
advancing rapidly and expected to turn into a trillion-dollar
industry by 2018 with employment of 2 million, exceeding
the industrial revolution impact [1]. This industry is posing
significant impacts on the environment, economy and society
worldwide. In turn, it is producing both encouraging and dis-
couraging responses from governments, researchers and
social media [2]. Due to their distinctive characteristics, this
field is advancing its applications in the diverse fields of biol-
ogy. Conversely, the use of nanoparticles is novel and
requires elaborative research in the area including tissue cul-
ture and medicinal plant biotechnology. Currently, the major-
ity of studies regarding plant growth, seed germination and
physiological responses are concerned about nanoparticles
toxicity [1,3]. It has been reported that seed germination is
accelerated in Glycine max by the applications of TiO
2
and
SiO
2
and they also enhanced activities like antioxidation and
nitrate reductase [4]. Similarly, Sharma et al. [5] reported that
antioxidative enzymes and growth of the seedlings is
improved by AgNPs. Contrarily, seed germination and root
development are inhibited by ZnONPs in various plants [1].
Furthermore, in Brassica oleracea, synthesis of chlorophyll,
metabolism and dry biomass are enhanced by the applica-
tion of TiO
2
NPs [6]. However, the phenomenon of plant
growth and development by these nanoparticles is still poor
and needs further research.
Prunella vulgaris (self heal/all heal) is thermophilic and
hygrophilous edible herb (family; Lamiaceae), that has been
utilized in North America, China and Europe as a potent
herbal medicine [7]. Recent studies emphasized on the
potentials of P. vulgaris as promising anti-HIV and against
herpes viruses [7]. However, in Unani, Korean and Chinese
traditional medicine, it has been widely used against cold
and sore throat, headache, oedema goiter and nephritis
[8–10]. Due to the occurrence of polyphenols in this plant
species, it also exhibits properties including, antitumor, anti-
viral and anti-inflammatory [11,12]. Plant-based phenolic and
flavonoids, and their various activities are associated with
CONTACT Nisar Ahmad ahmadn@uswat.edu.pk nisarbiotech@gmail.com Center for Biotechnology and Microbiology, University of Swat, Swat, 19200,
Pakistan; Dong-Qing Wei dqwei@sjtu.edu.cn Department of Bioinformatics and Biostatistics College of Life Sciences and Biotechnology, The State Key
Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, Minhang District, China
ß2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
2019, VOL. 47, NO. 1, 2553–2561
https://doi.org/10.1080/21691401.2019.1625913
antioxidant potential [13]. During extreme conditions, like
light fluctuation, osmotic stress and in defense mechanism
against injury, the plant antioxidant system plays a protective
role against these conditions. DPPH-based antioxidant activity
or others have a direct or linear relationship with species
resistance toward the stressors [14–16].
During unusual conditions, reactive oxygen species (ROS)
interact with biomolecules and therefore hinder the process of
growth and differentiation as well as effect secondary metabol-
ism [17]. To neutralize the adverse effects of these stressful
ROS, plant cells utilize endogenous enzymes such as POD and
SOD, or provoke non-enzymatic system to release polyphe-
nolics [18]. Endogenous enzymes are of great importance in
plant development, morphogenesis, catabolism of auxins and
differentiation [19]. Likewise, the non-enzymatic machinery
helpstoquenchthetoxicfreeradicals,motivatetheimmune
machinery, control apoptosis, activate gene expression, stimu-
late the biosynthesis of enzymes and cooperate with cell-cycle
arrest [20]. Generally, in plants, the production of bioactive
compounds results as a comeback to the biotic and abiotic
stresses [21]. Therefore, the exposure of plant cell culture to
biotic and abiotic elicitors stimulates the cultured cells to syn-
thesize metabolites in higher quantities [22–25].
Therefore, the major aim of this study was to explore the
possible effects of Ag and Au nanoparticles, as metallic elici-
tors, in combination with NAA on the development of cell
suspension culture in P. vulgaris. Moreover, the impact of
these nanoparticles on the biosynthesis of the content of
total protein (CTP) and polyphenolics was also investigated.
Also, enzymatic potentials (SOD and POD) were conducted
along with the determination of possible DPPH-based antioxi-
dant activities.
Methods
Establishment of cell suspension from calli cultures
In order to develop calli cultures, leaf explants were exploited
from in vitro grown plantlets of P. vulgaris. Here, 0.5–1cm
young leaf pieces were carefully harvested and placed on the
Murashige and Skoog [26] media. To obtain maximum callo-
genesis, the media was augmented with 2.0 mg l
1
naphtha-
lene acetic acid (NAA). Sucrose (30 g l
1
) as limiting substrate
and good carbon source was added to the cultures media,
followed by the addition of gelling agent (8 g l
1
agar;
Oxoid, England). The fluctuated pH of the culture media was
altered to 5.6–5.8 through the addition of weak acid or base
(pH meter, Eutech Instruments, Singapore). The sealed jars
containing media and plant growth regulators (PGRs) were
autoclaved for 25 min at 121 C (Systec, Germany). The inocu-
lated cultures were placed in growth room having an opti-
mum temperature (25 C) and 40–50 mmol m
2
s
1
light
intensity for better response. Finally, the 16/8 h photoperiod
was applied in growth room to get maximum calli cultures.
To established suspension culture, 30-days old calli was
shifted to MS basal media augmented with 2.0 mg l
1
NAA in
Erlenmeyer flask (500 ml). The cultured flask was placed on an
orbital shaker (120 rpm; Gallenkamp, England) at 25 Cforpro-
ducing stock cell suspension culture. This culture was filtered
after 14-days and transferred to 250 ml flasks for subsequent
experiments. Synthesized NPs were procured from the research
projectofRahmanetal.[27] Accurately, 30 mgl
1
of each NPs
along with NAA (2.0 mg l
1
) were added to MS media. MS
basal media augmented with AuNPs with NAA (T1), AgNPs
with NAA (T2), AgAu (1:2) with NAA (T3), AgAu (1:3) with NAA
(T4), AgAu (2:1) with NAA (T5), AgAu (3:1) with NAA (T6) and
NAA alone (T7) were placed on orbital shaker for the establish-
ment of cell suspension culture (Table 1).
Growth kinetics and biomass determination
The data for suspended cells growth kinetics were obtained
with 07 days interval for 07 wee ks period. From these data, the
suspended cells growth curve was developed upon exposure
to each ratio of metallic nanoparticles (1:2; 1:3; 2:1; 3:1) in com-
bination with 2.0 mg l
1
NAA. In order to investigate fresh
weight, the suspended cells were slowly washed with sterile
distilled water and slightly dried with filter paper and then
weighed. The harvested cells were then weighed (Sortorious;
Germany) for the estimation of FB. The suspended cells were
placed in an oven (55 C; Thermo Scientific; Germany) to inves-
tigate the dried biomass. Herein, the FW and DW of suspended
cells are represented in gram/100ml.
Determination of antioxidative enzymes
Antioxidative enzymes or stress enzymes in suspended cells
were determined according to the established protocol of
Nayyar and Gupta [28]. One gram fresh calli cultures in the
presence of 1% PVPP (pH 7) was carefully blended with 10 ml
of extraction buffer (50 mM) prepared from KH
2
PO
4
in auto-
claved tubes. The solution was centrifuged at 14,000 rpm under
chilling temperature (4 C) for approximately 20–30 min. After
centrifugation, the upper portion of the mixture was used for
the investigation of antioxidative enzymes. The protocol of
Lagrimini [29] was followed for the evaluation of peroxidase
(POD) activity with some modifications. Furthermore, for inves-
tigating superoxide dismutase (SOD), the recent method of
Ahmad et al. [14]wasused.
Determination of polyphenolics content
To prepare extract for polyphenolics content, the dried
suspended cells were powdered and mixed with ethanol
Table 1. Application of various treatments of metallic nanoparticles alone or
different ratios of silver and gold nanoparticles in combination with naphtha-
lene acetic acid [AuNPs þNAA (T1); AgNPs þNAA (T2); AgAuNPs 1:2 þNAA
(T3); AgAuNPs 1:3 þNAA (T4); AgAuNPs 2:1 þNAA (T5); AgAuNPs 3:1 þNAA
(T6) and control) for accumulation of biomass and biosynthesis of desirable
secondary metabolites in cell cultures of P. vulgaris.
Treatments MS þNanoparticles (30 mgl
-1
) Phytohormone (2.0 mg l
-1
)
T1 Au NAA
T2 Ag NAA
T3 AgAu (1:2) NAA
T4 AgAu (1:3) NAA
T5 AgAu (2:1) NAA
T6 AgAu (3:1) NAA
Control MS NAA
2554 H. FAZAL ET AL.
solvent. To obtain maximum extraction, the powdered solu-
tion was placed in a dark room for seven days period. The
supernatant was collected in a separate container after cen-
trifugation at 14,000 rpm for 10 min and the cells debris were
discarded. The collected supernatant was exploited for
polyphenolics determination. The polyphenolics content were
investigated in dried extracts according to the method of
Ahmad et al. [14]. Accurately, the supernatant (0.03 ml) from
each treatment was combined with 0.03 ml of FCR (2 normal
Folin–Ciocalteus reagent) for in vitro activity along with
2.55 ml sterile distilled water. Before filteration (through
45 mm membrane) the solution was centrifuged (10,000 rpm)
for 10 min. A wavelength of 760 nm was applied for phe-
nolics estimation through double-beam spectrophotometer
(UV visible; Shimadzu, Japan). A known concentration (1 to
10 mg/ml) ofGallic acid as standard for phenolics was
exploited for curve and regression (R
2
¼0.9878). The TPC
mean values were indicated as Gallic acid equivalent (GAE)
milligram/gram of dry weight (DW) using the following for-
mulae.
% Total phenolics ¼100 AbSC AbC
ðÞ
=CnFDnF
ðÞ
Here, Ab
SC
represents suspended cells absorbance; Ab
C
represents control absorbance, while the Cn
F
and Dn
F
repre-
sent the conversion and dilution factors from the calibra-
tion curve.
The Ahmad et al. [14] protocol was used for flavonoid deter-
mination. In this activity, 0.25 ml of cells solution and 1.25 –ml
of sterile distilled water was carefully combined with 0.075 –ml
of aluminum chloride (5%). The mixture was then combined
with 0.5ml of 1 M NaOH and placed in a dark room to minim-
ize the oxidation reaction. The mixture was centrifuged at
10,000 revolutions per min and the supernatant absorbance
was checked at 510 nm for the investigation of flavonoids con-
tent. The different known concentrations (1–10mg/ml) of Rutin
as standard for flavonoids were exploited for obtaining the cali-
bration curve. The regression obtained from the calibration
curve was R
2
¼0.9866. Finally, the flavonoid results were rep-
resented as Rutin equivalent mg/g-DW (milligram per gram of
dried extract).
Estimation of content of total protein
Prunella cell cultures were extracted through the methods of
Giri et al. [30]. Fresh and viable suspended cells from each
treatment (4 g) were grinded with liquid nitrogen. Accurately,
200 mg of each crushed sample was mixed with 1 ml of
methanol and then incubated for 5 min in a cold room. For
the removal of intracellular protein, the samples were
exposed to Sonicator (Toshiba, Japan) for 5 min. The solution
was then vortexed for approximately 5 min to get homogen-
ous metabolites. Centrifugation for 20 min at 13,000 rpm for
the removal of cell debris and the supernatant was used for
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5 T1
T2
T3
T4
T5
T6
T7
Cell suspension
g
rowth kinetics
Cell suspension biomass (g)
42 941028
35
2114
Culture period (da
y
s)
07
Figure 2. Establishment of growth curve (49 days period) for suspended cells
for exposed to different ratios of metallic nanoparticles along with NAA in cell
cultures of P. vulgaris. Mean data was taken on weekly basis. The data of mean
values and SE were collected from revised independent experiments for each
treatment. Mean data with common alphabets are significantly different
at p<.05.
Inoculum Lag phase Log phase Decline phase
Figure 3. Different phases of growth kinetics during cell suspension culture in P. vulgaris[Mismatch].
1
2
3
4
5
6
7
8
9
10
11
Dr
y
cells wei
g
ht (DCW-
g
/100 ml)
Fresh cells weight (FCW-g/100 ml)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
b
Nanoparticles and plant growth regulators (µg l-1; mg l-1)
a
b
b
b
cc
a
d
c
bb
ab
a
T7T6
T5
T4
T3T2
T1
Figure 1. Investigation of fresh and dry biomass (gram/100 ml) in suspended
cells of Prunella vulgaris exposed to different ratios of nanoparticles and NAA.
The data of mean values and SE were collected from revised independent
experiments for each treatment. Mean data with common alphabets are signifi-
cantly different at p<.05.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2555
the determination of CTP. The method of Lowry et al. [31]
was used for calculating total protein (CTP), following which
the supernatant was exposed to 650 nm wavelength in
Shimadzu UV-spectrophotometer. BSA-standard curve was
established from protein absorbance data.
Determination antioxidant activity in cell cultures
DPPH-radicals scavenging activity in suspended cells were
investigated according to the method of Ahmad et al. [32].
Herein, the pure extract from each treatment (2.0 ml) was
slowly combined with DPPH solution in a test tube. The test
tube containing mixture was placed in a dark condition to
minimize oxidation reactions. The DPPH solution was
obtained by the addition of 0.25 mg of powdered DPPH in
20–80 ml of HPLC grade ethanol. After incubation in dark, the
solution was applied for antioxidant activity. The readings of
the samples were taken at 517 nm using double-beam UV-
visible system (Shimadzu, Japan). The DPPH-based antioxi-
dant activity was investigated using the given formul:
% Antioxidant activity ¼100 1–ASC =ADRS
ðÞ
Here, A
SC
indicates suspended cells absorbance and A
DRS
represents DPPH radicals solution absorbance
Analysis of data
The data for each treatment was obtained from twice-
revised independent experiments. Here, one-way analysis of
variance (ANOVA) was used for obtaining the mean values
from each treatment. To obtain least significant difference
and SE (±), Statistix 8.1 (USA) was applied. Further, Origin
Lab (8.5; USA) software was applied for graphical presenta-
tion of the mean data.
Results
Production of biomass during growth kinetics in cell
suspension cultures
In the current study, combination of singlet NP (Ag or Au) with
NAA or a synergistic combination of NPs (AgAu) and NAA were
investigated for biomass accumulation. During cell suspension
culture development, maximum accumulation of fresh biomass
(9.25 g) was observed when liquid media were augmented
with a combination of AgAuNPs (3:1) and NAA after 30-days of
growth kinetics (Figure 1). The addition of AgAuNPs (1:3) with
NAA produced 8.77 g fresh and 0.64 g/100 ml dry biomass as
compared to control (6.67; 0.233 g). However, the combination
of AgAuNPs (3:1) and NAA did not show a positive correlation
with dry biomass accumulation.
The growth kinetics of cell suspension culture was investi-
gated for 49 days period with 7 days interval (Figures 2 and
3). The lag phases took maximum days (1–28) and the
growth was found relatively slower. The log phases take
14 days, while the decline phases were found shorter (7 days).
The color of cell culture showed variation at each growth
phase and become dark brown at the closing stages (Figure 3).
During growth kinetics, higher fresh biomass (20.14 g) was
observed at 42
nd
day on MS medium incorporated with AuNPs
in combination with NAA as compared to control (8.42g). The
application of different ratios of AgAuNPs (1:2; 1:3) with NAA
also improved fresh biomass accumulation (16.5 and 18g).
Moreover, other treatments also accumulated optimum quanti-
ties of biomass as compared to control (Figure 2).
-0.16
-0.08
0.00
0.08
0.16
0.24
0.32
0.40
Peroxidase activity (nM/min/mg-FW)
bb
dcc
a
a
Nano
p
articles and
p
lant
g
rowth re
g
ulators (
µg
l-1; m
g
l-1)
T7T6
T5
T4
T3T2
T1
(c)
0
50
100
150
200
250
300
350
Content of total protein (µg BSAE/mg-FW)
f
d
e
cd
b
c
a(a)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
bc
Superoxide dismutase activity (nM/min/mg-FW)
d
c
b
b
ab a(b)
Figure 4. Estimation of stress enzymes (superoxide dismutase and peroxidases)
and protein content in suspended cells of P. vulgaris exposed to metallic nano-
particles along with naphthalene acetic acid. Mean values were collected from
three replicates and also containing ± standard error. Bars with common alpha-
bets are significantly different at p<.05.
2556 H. FAZAL ET AL.
Effect of NPs on total protein content and stress
enzymes in suspended cells
Herewith, peroxidase enzymes, superoxide dismutase enzymes
and content of total protein were determined in cell suspen-
sion cultures of Prunella exposed to combinations of NAA and
NPs (Figure 4). In cell suspension cultures, the combination of
AgAuNPs (3:1) with NAA (2.0 mgl
1
) induced maximum CTP
(326 mg BSAE/mg-FW) as compared to control (Figure 4(a)).
Higher production of SOD enzym e (0.59 nM/min/mg-FW) was
observed when AgAuNPs (2:1) with 2.0 mg l
1
NAA was
applied (Figure 4(b)). Significantly similar enzyme production
(0.56 nM/min/mg-FW) was observed when AuNPs (30 mgl
1
)
with NAA (2.0 mg l
1
) was applied as compared to control
(0.32 nM/min/mg-FW) and other treatments. An increment in
POD enzyme production (0.33 and 0.334 nM/min/mg-FW) was
observed on medium containing combinations of AgNPs and
AgAuNPs (1:2) along with NAA, which is comparatively higher
than control (0.005 nM/min/mg-FW) (Figure 4(c)). Herewith, the
stress enzymes showed autonomous behavior and hence sug-
gest that they are not restricted to the application of single
type of NP and plant growth regulators, but varied upon each
exposure. In contrast, SOD enzyme did not showed linear cor-
relation with POD production. Similarly, the CTP did not show
positive correlation with stress enzymes production (Figure 5).
Effect of NPs on polyphenolics content and antioxidant
activity in suspended cells
Herewith, TPC, TFC and DPPH-radical scavenging activity
(DRSA) were investigated in cell suspension cultures in
response to various combinations of NPs þNAA (Figure 6).
Maximum accumulation of TPC (7.62 GAE-mg/g-DW) was
observed with the combination of AgAuNPs (1:3; 30-mgl
1
)
and 2.0 mg l
1
NAA supplemented to MS medium (Figure
6(a)). The same combination of NPs and NAA enhanced TFC
production (0.61 RE-mg/g-DW) in cell suspension culture of P.
vulgaris (Figure 6(b)). In contrast, maximum DRSA (87.8%)
was observed on medium containing AgAuNPs (2:1) as com-
pared to control (83.64%), as shown in Figure 6(c). Here, we
observed a clear correlation between TPC and TFC produc-
tion when exposed to the same NPs combination as shown
in Figure 7. In contrast, we did not observe correlation
between TPC and free radicals scavenging activity (Figure 7).
Discussion
Application of nanoparticles significantly influenced the
establishment of cell cultures in P. vulgaris. The literature is
still limited that know the effect of nanoparticles on the pro-
gression and development of different plant tissues and cells.
The effect of nanoparticles not only restricted to growth
regulation but also prominently affected the pathways of sec-
ondary metabolism. Here, the combination of nanotechnol-
ogy and plant tissue culture produce promising results in
suspended cells of P. vulgaris that further need insight view
of molecular mechanism. In some plant species, the applica-
tion of nanoparticles produced positive results but their accu-
mulation in plant tissues and its subsequent release to the
environment are still contradictory [2]. The effect of nanopar-
ticles varies with plant species, with age and type of tissues
selected for applications [33]. Previously, in plant species like
Hordeum vulgare and Linum usitatissimum, AgNPs negatively
affected the process of seed germination [5,34]. Contrarily,
the growth of Zea mays and Phaseolus vulgaris were
-100
-50
0
50
100
150
200
250
300
350
Superoxide dismutase activity (nM/min/mg-FW)
Peroxidase activity (nM/min/mg-FW)
Content of total protein (µg BSAE/mg-FW)
bbb
d
c
b
Nano
p
articles and
p
lant
g
rowth re
g
ulators (
µg
l-1; m
g
l-1)
T7T6
T5
T4
T3T2
T1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
b
b
a
ab
a
f
b
d
e
cd
a
b
c
a
Figure 5. Different ratios of nanoparticles induced correlation of protein content with superoxide dismutase and peroxidase enzymes in suspended cells of P. vulgaris.
Common alphabets on each bar with mean values are significantly different at p<.05.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2557
enhanced using the same NPs [35,36]. Herewith, the synergis-
tic application of Ag and Au NPs along with NAA showed
positive effects on cell culture development. Moreover, the
same combinations during phases of growth kinetics (lag,
log, stationary and decline) enhanced fresh and dry biomass
biosynthesis. On 30–42 days of cell cultures, biomass accumu-
lation as compared to control was found maximum on media
with variable ratios of nanoparticles and NAA.
Inside plant morphogenesis, the unconditional release of
reactive oxygen species (ROS) reduced cell dedifferentiation
and redifferentiation, which directly or indirectly produced
toxic metabolites [36]. Plants produce tissue-specific stress
enzymes to avoid such oxidative stress conditions [37]. These
enzymes form an intervertebral protection system, which pro-
tect plant tissues from mono-oxygen related species [36]. In
the current experiment, the exposure of suspended cells to
different metallic NPs improved the production of these
stress enzymes in comparison to control, but here we did
not observe linear correlation between SOD and POD
enzymes. Similar reports regarding the stress enzymes (SOD
and POD) are widely available in various species including
Prunus and Solanum [38,39]. In Brassica juncea the application
of silver NPs enhanced the synthesis of stress enzymes [5].
Furthermore, the exposure of Glycine max to different NPs
improved germination rate and stimulate the production of
stress enzymes [4]. It is also reported that Acanthophyllum
sordidum callus cultures produce small amount of stress
enzyme (SOD) during dedifferentiation but gradually
increases in redifferentiation [40]. Moreover, the role of stress
enzymes (SOD and POD) and their production are widely
reported in many elite plant species [41,42]. During the stress
situation, singlet free radicals are released in plant cells,
which have the ability to react with macromolecules and
finally reduce the developmental processes of various plant
tissues [43]. In response to highly singlet reactive radicals,
selected plant tissues stimulate the production of polyphe-
nolics, which scavenge free radicals for further reaction [44].
In this study, the addition of nanoparticles to broth cul-
tures induced the accumulation of biomass as well as the
biosynthesis of secondary metabolites in suspended cells of
P. vulgaris. This study is not restricted to Prunella but helpful
for the synthesis of novel or other important secondary cell
products in high-valued medicinal species. Information of
nanoparticle-induced biosynthesis of secondary metabolites
in liquid cultures of medicinal plants are limited in the
already published work; however, the biosynthesis of second-
ary metabolites in various elite medicinal species is widely
reported. In this context, the addition of exogenous biotic or
0
2
4
6
8
10
12
Total Flavonoid Content (TFC-mg/g-DW)
ede
de
b
a
d
c
(b)
(a)
0
1
2
3
4
5
6
7
8
cd
d
c
a
b
d
e
Total Phenolic Content (TPC-mg/g-DW)
50
60
70
80
90
100
Free radical scavenging activity (DRSA-%)
cd
b
e
c
bc
a
d
Nanoparticles and plant growth regulators (µg l-1; mg l-1)
T7T6
T5
T4
T3T2
T1
(c)
Figure 6. Effects of gold and silver nanoparticles on polyphenolics (a; phenolics,
b; flavonoids) and antioxidant activity (c) in suspended cells of P. vulgaris. Mean
results in bars with common letters are significantly different at p<.05.
60.0
62.5
65.0
67.5
70.0
72.5
75.0
77.5
80.0
82.5
85.0
87.5
90.0
Total Flavonoid Content (TFC-mg/g-DW)
Total Phenolic Content (TPC-mg/g-DW)
de
b
b
Free radical scavenging activity (DRSA-%)
e
de
d
d
cd
cd
b
e
d
c
c
c
bc
a
e
d
a
a
Nanoparticles and plant
g
rowth re
g
ulators (
µg
l-1; m
g
l-1)
T7T6
T5
T4
T3T2
T1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Figure 7. Different ratios of nanoparticles induced correlation of antioxidant
activity with polyphenolics in suspended cells of P. vulgaris. Common alphabets
on each bar with mean values are significantly different at p<.05.
2558 H. FAZAL ET AL.
abiotic elicitors and growth regulators not only effect the
architecture development of plant but also regulate the bio-
synthesis and release of high valued secondary cell products
[45]. In continuation, the calli cultures of Cicer arietinum accu-
mulate higher quantities of secondary cell products than
other plant in vitro tissues [46]. A similar variation in the syn-
thesis and release of secondary cell products were also docu-
mented in two species of Zingiber officinale [13]. Therefore,
the current research showed a clear picture of nanobiotech-
nology that enhanced the biosynthesis secondary cell prod-
ucts by regulating the growth behavior of various tissues.
Different ratios of NPs and NAA significantly alter the DRSA
in cell suspension culture. Various biotechnological techni-
ques have been used for the biosynthesis of valuable bio-
active compounds [47]. Here, the applications of NPs along
with NAA have displayed maximum DRSA in suspended cells
than control. Previous literature also proved that NPs-induced
antioxidant system enhanced malon-di-aldehyde content in
medicinal plants [48].
It is believed that the movement of nanoparticles in solid
medium is comparatively slower to interact with the cells to
enhance the secondary metabolism. However, the previous
study of Riaz et al. [49] confirmed that the addition of mela-
tonin to the solid culture media modulate the synthesis of
ZnONPs in callus cultures of Catharanthus roseus. In this
study, we directly exposed the suspended cells of P. vulgaris
to the silver and gold nanoparticle to investigate its possible
mechanism on secondary metabolites biosynthesis in liquid
medium rather than solid one. At this stage, it is very difficult
to understand the overall mechanism that how nanoparticle
modulates the secondary metabolism at the molecular level.
However, some previous studies endorse that nanoparticles
interact with plant cell and provoke the defense system of
plant by releasing higher quantities of secondary metabolites
[50]. In the direct interaction of nanoparticles with suspended
cells in liquid media, it raised the level of cytoplasmic cal-
cium, ROS and the expression of mitogen-activated protein
kinas cascades (Figure 8). According to Sosan et al. [51], the
nanoparticles bind with the cell membrane of Arabidopsis
thaliana and triggered ROS activation and calcium burst.
Mirzajani et al. [52] proved the expression of calcium and
other factors associated with biosynthetic pathways of sec-
ondary metabolism in response to nanoparticles in O. sativa
roots. Therefore, these calcium spike, elevated ROS and
MAPK are involve in the reprogramming of secondary metab-
olism that activate the defense system of plant cell to release
higher amount of secondary metabolites to cope with the
oxidative stress [50,53–55].
Conclusion
In conclusion, NPs are not only modulating biomass accumu-
lation but also enhance the production of polyphenolics con-
tent in suspended cells of P. vulgaris. This is one of the best
applications of Nanobiotechnology for the synthesis of high-
valued secondary metabolites in liquid cultures and can be
easily scaled up to bioreactor for enhanced productivity for
pharmaceutical and nutraceuticals industries. However, the
molecular mechanism of nanoparticles in various plants tis-
sues, their translocation to other parts and its effect on mod-
ulating the chemistry of other secondary metabolites are yet
to be explore and need further research.
Figure 8. The effect of nanoparticles on the synthesis of secondary metabolites in cell cultures (Adopted from Marslin et al. [50]). After the supplementation of sil-
ver and gold nanoparticles to the culture media, the NPs directly interact with the suspended cells producing pores in the cell membrane and provoke the antioxi-
dant system by releasing reactive oxygen species. The NPs induced ROS mediate mitogen-activated protein kinase cascade and calcium spikes, which possibly
modulate the transcription of secondary metabolism. In response to ROS, the plant cell activates the defensive machinery to prevent ROS damage. In this way, the
quantity of secondary metabolites increases as compared control cultures having no nanoparticles. The nanoparticle-secondary metabolites complexes exit the plant
cell or sometimes persist inside the cell in the form of intracellular metabolites. However, the effect of nanoparticles on alteration of metabolites, plant growth and
yield and its interaction with environment is still unknown.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2559
Acknowledgements
We acknowledge the support of Latif-ur-Rahman team for providing dif-
ferent nanoparticles for the experiment.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work is supported by the grants from the Key Research Area Grant
[2016YFA0501703] of the Ministry of Science and Technology of China,
the National Natural Science Foundation of China [Contract no.
61832019, 61503244], the Natural Science Foundation of Henan Province
[162300410060] and Joint Research Funds for Medical and Engineering
and Scientific Research at Shanghai Jiao Tong University [YG2017ZD14].
ORCID
Bilal Haider Abbasi http://orcid.org/0000-0002-6529-2134
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