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Molecules 29 05263

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24 views14 pages

Molecules 29 05263

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Giorgio Vilardi
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© © All Rights Reserved
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Article

Effect of Ag+ and Cd2+ Elicitation on Polyphenol Production in


Shoot Culture of Dracocephalum ruyschiana L.
Izabela Weremczuk-Jeżyna 1, *, Jan Gomulski 1 , Anna K. Kiss 2 and Izabela Grzegorczyk-Karolak 1

1 Department of Biology and Pharmaceutical Botany, Medical University of Lodz, 90-151 Lodz, Poland;
[email protected] (J.G.); [email protected] (I.G.-K.)
2 Department of Pharmaceutical Biology, Medical University of Warsaw, 02-097 Warsaw, Poland;
[email protected]
* Correspondence: [email protected]

Abstract: Abiotic elicitation with heavy metals has demonstrated considerable potential to stimulate
the production of industrially important secondary metabolites in plant in vitro cultures. The present
study investigates the effect of exogenous silver nitrate and cadmium chloride supplementation
on flavonoid and phenolic acid production, as well as other indicators of oxidative stress, in shoot
cultures of Dracocephalum ruyschiana L. Owing to the presence of bioactive polyphenolic compounds,
this Mongolian medicinal plant is traditionally used as an anti-inflammatory, antibacterial and
antipyretic agent. The shoots were cultured for three weeks, and then, cadmium (Cd2+ ) and silver
(Ag+ ) ions (50 or 100 µM) were added to the medium. The maximum proliferation rate was observed
in the presence of 100 µM Ag+ (almost 5), the highest chlorophyll content in the presence of 100 µM
Cd2+ (0.6 mg/g FW) and the highest biomass was observed with both these treatments (73.4–75.7 g
FW and 7.53–7.72 g DW). UPLC-PDA-ESI-MS analysis revealed four phenolic acids and five flavonoid
derivatives in the hydromethanolic extract of D. ruyschiana shoots. All treatments stimulated the
production of rosmarinic acid (RA), which was the dominant compound in the analyzed culture;
the highest level of RA, i.e., about three times higher than the control, was noted in shoots exposed
to 50 µM Cd2+ (14.72 mg/g DW), whereas the level of most flavonoids in the culture increased
most significantly when exposed to Cd2+ at a concentration of 100 µM. Moreover, the shoots grown
Citation: Weremczuk-Jeżyna, I.; in the presence of 100 µM Cd2+ exhibited significantly higher antioxidant potential in comparison
Gomulski, J.; Kiss, A.K.; to the control. Our findings indicate that heavy metals are able to stimulate phenolic compound
Grzegorczyk-Karolak, I. Effect of Ag+ biosynthesis in Dracocephalum shoots without any negative impact on their growth. These results
and Cd2+ Elicitation on Polyphenol could be of significant importance for the medical, nutraceutical and agronomic industries.
Production in Shoot Culture of
Dracocephalum ruyschiana L. Molecules Keywords: abiotic elicitors; apigenin glycosides; antioxidant activity; antioxidant enzymes; heavy
2024, 29, 5263. https://doi.org/
metals; rosmarinic acid
10.3390/molecules29225263

Academic Editor: Cristina Forzato

Received: 21 October 2024


1. Introduction
Revised: 3 November 2024
Accepted: 5 November 2024
Medicinal plants are sources of a wealth for valuable bioactive metabolites and there is
Published: 7 November 2024 growing interest in identifying ways to increase their yield. Particularly rich in polyphenols
are species from the Lamiaceae family, including those belonging to the genus Dracocephalum
such as D. ruyschiana. This rare medicinal plant migrated from the Russian and Asian
steppes to Europe where it is found locally from the boreal regions to the steppes and
Copyright: © 2024 by the authors. European mountains [1,2]. D. ruyschiana is reported as a traditional medicinal plant in
Licensee MDPI, Basel, Switzerland. Mongolia. Its aerial parts are used as a natural drug for the treatment of rheumatoid
This article is an open access article
arthritis, laryngitis and acute respiratory infection, headache, diarrhea and gastric ulcers.
distributed under the terms and
This plant also possesses antimicrobial, antioxidative, antispasmodic and hepatoprotective
conditions of the Creative Commons
effects [3,4]. The aerial parts are known to accumulate many secondary metabolites,
Attribution (CC BY) license (https://
including flavonoids and their mono- and tetraglycosides, phenolic acids (chlorogenic acid
creativecommons.org/licenses/by/
and caffeoylquinic acids), benzyl alcohol glycosides and essential oil components [4,5].
4.0/).

Molecules 2024, 29, 5263. https://doi.org/10.3390/molecules29225263 https://www.mdpi.com/journal/molecules


Molecules 2024, 29, 5263 2 of 14

However, the biosynthesis of bioactive compounds in plants is typically relatively


low and variable. Moreover, the population of D. ruyschiana, like many other species,
is gradually decreasing due to the overexploitation of natural resources. Consequently,
alternative methods for producing valuable phytochemicals are being sought. A promising
approach to support and enhance the production of bioactive compounds involves in vitro
plant cultures [6]. Biotechnological methods not only make cultivation independent of
geographical location and seasonal variation but also enable the stimulation of biosynthesis
through the controlled regulation of cultivation conditions.
One effective biotechnological strategy is based on elicitation with biotic or abiotic
substances [7]. A particularly interesting set of abiotic elicitors are heavy metal ions. Metal
ions, such as Ag+ and Cd2+ , act as stressors to induce primary metabolism and accelerate
the production of secondary metabolites, especially those with antioxidant activity [8,9]. It
has been reported that cadmium-stimulated culture of Carthamus tinctorius L. accumulated
several times higher flavonoid levels in comparison to a control [10], while titanium dioxide
increased flavonoid biosynthesis in Salvia officinalis L. up to 50% [11]. In other studies,
a large increase in phenolic and anthocyanin contents was observed in Salvia sclarea L.
exposed to cadmium [12]. Rosmarinic acid (RA) production increased threefold following
the addition of vanadyl sulfate to Lavandula vera DC. culture [13], and lithospermic acid
B increased fourfold in the hairy roots of Salvia miltiorrhiza Bunge under the influence of
silver thiosulfate [14].
Under stressful conditions, such as heavy metal treatment, plant tissues show elevated
levels of reactive oxygen species (ROS) [7,15]. Excessive ROS accumulation can result in
oxidative stress, causing DNA, RNA and protein damage and membrane lipid peroxidation.
In response to oxidative stress, organisms activate various types of defense mechanisms:
these include the increase in activity of antioxidant enzymes such as superoxide dismu-
tase (SOD), peroxidase (POD) and catalase (CAT), and the production of non-enzymatic
compounds, such as polyphenols, that bind and neutralize ROS [15].
The aim of the study was to elicit the biosynthesis of polyphenolic compounds in D.
ruyschiana shoots. For this purpose, the culture was treated with silver and cadmium ions
at a concentration of 50 and 100 µM. The level of bioactive compounds accumulated in
shoots were determined by HPLC. The study also examines the influence of abiotic stress
on the shoot growth, the activity of antioxidant enzymes and the antioxidant potential of
the culture.

2. Results
2.1. Effect of Ag+ or Cd2+ on Growth and Development of D. ruyschiana Shoot Culture
Nodal segments of in vitro cultivated shoots were used for the propagation of D. ruyschi-
ana. The cultures were obtained from the shoot tips of seedlings germinated in vitro from
the sterilized seeds. The D. ruyschiana shoots were grown in basal MS [16] liquid medium
with 0.5 mg/L 6-bezyloamino purine (BAP) and 0.2 mg/L indole-3-acetic acid (IAA). After
three weeks of culture, the medium was supplemented with Ag+ or Cd2+ ions at a concen-
tration of 50 or 100 µM. After another two weeks of cultivation, the parameters of culture
growth, development and biochemical response were determined.
The highest proliferation rate was observed for the culture exposed to 100 µM of Ag+
(4.9 shoot/explant) followed by 100 µM of Cd2+ (4.3 shoot/explant) (Figures 1 and 2). No
significant differences in shoot regeneration were found between the 50 µM treatments and
the control.
Molecules 2024, 29, x FOR PEER REVIEW
Molecules 2024, 29, 5263
3
3 of 14

Figure 1. D. ruyschiana shoot culture grown in liquid basal MS medium with 0.5 mg/
Figure
mg/L
Figure 1.1.D.D.
IAA; (A)ruyschiana
inoculum
ruyschiana shoot
shoot (oneculture
culture day
growngrown in liquid
ofinculture),
liquid basal
(B)MS
basal and MS
(C) medium
medium control with 0.5
shoots
with 0.5 mg/L BAP mg/L
after BAPwe
and five an
mg/L
0.2 mg/L
shoots IAA;IAA;
grown (A)(A)
ininoculum
inoculum
basal (one
(one day
medium of culture),
daywith culture),
addition (B)and
(B) and
of (C)
(C)
(D) control
control
50 µM shoots
shoots
Ag (E)after
+,after five five
100weeksµMweeks of cu
of +, (F)
Ag 50
shoots
culture; grown
shoots in
grown basal medium
in basal medium with addition
with additionofof (D) 50
(D) 50 µM
µM Ag
Ag + ,+(E)
, (E) 100
100 µMµMAgAg+ , (F)
+, (F) 50
50 µM µM Cd2
(G) 2+100 Cd 2+ µM 2+after five weeks of culture (two weeks after the application of heav
2+
(G) 100
Cd Cd 100µM
and (G) Cd after
µM five
after weeks of culture
five weeks of culture(two weeksafter
(two weeks after
thethe application
application of heavyof heavy
metal metal
Bar 1 cm.
Bar 1Bar
ions). cm.1 cm.

Figure 2. Effect of Ag+ and Cd2+ at concentrations of 50 or 100 µM on proliferation rate of D.


chiana2.2.
Figure
Figure culture.
Effect
Effect The
of Ag + given
of and
AgCd
2+values
+ and Cd2+represent
at concentrations of means
50 or 100±µM
at concentrations SDof
onof50three independent
proliferation
or 100 rate
µMofon experimental r
D. ruyschiana
proliferation
cates. Means
culture. marked
The given with themeans
values represent same ± letter
SD ofwere
three not significantly
independent different
experimental (p < 0.05).
replicates. Means
chiana culture. The given values represent means ± SD of three independent exper
marked with the same letter were not significantly different (p < 0.05).
cates. Means marked with the same letter were not significantly different (p < 0.05).
The treated D. ruyschiana culture with the higher concentration of heavy metal
The treated D. ruyschiana culture with the higher concentration of heavy metal ions
yieldeda significantly
yielded
a significantly larger
larger
quantity
quantity culture
of fresh biomass
of fresh biomass
than thethecontrol;
than higher
the control;
the fresh we
fresh weightof heav
after The
two treated
weeks D.exposure
of ruyschiana was withmg/vessel
367.2–378.5 the concentration
(Figure 3), i.e., a 60-fold incr
after two weeks of exposure was 367.2–378.5 mg/vessel (Figure 3), i.e., a 60-fold increase
yielded
compared a
comparedto tosignificantly
thethe fresh
fresh larger
weight
weight quantity
of theofinoculum. of
the inoculum.fresh
A higher biomass
Adry
higher than the
dryof weight
weight the culture control;
of the the
was culture
after twoinweeks
achieved
achieved shoots
in shootsofcultivated
exposure
cultivated was 367.2–378.5
in theinpresence of both of
the presence metalsmg/vessel
both atmetals
100 µMat (Figure
and CdµM
100 2+ at3), i.e.,
Cd2+aat60-
50 µM,
and 50
i.e., almost 30% higher than the control value (Figure 3).
compared to the fresh weight of the inoculum. A higher dry weight of the
i.e., almost 30% higher than the control value (Figure 3).
achieved in shoots cultivated in the presence of both metals at 100 µM and C
i.e., almost 30% higher than the control value (Figure 3).
Molecules 2024, 29, x FOR PEER REVIEW
Molecules 2024, 29, 5263 4 of 14

Figure 3. Effect of Ag+ and Cd2+ at concentrations of 50 or 100 µM on biomass of D. ru


ture. The given values represent means ± SD of three independent experimental repli
marked with the same letter were not significantly different (p < 0.05).
Figure 3.3.Effect + 2+ D. ruyschiana
Figure Effectofof
AgAgand Cd Cd
+ and at2+concentrations of 50 or
at concentrations of100
50 µM on biomass
or 100 µM onofbiomass of D. ruysch
culture.
The
ture. The given
Thepresence values
given values represent
of cadmium means
represent means± SD of three
or silver
± SD in independent
of culture experimental replicates.
mediumexperimental
three independent increased Means
the total
replicates
marked with the same letter were not significantly different (p < 0.05).
content
markedin shoots
with of letter
the same D. ruyschiana, with cadmium
were not significantly different stimulating
(p < 0.05). chlorophyll b
more The
intensely
presencethan silver; orinsilver
of cadmium addition, better
in culture mediumresults were
increased theobserved
total chloro-at 100
phyll The presence
content in shootsofofcadmium or
D. ruyschiana, silver
with in culture
cadmium medium
stimulating increased
chlorophyll the100
totalµM
biosynthe- chloo
µM (Figure 4). The maximum chlorophyll level was achieved with
sis more intensely than silver; in addition, better results were observed
content in shoots of D. ruyschiana, with cadmium stimulating chlorophyll biosy at 100 µM than
mg/g
50 µMFW), i.e.,4).75%
(Figure The higher
maximum than the control.
chlorophyll level was achieved with 100 µM of Cd2+
more intensely than silver; in addition, better results were observed at 100 µM
(0.59 mg/g FW), i.e., 75% higher than the control.
µM (Figure 4). The maximum chlorophyll level was achieved with 100 µM of Cd
mg/g FW), i.e., 75% higher than the control.

Figure 4. Effect of Ag+ or Cd2+ at concentrations of 50 or 100 µM on chlorophyll content in D. ruyschi-


Figure 4. Effect
ana culture. of Ag
The given
+ or Cd2+ at concentrations of 50 or 100 µM on chlorophyll conte
values represent means ± SD of three independent experimental replicates.
chiana
Meansculture. Thethegiven
marked with same values
letter wererepresent means
not significantly ± SD
different (p <of three independent experi
0.05).
Figure 4. Effect of Ag+ or Cd2+ at concentrations of 50 or 100 µM on chlorophyll content in
cates. Means marked+ with
2+ the same letter were not significantly different (p < 0.05).
chiana culture.
2.2. Effect TheCd
of Ag and given values represent
on Production means
of Phenolic ± SDinofD.three
Compounds independent
ruyschiana experiment
Shoot Culture
cates.Nine
Means markedwere
compounds withdetected
the same in letter
the 80%were not significantly
methanol extract from different (p <shoots.
D. ruyschiana 0.05).
2.2. Effect
Four of Ag
of them,
+ and Cd2+ on Production of Phenolic Compounds in D. ruyschiana S
corresponding to peaks 1, 2, 4 and 9 on the UPLC-PDA-ESI-MS chromatogram
(Figure 5) were identified by2+UV-Vis spectra and
2.2. Effect
Nine of Ag + and Cd
compounds were detectedofMS
on Production in fragmentation
Phenolic patterns
Compounds
the 80% methanol inasD.polyphenolic
ruyschiana
extract Shoot
from D
acids. Peak 1 was characterized as chlorogenic acid (CA), Peak 2 as dicaffeoylquinic acid,
shoots. Four
Peak 4Nine
as of them,
compounds
rosmarinic corresponding
were
acid (RA) and detected tomethyl
Peak 9 as itsin peaks
the 80% 1,(Table
ester 2, 4 1).
methanoland 9 on the
extract
IdentificationfromUPLC-P
was D. ruy
shoots. Four
chromatogram
performed of(Figure
them, with
by comparison corresponding
5) authentic to peaks
were identified
standards andby1, UV-Vis
2, 4 and
literature 9 on the and
data spectra
[17–20]. UPLC-PDA-
MS fra
chromatogram
patterns (Figure 5)
as polyphenolic werePeak
acids. identified by UV-Vis spectra
1 was characterized and MS fragme
as chlorogenic acid (
aspatterns as polyphenolic
dicaffeoylquinic acid, acids.
Peak Peak
4 as 1rosmarinic
was characterized
acid (RA) as chlorogenic
and Peak 9acid as (CA)
its m
as dicaffeoylquinic acid, Peak 4 as rosmarinic acid (RA) and
(Table 1). Identification was performed by comparison with authentic standa Peak 9 as its meth
(Table 1). Identification was performed by comparison with authentic standards
erature data [17–20].
erature data [17–20].
Molecules 2024, 29, x FOR PEER REVIEW 5 of 15
Molecules 2024, 29, 5263 5 of 14

Chromatogram ofofextract
Figure5.5.Chromatogram
Figure of D.
extract of ruyschiana shootsshoots
D. ruyschiana grown grown
in MS medium
in MS with 0.5 mg/L
medium withBAP
0.5 mg/L
andand
BAP 0.2 mg/L IAA: (1)
0.2 mg/L chlorogenic
IAA: acid, (2) dicaffeoylquinic
(1) chlorogenic acid, (3) acacetinacid,
acid, (2) dicaffeoylquinic rhamnosyl-trihexoside,
(3) acacetin rhamno-
(4) rosmarinic acid,
syl-trihexoside, (4) (5)rosmarinic
apigenin caffeoyl-rhamnoside,
acid, (5) apigenin (6) apigenin p-coumaroyl-rhamnoside
caffeoyl-rhamnoside, (6) (I),
apigenin
p-coumaroyl-rhamnoside (I), (7) acacetin
(7) acacetin acetyl-rhamnosyl-trihexoside, (8) apigeninacetyl-rhamnosyl-trihexoside,
p-coumaroyl-rhamnoside (II) and (9)(8) apigenin
methyl
p-coumaroyl-rhamnoside
rosmarinate. (II) and (9) methyl rosmarinate.

Table 1.1.MS
Table MSfragmentation of compounds
fragmentation of compoundsfrom
from extracts
extracts ofruyschiana
of D. D. ruyschiana
shootshoot culture.
culture.

Ion Mode
Ion Mode
Peak
Rt [min] Rt [min]
Peak Number [M − H] /[M

[M −+H]
H]/[M
− + * + H]+ * Tentative
Tentative Assignation
Assignation
Number Main Fragments
Main Fragments
(m/z) (m/z)
1 1 17 17 353 353 191 191 Chlorogenic
Chlorogenic acidacid
2 34.9 515 353, 191 Dicaffeoylquinic acid
2 3 34.9 37 515 917 * 353, 191
771, 447, 285 Dicaffeoylquinic
Acacetin acid
rhamnosyl-trihexoside
3 4 37 37.8 917 * 359 771, 447, 285161
197, 179, Acacetin rhamnosyl-trihexoside
Rosmarinic acid
4 5 37.8 39.3 359 577 415, 269,
197, 179, 161161 ApigeninRosmarinic
caffeoyl-rhamnoside
acid
6 41.3 561 397, 163 Apigenin p-coumaroyl-rhamnoside (I)
5 7 39.3 42.1 577 959 * 415,813,
269,
651,161447, 285 Apigenin
Acacetin caffeoyl-rhamnoside
acetyl-rhamnosyl-trihexoside
6 8 41.3 43.3 561 561 397, 163
415, 397, 163 Apigenin p-coumaroyl-rhamnoside
Apigenin p-coumaroyl-rhamnoside (II) (I)
7 9 42.1 43.8 959 * 373 179
813, 651, 447, 285 Methyl rosmarinate
Acacetin acetyl-rhamnosyl-trihexoside
Peak numbers refer to those used in Figure 5. * Positive ion mode [M+H]+ (m/z); in bold—the most abundant
8 43.3 561
fragmentation ion.
415, 397, 163 Apigenin p-coumaroyl-rhamnoside (II)
9 43.8 373 179 Methyl rosmarinate
Peak numbers refer to five
The remaining those usedwere
peaks in Figure 5. * Positive
identified ion mode
as flavonoid [M+H]+with
derivatives, (m/z);peaks
in bold—the
3 and 7 most
abundant
showingfragmentation
a fragmentation ion. +
ion [M + H] at m/z 285 (in positive mode) assigned to an acacetin
derivative [4] (Table 1). Based on the MS fragmentation data and literature data [21–24],
The remaining
Compound five peaks
3 was identified were identified
as acacetin as flavonoid and
rhamnosyl-trihexoside, derivatives, with peaks 3 and
7 as acetyl-rhamnosyl-

[M − H]mode)
7 showing
trihexoside. a fragmentation
Peaks 5, 6 and ion [M + H]
8, showing at m/z 285 (in
a fragmentation
+ ionpositive assigned
at m/z 269 (in to an
negative mode), were identified as representing apigenin derivatives
acacetin derivative [4] (Table 1). Based on the MS fragmentation data and literature (Table 1). Peak 5 data
was tentatively assigned to caffeoyl-rhamnoside, and 6 and 8 to p-coumaroyl-rhamnosides
[21–24], Compound 3 was identified as acacetin rhamnosyl-trihexoside, and 7 as ace-
(I and II).
tyl-rhamnosyl-trihexoside. Peaks 5, 6 and 8, showing a fragmentation ion [M − H]− at m/z
The levels of individual polyphenolic compounds in shoots of D. ruyschiana depended
269 (in negative mode), were identified as representing apigenin derivatives (Table 1).
on the presence and concentration of heavy metals added to the medium. The highest total
Peak 5 was
phenolic tentatively
compound content wasassigned
found intoshoots
caffeoyl-rhamnoside,
cultivated on medium and 6 and 8 to
supplemented
p-coumaroyl-rhamnosides
2+ (I and II).
with 100 µM Cd (25.9 mg/g DW) (Table 2), with values over twice as high as the control.
TheThe levelsofof
addition theindividual
elicitor to thepolyphenolic compounds
medium stimulated in shootsof of
the production D. ruyschiana
rosmarinic acid de-
2+ resulted in levels of
pended on the presence
and chlorogenic acid most and concentration
intensely. Treatmentof with
heavy 100metals
µM Cdadded to the medium. The
10.95 mg/g
highest total DW for RAcompound
phenolic and 7.66 mg/g DW for
content wasCA, with in
found these respective
shoots valueson
cultivated being
medium
about two and three times higher than the control. Interestingly, the
supplemented with 100 µM Cd2+ (25.9 mg/g DW) (Table 2), with values over twice as high RA biosynthesis
as the control. The addition of the elicitor to the medium stimulated the production of
rosmarinic acid and chlorogenic acid most intensely. Treatment with 100 µM Cd2+ re-
sulted in levels of 10.95 mg/g DW for RA and 7.66 mg/g DW for CA, with these respective
values being about two and three times higher than the control. Interestingly, the RA bi-
osynthesis was more intensely stimulated by lower concentrations of Ag and Cd, with 50
maining treatments increased the level of apigenin derivatives only slightly or did not
change it compared to that obtained for the control. In contrast, the treatments inhibited
the biosynthesis of acacetin rhamnosyl-trihexoside; however, 100 µM Cd2+ increased the
accumulation
Molecules 2024, 29, 5263 of acacetin acetyl-rhamnosyl-trihexoside twofold compared to the control 6 of 14
(Table 2).
2+
Table 2. The effect was more metals
of heavy intensely
onstimulated by lowerofconcentrations
the accumulation of Ag and
phenolic compounds Cd, with
(mg/g DW) 50
in µMD. Cd
inducing
ruyschiana shoot culture. Theconcentrations as high with
shoots were treated as 14.72 mg100
50 or perµMg DW.
Ag+However,
and Cd2+a. higher concentration of
cadmium ions enhanced flavonoid production more intensely; with exposure resulting in
a twofold increase in total apigenin
Treatmentderivative level (Table 2). The remaining treatments
mpounds increased the level of apigenin derivatives only slightly or did not change it compared
Ag+ 50 µM Ag+ 100 µM Cd2+ 50 µM Cd2+ 100 µM Control
to that obtained for the control. In contrast, the treatments inhibited the biosynthesis of
rogenic acid 6.54 ± 0.06acacetin5.12
a ± 0.17 b 4.59 ±however,
rhamnosyl-trihexoside; 0.07 c
100 7.66
µM Cd± 0.98
2+ increased2.74
a
the ± 0.02 d
accumulation of
oylquinic acid 0.84 ± 0.05acacetin
b 0.59 ± 0.01 c 0.54 ± 0.08twofold
acetyl-rhamnosyl-trihexoside c 0.99 ± 0.06
compared a
to the 0.41(Table
control ± 0.042).d
mnosyl-trihexoside 0.32 ± 0.03 c 0.22 ± 0.01 d 0.29 ± 0.02 c 0.50 ± 0.03 b 0.73 ± 0.06 a
Table 2. The effect of heavy metals on the accumulation of phenolic compounds (mg/g DW) in D.
marinic acid 13.25 ± 0.05 b 8.88 ± 0.22 d 14.72 ± 0.31 a 10.95 ± 0.33 c 5.15 ± 0.14 e
ruyschiana shoot culture. The shoots were treated with 50 or 100 µM Ag+ and Cd2+ .
ffeoyl-rhamnoside 0.32 ± 0.03 b 0.21 ± 0.03 c 0.27 ± 0.02 b,c 0.43 ± 0.03 a 0.23 ± 0.03 c
Apigenin Compounds
Treatment
0.79 ± 0.02 c 0.57 ±+ 0.01 e 0.63
+
± 0.02 d 2.26 ± 0.11 a 2+ 1.15 ± 0.04 b
yl-rhamnoside (I) Ag 50 µM Ag 100 µM Cd2+ 50 µM Cd 100 µM Control
acetin ace- Chlorogenic acid 6.54 ± 0.06 a
5.12 ± 0.17 b 4.59 ± 0.07 c 7.66 ± 0.98 a
2.74 ± 0.02 d
0.75 ± 0.02 b 0.62 ± 0.02
0.84 ± 0.05
c
b 0.45 ± 0.02
0.59 ± 0.01 c d 1.14
0.54 ± 0.08 ±c 0.04 a 0.46
0.99 ± 0.06 a ± 0.01 d
0.41 ± 0.04 d
nosyl-trihexosideDicaffeoylquinic acid
Acacetin rhamnosyl-trihexoside 0.32 ± 0.03 c 0.22 ± 0.01 d 0.29 ± 0.02 c 0.50 ± 0.03 b 0.73 ± 0.06 a
Apigenin ± 0.05c b d ± 0.31 a ± 0.33 c 5.15 ± e
Rosmarinic
0.19acid
± 0.02 a 13.25
0.11 ± 0.02 0.14±±0.22
8.88 0.01c
b 14.720.15 ±b,c0.0310.95
ab 0.11
a
± 0.01 c 0.14
c
yl-rhamnoside (II)
Apigenin caffeoyl-rhamnoside 0.32 ± 0.03 b 0.21 ± 0.03 0.27 ± 0.02 0.43 ± 0.03 0.23 ± 0.03
± c ± e d ± a b
Apigenin
l rosmarinate p-coumaroyl-rhamnoside
0.93 ± 0.04 b (I) 0.79 0.02
0.28 ± 0.05 cb 0.57 0.01
1.11 ± 0.15 b 0.63 ± 0.02
1.82 ± 0.24 a 2.26 0.11 1.15
1.02 ± 0.05 b ± 0.04
Acacetin acetyl-rhamnosyl-trihexoside 0.75 ± 0.02 0.62 ± 0.02 c 0.45 ± 0.02 d 1.14 ± 0.04 a 0.46 ± 0.01 d
l phenolics 23.93 ± 0.04 b (II) 16.6±
Apigenin p-coumaroyl-rhamnoside 0.06
0.19 ±
c
0.02 a 22.81
0.11 ± ±0.02
0.08
c b
0.14 25.90
± 0.01 ±b 0.39 a 12.00
0.15 ± 0.03 ab ± 0.40 d
0.11 ± 0.01 c
± bSD of0.28 c 0.24 a
The given
Methyl values represent 0.93
rosmarinate means
± 0.04 three independent
± 0.05 0.15 b
1.11 ± experimental
1.82 ±replicates. ± 0.05 b
Means
1.02
b c b a
Total phenolics
marked 23.93 ±
with the same letter were 0.04
not 16.6± different
significantly 0.06 22.81
(p ± 0.08
< 0.05). 25.90 ± 0.39 12.00 ± 0.40 d
The given values represent means ± SD of three independent experimental replicates. Means marked with the
same letter were not significantly different (p < 0.05).
2.3. Antioxidant Response of D. ruyschiana Shoots Under Ag+ and Cd2+ Elicitation
The antioxidant activity ofResponse
2.3. Antioxidant the shoots of D. ruyschiana
of D. ruyschiana wasAgevaluated
Shoots Under using DPPH,
+ and Cd2+ Elicitation

ABTS and FRAP tests The(Figure 6). The


antioxidant strongest
activity antiradical
of the shoots activitywas
of D. ruyschiana was observed
evaluated forDPPH,
using
shoots exposed to 100and
ABTS µMFRAPCd2+tests
and(Figure
Ag+, with
6). Therespective IC50 values
strongest antiradical of 55.55
activity and 64.22
was observed for shoots
µg/mL (DPPH) and exposed
IC50 to
of 100 µMand
46.22 Cd2+55.19
and Ag + , with respective IC values of 55.55 and 64.22 µg/mL
µg/mL (ABTS) (Figure 50 6A). Compared to the
(DPPH) and IC 50 of 46.22 and 55.19 µg/mL
untreated shoots, these values were 50% lower following the DPPH (ABTS) (Figure 6A). Compared
test, and to the untreated
22–35%
shoots, these values were 50% lower following the DPPH test, and 22–35% lower following
lower following the ABTS test. Additionally, D. ruyschiana shoots exposed to 100 µM Cd²⁺
the ABTS test. Additionally, D. ruyschiana shoots exposed to 100 µM Cd2+ exhibited nearly
exhibited nearly aa two-fold higherreduction
two-fold higher reduction potential
potential following
following the FRAP
the FRAP test compared
test compared to the control
to the control shoots (Figure
shoots (Figure6B).
6B).

Figure 6. Effect of Figure


50 or 100 µMofAg
6. Effect 50+orand
100 µM + and
Cd2+Agon Cd2+ on antioxidant
antioxidant activity activity
of the extract fromfrom
of the extract D. D.ruys-
ruyschiana
chiana culture (ABTS, DPPH
culture (A)DPPH
(ABTS, and FRAP (B)
(A) and assays).
FRAP The given
(B) assays). values
The given represent
values means
represent means± ±SDSDofof three
three independent independent
experimental replicates.replicates.
experimental Means marked with the
Means marked withsame letterletter
the same were notnot
were signifi-
significantly
cantly different (p <different
0.05). Butylated hydroxytoluene
(p < 0.05). Butylated (BHT) (BHT)
hydroxytoluene was used as a as
was used positive reference.
a positive reference.
9, x FOR PEER REVIEW 7 of 15
Molecules 2024, 29, 5263 7 of 14

SOD and POD activity


SOD anddemonstrated different responses
POD activity demonstrated to heavy to
different responses metal
heavyexposure.
metal exposure.
SupplementationSupplementation
with 50 µM Agwith + or 50 µM Cd +2+ had similar2+effects on
50 µM Ag or 50 µM Cd had similar effects POD activity,
on PODre-activity,
sulting in a 35% increase
resulting in aactivity compared
35% increase to non-elicited
in activity culture (Figure
compared to non-elicited 7A).
culture (Figure 7A).

7. Effect of+50 or Cd
1002+µM + 2+
Figure
Figure 7. Effect of 50 or 100 µM Ag and onAg and Cd enzyme
antioxidant on antioxidant enzyme
activities: POD activities:
(A) andPOD SOD(A) and
SOD (B) in D. ruyschiana shoots. The given values represent means ±
(B) in D. ruyschiana shoots. The given values represent means ± SD of three independent experi-SD of three independent
experimental
mental replicates. Means replicates.
marked with Means marked
the same withwere
letter the same
not letter were not significantly
significantly different (p different (p < 0.05).
< 0.05).
POD—peroxidase, SOD—superoxide
POD—peroxidase, SOD—superoxide dismutase. dismutase.

In contrast, silver increased SOD activity much more intensely than cadmium. The
In contrast, silver increased
SOD activity SODexposed
in shoots activity
to much
50 µM Agmore
+ wasintensely
2.5 times than
highercadmium.
than in thoseTheexposed
SOD activity in shoots
to 50 µM 2+
exposed
Cd , and to five
50 µM Ag
times + wasthan
higher 2.5 in
times higher(Figure
the control than 7B).
in those exposed
Increasing the concen-
to 50 µM Cd2+, andtration
fiveoftimes
metalshigher
to 100 µM decreased
than the SOD activity
in the control (Figureby7B).
1.6 times in the case
Increasing theofcon-
silver and
two times in the case of cadmium. The SOD level in shoots exposed
centration of metals to 100 µM decreased the SOD activity by 1.6 times in the case of sil- to 100 µM Cd2+ was
similar to that recorded in the control.
ver and two times in the case of cadmium. The SOD level in shoots exposed to 100 µM
Cd2+ was similar to that recorded in the control.
3. Discussion
The impact of heavy metals on plant health is influenced by the metal and its concen-
3. Discussion tration and the sensitivity and resistance of the plant species to metal-induced oxidative
The impact ofstress.
heavyWhereas
metals some
onspecies
plant demonstrate stunted growth
health is influenced or development
by the metal and its in response
con- to
heavy metal exposure, others have evolved defense mechanisms. For example, the addition
centration and the sensitivity and resistance of the plant species to metal-induced oxida-
of both cadmium and silver ions at a concentration of 100 µM reduced the biomass of the
tive stress. Whereas some
Phoenix species
dactylifera demonstrate
L. culture by morestunted growth
than twofold [25].or development
Exposure to cadmium in re-
reduced
sponse to heavy metal
the dryexposure,
weight of S. others
sclareahave
shootsevolved
by aboutdefense
15% [14],mechanisms.
and exposure to For example,
silver ions reduced
+ promoted the biomass
the addition of both cadmium
Catharanthus and
roseus silver
(L.) G. Don.ionsby at
upatoconcentration of 100AgµM
30% [26]. In contrast, reduced the
of hairy root of Salvia castanea Diels. even with a 25%
biomass of the Phoenix dactylifera L. culture by more than twofold [25]. Exposure increment over the control
to cad-[27]. In
addition, silver stimulated Solanum nigrum shoot proliferation intensely, increasing the
mium reduced the dry weight of S. sclarea shoots by about 15% [14], and exposure to
number by more than threefold compared to the control; this increase was particularly
silver ions reduced Catharanthus roseus (L.) G. Don. by up to 30% [26]. In contrast, Ag+
apparent in the range of 200–600 µM [28]. Moreover, 90 µM Cd2+ stimulated the growth of
promoted the biomass of hairyFranch
Arabis paniculata root of Salvia castanea
significantly, and evenDiels. even concentration
the highest with a 25% of increment
ions used in that
over the control experiment
[27]. In addition, silver
led to culture stimulated
growth Solanum
comparable to thatnigrum shoot Cd
of the control; proliferation
treatment also did
intensely, increasing the chlorophyll
not affect number byconcentrations
more than regardless
threefoldofcompared to the[29].
the concentration control; this 100 to
In contrast,
µM silver increased the chlorophyll level in Solanum
increase was particularly apparent in the range of 200–600 µM [28]. Moreover, 90 µM [30].
200 shoots by more than twofold
In our present study, two concentrations (50 and 100 µM) of cadmium chloride and
Cd2+ stimulated the growth of Arabis paniculata Franch significantly, and even the highest
silver nitrate were used. These values were chosen based on other elicitation experiments
concentration of ions used in
that used a wide that experiment
range led to culture
of metal concentrations; forgrowth
example,comparable
with cadmium to concentrations
that of
the control; Cd treatment
from 0.5 toalso
500 µMdid[14,31–33].
not affect chlorophyll concentrations regardless of the
concentration [29]. InPlants
contrast, 100 tomanage
can usually 200 µM silver
metal increased
stress the concentrations.
up to certain chlorophyll level in that
The way
Solanum shoots bygrowth
moreisthan
stimulated
twofoldat low
[30].doses and inhibited at high doses is referred to as the hormetic
dose
In our present response
study, two[34]. However, these
concentrations (50doses
and may
100 vary
µM) greatly betweenchloride
of cadmium species due andto their
different sensitivities. For example, cadmium drastically reduced the proliferation of Albizia
silver nitrate were used. These values were chosen based on other elicitation experiments
lebbeck (L.) Benth. at a concentration of 5 µM [35], but did not reduce the production of
that used a wide range of metal concentrations; for example, with cadmium concentra-
tions from 0.5 to 500 µM [14,31–33].
Plants can usually manage metal stress up to certain concentrations. The way that
growth is stimulated at low doses and inhibited at high doses is referred to as the hor-
metic dose response [34]. However, these doses may vary greatly between species due to
Molecules 2024, 29, 5263 8 of 14

biomass of Trigonella foenum-graecum L. at 500 µM [33]. Also, in the present study, the shoots
of D. ruyschiana showed a high tolerance to Cd2+ and Ag+ with increased proliferation,
chlorophyll level and biomass accumulation, especially at a concentration of 100 µM.
The data suggest that heavy metal tolerance and protection may operate through
various modes. One is based on limiting the entry of the metals into the cytoplasm, i.e., by
limiting their uptake by the plant or by increasing their accumulation in the cell wall [36].
Plants can also enhance tolerance to heavy metal stress through osmoregulation, which
involves the increased production of osmolytes such as sugars and proteins; these act as
osmoprotectants, helping to maintain cellular osmotic balance and protect against oxidative
damage and metal-induced dehydration. Increased sugar concentrations also enhance
plant tolerance to abiotic stress by altering signaling pathways, triggering the production of
repair enzymes, and increasing ROS scavenging efficiency [37]. In C. tinctorius, exposure to
metal, particularly high doses, elevates protein and sugar levels and increased biomass [10].
As such, the accumulation of shoot biomass in contaminated environments can represent a
survival strategy by the plant. The plant accumulates sugar by increasing photosynthesis,
and the first visible manifestation of this may be an increase in photosynthetic pigment pro-
duction [10,30]; it was noted in D. ruyschiana following heavy metal stimulation, especially
at higher metal concentrations and particularly so in the presence of cadmium.
On the other hand, some studies suggest that increased plant growth and proliferation
may be also associated with the inhibition of the ethylene molecule, which has a negative
effect on the chlorophyll content [38]. Silver nitrate turned out to be a potent inhibitor of
ethylene action, blocking or reducing the capacity of its ETR1 receptor [39]. Studies on
Solanum tuberosum L. indicated that the silver presence prevented the binding of copper,
a cofactor required for ethylene activity, resulting in an increase in the total chlorophyll
content, especially at high silver concentrations (100–200 µM) [30]. However, Ali et al. [40]
proposed that the growth stimulation of Caralluma tuberculata N.E.Br. culture observed
following exposure to silver could have resulted from enhanced nutrient uptake from the
culture medium due to partial damage to the cell wall and increased permeability.
Other physiological adaptations include the secretion of enzymatic and non-enzymatic
antioxidant compounds, which reduced the production of ROS and neutralized them [41].
The plant employs various enzymatic antioxidant defense mechanisms [42], including SOD,
which converts superoxide radical to H2 O2 and O2 , and POD for scavenging H2 O2 [43].
Our present findings indicate that D. ruyschiana shoots demonstrated elevated POD
activity when exposed to cadmium and silver at 50 µM, but not at higher concentrations.
In contrast, higher SOD levels induced greater increases in SOD activity, and significantly
higher levels were noted in the presence of Ag+ than in the presence of Cd2+ . The possible
reason could be associated with the consumption of existing enzyme stock, needed to
neutralize the increased levels of free radicals [10,44]. Although antioxidant enzyme
activity has generally been reported to increase with metal concentration [40,45], some
reports have shown that increased heavy metal stress was associated with lower antioxidant
enzyme activity [10,44,46]. This indicates that above certain concentrations, metals could
inhibit enzyme systems, for example by damaging and/or deactivating them. It was
previously noticed that the activity of POD increased as a response to oxidative stress
induced by 50 and 100 µM Cu in the leaves of tomatoes, but broke down at higher metal
concentrations [44]. Similarly, SOD activities in C. tinctorius increased significantly at
150 µM Cd2+ , but decreased when the concentration was increased to 200 µM, and CAT
activity peaked at 100 µM and decreased at higher concentrations [10].
The activity of antioxidant enzymes could also vary depending on the length of expo-
sure [47]. For example, in Solanum lycopersicum L., oxidase activity increased significantly
in the first day after copper supplementation, but started to decrease after the second day,
and dropped drastically over the next three days. The authors attribute this to metabolism
disruption caused by Cu toxicity [44]. It is therefore possible that during the two-week
exposure in the present study, the activity of SOD and POD in the D. ruyschiana shoots
changed, with the final level reflecting their response to the elevated stress caused by
Molecules 2024, 29, 5263 9 of 14

the higher concentration of the metals and the higher toxicity of cadmium in comparison
to silver.
Non-enzymatic secondary metabolites such as polyphenols also play a supporting
role in protecting against ROS. The exogenous application of silver and cadmium ions
has been reported to induce the biosynthesis of such compounds as a result of oxidative
injury [48,49]. The compounds can form stable complexes with heavy metal ions, thus
preventing the development of oxidative stress [50]. Cadmium used at concentrations from
10 to 200 µM increased the level of all flavonoids identified in regenerated shoots of C.
tinctorius [10], while the amount of chlorogenic acid in Vaccinium corymbosum L. increased
to 15% following cadmium treatment [51]. Also, the silver ions promoted the biosynthesis
of polyphenols in several species; Lam et al. [52] reported that the amount of acacetin and
acacetin glucosides in Agastache rugosa Kuntze exposed to 100 µM silver nitrate were about
10% higher than in untreated plants, and 50 µM stimulated rosmarinic acid production in
Thymus lotocephalus G. López and R. shoots by 25% [53].
The influence of heavy metals on the biosynthesis of secondary metabolites in the
shoots of D. ruyschiana is described in the current study. Some of the phenolic metabo-
lites identified in shoot culture such as chlorogenic acid, acacetin rhamnosyl-trihexoside,
acacetin acetyl-rhamnosyl-trihexoside, apigenin p-coumaroyl-rhamnoside (II) have previ-
ously been detected in aerial parts of this species growing in the field [4,5]. Rosmarinic acid,
methyl rosmarinate apigenin caffeoyl-rhamnoside, apigenin p-coumaroyl-rhamnoside and
dicaffeoylquinic acid, were detected for the first time in D. ruyschiana shoots, but these
compounds are known in other Dracocephalum species; rosmarinic acid, and methyl ros-
marinate were found in aerial parts of D. moldavica L., D. heterophyllum Benth., D. foetidum
Bunge. and D. forrestii W.W. Smith [54–57], while the dicaffeoylquinic acid and apigenin
caffeoyl–rhamnoside were identified in transformed shoots of D. forrestii [24].
In the present study, the metals stimulated a high level of biosynthesis of the predomi-
nant compound in the extract, rosmarinic acid, with the strongest effect observed for 50 µM
Cd2+ . This may indicate that rosmarinic acid, the main polyphenol of the plant, performs
the main defensive functions in situations of oxidative stress. The levels of chlorogenic acid
and flavonoids in D. ruyschiana shoots were also modified by heavy metals, with the higher
concentration of cadmium having the greatest effect.
The presence of a high polyphenol content has been associated with increased antioxi-
dant potential in elicited cultures of A. rugosa, A. annua or C. tinctorius [12,52,58]. A similar
effect was also observed for D. ruyschiana shoots, where the extract from shoots exposed
to 100 µM Cd2+ demonstrated both the strongest antioxidant potential and the highest
polyphenol content.

4. Materials and Methods


4.1. Plant Material
D. ruyschiana shoot culture was established from seeds provided by the Innsbruck Uni-
versity Botanical Garden. Surface sterilization of the seeds was performed with 70% ethanol
for 10 s and subsequently by 1% (v/v) sodium hypochlorite for 2 min and three washings
with sterilized distilled water. The seeds were placed on Murashige and Skoog (MS) [16]
agar (0.7%) medium to produce aseptic seedlings. The apical buds of four-week-old
seedlings were placed on the basal MS agar medium with 0.5 mg/L 6-benzylaminopurine
(BAP) and 0.2 mg/L indole-3-acetic acid (IAA) for culture establishment. The previous
study showed that this type and concentration of phytohormones was optimal for the
cultivation of another Dracocephalum species [20]. The shoots were subcultured every
five weeks.

4.2. Culture Conditions


Fragments with a single nodal segment (about 0.5–0.7 cm in length) were taken from
the five-week-old shoots of D. ruyschiana (from 15–17 subculture) as the explants. Groups
of five explants (initial fresh weight about 6.5 mg) were placed into a glass growth vessel
Molecules 2024, 29, 5263 10 of 14

with 25 mL liquid MS medium containing BAP (0.5 mg/L) and IAA (0.2 mg/L). In order
to avoid the complete submersion of explants in the liquid medium, polyurethane foam
(5 cm × 5 cm × 0.7 cm) (EuroFoam, Zgierz, Poland) was placed as support at the bottom
of the vessel [59]. Polyurethane foam is inert to plant material, does not absorb medium
components, and can be repeatedly sterilized in an autoclave (17 min, 121 ◦ C) without
changes to its physical or chemical properties.
After three weeks of culture growth, aqueous solutions of argentum nitricum (AgNO3 )
or cadmium chloride (CdCl2 ) were added into the growth medium using Sterile Syringe
Filters (0.22 µm) to a final concentration 50 or 100 µM. The effects of metal ion treatments
were evaluated after two consecutive weeks of growth. Shoots cultivated in medium sup-
plemented with distilled water without the addition of heavy metals were used as controls.
The shoots were grown under a 16 h photoperiod (light intensity 50 µM/m2 /s) at
26 ± 2 ◦ C. The experiment was conducted in three replicates including twenty explants
each. After two weeks of exposure to heavy metals, the proliferation rate, i.e., the mean
number of new buds (<0.5 cm long) and/or shoots (≥0.5 cm long) on an explant, and their
fresh (FW) and dry weight (DW) (mg/growth vessel) were recorded.

4.3. Measurement of Chlorophyll Content


The chlorophyll content was determined spectrophotometrically according to the
method described by Wellburn [60]. Chlorophyll content was expressed in mg/g FW as the
sum of chlorophyll a and chlorophyll b. Absorbance was measured at 664 nm (chlorophyll
a) and 647 nm (chlorophyll b).

4.4. Shoot Extraction


Lyophilized shoots (100 mg for the phytochemical analysis and 300 mg for the biologi-
cal assays) were pulverized and extracted three times with 20 mL methanol:water (8:2 v/v)
solution for 15 min in an ultrasonic bath (UD-20 ultrasonic disintegrator; Techpan, Warsaw,
Poland). The extracts were combined and evaporated under reduced pressure.

4.5. Qualitative UPLC-PDA-ESI-MS Analysis


The compounds present in the extract were identified using UPLC-PDA-ESI-MS using
a UPLC-3000 RS apparatus (Dionex, Germering, Germany) with DAD detection and an
AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonik GmbH,
Bremen, Germany) with a Zorbax SB-C18 column (150 × 2.1 mm, 1.9 µm) (Agilent, Santa
Clara, CA, USA). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1%
formic acid in acetonitrile (B). The course and details of the analysis have been described
earlier [61]. The metabolites were tentatively identified by comparison of their UV-Vis
spectra, mass spectra and the retention times with those for standard compounds and
literature data [4,20,24].

4.6. Qualitative HPLC Analysis


The samples were dissolved in 2 mL of 80% (v/v) methanol:water solution and filtered
(PTFE syringe filter; 0.22 µm). The phenolic metabolite contents were determined using an
Agilent Technologies 1290 Infinity HPLC apparatus (Santa Clara, CA, USA) with a diode
array detector (DAD) and Eclipse XDB-C18 column (150 × 4.6 mm, 5 µm). The mobile
phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B).
The solvent system used for elution was: 0–1 min (95% B); 1–10 min (95–90% B); 10–11 min
(90–85% B); 11–40 min (85–80% B); 40–45 min (80–50% B); 45–50 min (50–0% B); 50–53
(0% B). All gradients were linear. The flow rate was 1.6 mL/min. Post-run, the initial phase
composition was used for 7 min. To quantify the phenolic compounds, calibration curves
were prepared by plotting the peak area of the standard compounds at each level against
the concentration of the sample. The experiment used reference standards purchased
from Sigma Aldrich (Darmstadt, Germany): 3,5-di-O-caffeoylquinic acid, rosmarinic acid,
acacetin, apigenin 7-O-glucoside and chlorogenic acid. When an authentic standard was
Molecules 2024, 29, 5263 11 of 14

not available, the phenolic compounds were quantified according to the calibration curve of
an appropriate similar standard: methyl rosmarinate as rosmarinic acid, acacetin glycosides
(Compounds 3 and 7) as acacetin, apigenin derivatives (Compounds 5, 6 and 8) as apigenin
glucoside. The content of the identified compounds and the total phenolic content, i.e., the
sum of all identified phenolics in the sample, were expressed as mg/g DW.

4.7. Antioxidant Activity of Extracts


A ferric reducing antioxidant power (FRAP) assay was performed according to
Grzegorczyk-Karolak et al. [62]. The samples containing a hydromethanolic extract, water
and FRAP reagent were incubated for 30 min at 37 ◦ C. The absorbance was measured at
595 nm. The activity was determined against a standard calibration curve of 0–2000 µM
ferrous sulfate. The results were expressed as µM Fe(II)/g of dry weight of the extract.
A DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was carried out according to Grzegorczyk-
Karolak and Kiss [63]. Samples containing 2 mL of extract and 2 mL DPPH (0.2 mM solution
of DPPH in methanol) were incubated for 30 min in the dark at room temperature. The
antiradical activity was indicated at 517 nm.
The ABTS (2,2′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) scavenging proper-
ties were measured according to Grzegorczyk-Karolak et al. [62]. Briefly, 2 mL of extracts
were mixed with 2 mL of freshly prepared ABTS solution (mixture of 7 mM ABTS and
2.45 mM potassium persulfate). After 10 min of incubation at 25 ◦ C in the dark, the
absorbance of the solutions was measured at 735 nm.
For the antiradical tests (DPPH and ABTS), results were expressed as the half-maximal
inhibitory concentration (IC50 ) (µg/mL). Butylated hydroxytoluene (BHT) was used as a
positive control in all antioxidant assays.

4.8. Activity of Antioxidant Enzymes


The fresh shoots (0.5 g) were homogenized in phosphate buffer (pH = 7.5) and EDTA
(4◦ C) and centrifuged. The obtained supernatant was then tested for sodium dismutase
(SOD) and peroxidase (POD) activity. SOD activity was evaluated by adding the extract to
the following mixture: phosphate buffer (pH = 7.8), riboflavin, nitro blue tetrazolium (NBT),
L-methionine and EDTA [64]. The absorbance of the samples was measured at 560 nm.
POD activity was determined by adding the extract to a mixture containing phosphate
buffer (pH = 7), guaiacol and hydrogen peroxide, and the absorbance was analyzed at
470 nm [65]. In both cases, the results were expressed as enzyme units per mg of fresh mass.
A UV-1800 spectrophotometer (Beijing Rayleigh Corp., Beijing, China) was used for
all spectrophotometric analyses.

4.9. Statistical Analysis


Results represent means ± SD (standard deviation) of three independent experimental
replicates calculated with Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA,
USA). The means were compared using the ANOVA test, followed by Tukey’s post hoc
test (p < 0.05). The statistical analysis was conducted with Statistica 13.1 PL (StatSoft Inc.,
Krakow, Poland).

5. Conclusions
The present study evaluated the effect of the heavy metals Ag+ and Cd2+ on the
proliferation of D. ruyschiana shoots and the accumulation of bioactive compounds in the
culture. Our findings indicate that the species has a high tolerance to the above metals.
No heavy metal treatment inhibited shoot growth, and in some cases, the treatment even
stimulated it. This may indicate that the treatment stimulated the biosynthesis of proteins
and sugars, which are part of the osmoprotective mechanism. Some treatments also
stimulated the production of polyphenols, particularly rosmarinic acid, whose content
in samples treated with 50 µM Cd2+ was three times that of the control. The production
of antioxidant compounds was accompanied by changes in the activity of antioxidant
Molecules 2024, 29, 5263 12 of 14

enzymes such as POD and SOD, thus significantly increasing the culture antioxidant
potential. In conclusion, the stress response in D. ruyschiana is associated with metal-
stimulated growth and the accumulation of non-enzymatic and enzymatic antioxidants.
Our findings suggest that D. ruyschiana uses various strategies to protect against heavy
metal stress depending on the type of metal and its concentration. Therefore, further
research to clarify the mechanisms of this protection would be advisable.

Author Contributions: Conceptualization, I.W.-J.; formal analysis, I.W.-J., A.K.K. and I.G.-K.; in-
vestigation, I.W.-J., J.G., A.K.K. and I.G.-K.; resources, I.W.-J.; writing—original draft preparation,
I.W.-J. and I.G.-K.; writing—review and editing, A.K.K. and I.G.-K.; visualization, I.W.-J.; supervision,
I.G.-K.; project administration, I.W.-J.; funding acquisition, I.W.-J. and I.G.-K. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by the Medical University of Lodz, grant No. 503/3-012-01/503-
31-001.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original results presented in the study are included in the article.
Conflicts of Interest: The authors declare no conflicts of interest.

References
1. Lazarević, P.; Lazarević, M.; Krivošej, Z.; Stevanović, V. On the distribution of Dracocephalum ruyschiana (Lamiaceae) in the Balkan
Peninsula. Phytol. Balc. 2009, 15, 175–179.
2. Kleven, O.; Endrestøl, A.; Evju, M.; Stabbetorp, O.E.; Westergaard, K.B. SNP discovery in the northern dragonhead Dracocephalum
ruyschiana. Conserv. Genet. Resour. 2019, 11, 431–435. [CrossRef]
3. Ligaa, U. Medicinal Plants of Mongolia Used in Western and Eastern Medicine; World Health Organization: Ulaanbaatar, Mongolia,
2006; p. 528.
4. Selenge, E.; Murata, T.; Kobayashi, K.; Batkhuu, J.; Yoshizaki, F. Flavone tetraglycosides and benzyl alcohol glycosides from the
Mongolian medicinal plant Dracocephalum ruyschiana. Nat. Prod. 2013, 76, 186–193. [CrossRef] [PubMed]
5. Okhlopkova, Z.M.; Razgonova, M.P.; Pikula, K.S.; Zakharenko, A.M.; Piekoszewski, W.; Manakov, Y.A.; Ercisli, S.; Golokhvast,
K.S. Dracocephalum palmatum S. and Dracocephalum ruyschiana L. Originating from Yakutia: A high-resolution mass spectrometric
approach for the comprehensive characterization of phenolic compounds. Appl. Sci. 2022, 12, 1766. [CrossRef]
6. Isah, T.; Umar, S.; Mujib, A.; Sharma, M.P.; Rajasekharan, P.E.; Zafar, N.; Frukh, A. Secondary metabolism of pharmaceuticals in
the plant in vitro cultures: Strategies, approaches, and limitations to achieving higher yield. Plant Cell Tissue Organ Cult. 2018,
132, 239–265. [CrossRef]
7. Narayani, M.; Srivastava, S. Elicitation: A stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary
metabolite production. Phytochem. Rev. 2017, 16, 1227–1252. [CrossRef]
8. Khalili, M.; Hasanloo, T.; Tabar, S.K.K.; Rahnama, H. Influence of exogenous salicylic acid on flavonolignans and lipoxygenase
activity in the hairy root cultures of Silybum marianum. Cell Biol. Inter. 2009, 33, 988–994. [CrossRef]
9. Narula, A.; Kumar, S.; Srivastava, P.S. Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures. Plant Cell
Rep. 2005, 24, 250–254. [CrossRef]
10. Ejaz, B.; Mujib, A.; Syeed, R.; Mamgain, J.; Malik, M.Q.; Birat, K.; Dewir, Y.H.; Magyar-Tábori, K. Phytocompounds and regulation
of flavonoids in in vitro-grown safflower plant tissue by abiotic licitor CdCl2 . Metabolites 2024, 14, 127. [CrossRef]
11. Ghorbanpour, M. Major essential oil constituents, total phenolics and flavonoids content and antioxidant activity of Salvia
officinalis plant in response to nano-titanium dioxide. Indian J. Plant Physiol. 2015, 20, 249–256. [CrossRef]
12. Dobrikova, A.G.; Apostolova, E.L.; Hanć, A.; Yotsova, E.; Borisova, P.; Sperdouli, I.; Moustakas, M. Cadmium toxicity in Salvia
sclarea L.: An integrative response of element uptake, oxidative stress markers, leaf structure and photosynthesis. Ecotoxicol.
Environ. Safe 2021, 209, 111851. [CrossRef] [PubMed]
13. Georgiev, M.; Pavlov, A.; Ilieva, M. Selection of high rosmarinic acid producing Lavandula vera MM cell lines. Process Biochem.
2006, 41, 2068–2071. [CrossRef]
14. Xiao, Y.; Gao, S.; Di, P.; Chen, J.; Chen, W.; Zhang, L. Lithospermic acid B is more responsive to silver ions (Ag+ ) than rosmarinic
acid in Salvia miltiorrhiza hairy root cultures. Biosci. Rep. 2010, 30, 33–40. [CrossRef]
15. Kruk, J.; Aboul-Enein, B.H.; Duchnik, E.; Marchlewicz, M. Antioxidative properties of phenolic compounds and their effect on
oxidative stress induced by severe physical exercise. J. Physiol. Sci. 2022, 72, 19. [CrossRef]
16. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 1962, 15,
473–497. [CrossRef]
Molecules 2024, 29, 5263 13 of 14

17. Chen, F.; Long, X.; Liu, Z.; Shao, H.; Lu, L. Analysis of phenolic acids of Jerusalem Artichoke (Helianthus tuberosus L.) responding
to salt-stress by liquid chromatography/tandem mass spectrometry. Sci. World J. 2014, 2014, 568043.
18. Catarino, M.D.; Silva, A.M.S.; Saraiva, S.C.; Sobral, A.J.F.N.; Cardoso, S.M. Characterization of phenolic constituents and
evaluation of antioxidant properties of leaves and stems of Eriocephalus africanus. Arab. J. Chem. 2018, 11, 62–69. [CrossRef]
19. Ncube, N.; Mhlongo, M.I.; Piater, L.A.; Steenkamp, P.A.; Dubery, I.A.; Madala, N.E. Analyses of chlorogenic acids and related
cinnamic acid derivatives from Nicotiana tabacum tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision
induced dissociation method. Chem. Cent. J. 2014, 8, 66–76. [CrossRef]
20. Weremczuk-Jeżyna, I.; Kuźma, Ł.; Kiss, A.K.; Grzegorczyk-Karolak, I. Effect of cytokinins on shoots proliferation and rosmarinic
and salvianolic acid B production in shoot culture of Dracocephalum forrestii W. W. Smith. Acta Physiol. Plant. 2018, 40, 189.
[CrossRef]
21. Lin, L.L.; Harmly, J.M. Identification of the phenolic components of chrysanthemum flowers (Chrysanthemum morifolium Ramat).
Food Chem. 2010, 120, 319–326. [CrossRef]
22. Bakr, R.O.; El Bishbishy, M.H. Profile of bioactive compounds of Capparis spinosa var. aegyptiaca growing in Egypt . Rev. Bras.
Farmacogn. 2016, 26, 514–520. [CrossRef]
23. Wu, W.; Liu, Z.; Song, F.; Lin, S. Structural analysis of selected characteristic flavones by electrospray tandem mass spectrometry.
Anal. Sci. 2004, 20, 1103–1105. [CrossRef] [PubMed]
24. Weremczuk-Jeżyna, I.; Skała, E.; Kuźma, Ł.; Kiss, A.K.; Grzegorczyk-Karolak, I. The effect of purine-type cytokinin on the
proliferation and production of phenolic compounds in transformed shoots of Dracocephalum forrestii. J. Biotechnol. 2019, 306,
125–133. [CrossRef] [PubMed]
25. Al-Khayri, J.M.; Poornananda, M.N. Elicitor-induced production of biomass and pharmaceutical phenolic compounds in cell
suspension culture of date palm (Phoenix dactylifera L.). Molecules 2020, 25, 4669. [CrossRef]
26. Paeizi, M.; Karimi, M.; Razavi, K. Changes in medicinal alkaloids production and expression of related regulatory and biosynthetic
genes in response to silver nitrate combined with methyl jasmonate in Catharanthus roseus in vitro propagated shoots. Plant Physiol.
Biochem. 2018, 132, 623–632. [CrossRef]
27. Li, B.; Wang, B.; Li, H.; Peng, L.; Ru, M.; Liang, Z.; Zhu, Y. Establishment of Salvia castanea Diels f. tomentosa Stib. hairy root
cultures and the promotion of tanshinone accumulation and gene expression with Ag+, methyl jasmonate, and yeast extract
elicitation. Protoplasma 2016, 253, 87–100. [CrossRef]
28. Geetha, G.; Harathi, K.; Naidu, C.V. Role of silver nitrate on flowering and shoot regeneration of Solanum nigrum—Important
multipurpose medicinal plant. Am. J. Plant Sci. 2016, 7, 1021–1032. [CrossRef]
29. Qiu, R.L.; Zhao, X.; Tang, Y.T.; Yu, F.M.; Hu, P.J. Antioxidative response to Cd in a newly discovered cadmium hyperaccumulator,
Arabis paniculata F. Chemosphere 2008, 74, 6–12. [CrossRef]
30. Rostami, F.; Ehsanpour, A. The effect of silver thiosulfate (STS) on chlorophyll content and the antioxidant. J. Cell Mol. Res. 2010,
2, 29–34.
31. Wiszniewska, A.; Hanus-Fajerska, E.; Muszyńska, E.; Smoleń, S. Comparative assessment of response to cadmium in heavy
metal-tolerant shrubs cultured in vitro. Water Air Soil Pollut. 2017, 228, 304. [CrossRef]
32. Açıkgoz, M.A. Establishment of cell suspension cultures of Ocimum basilicum L. and enhanced production of pharmaceutical
active ingredients. Ind. Crop. Prod. 2020, 148, 112278. [CrossRef]
33. De, D.; De, B. Elicitation of diosgenin production in Trigonella foenum-graecum L. seedlings by heavy metals and signaling
molecules. Acta Physiol. Plant. 2022, 33, 1585–1590. [CrossRef]
34. Calabrese, E.J.; Blain, R. Hormesis and plant biology. Environ. Pollut. 2009, 157, 42–48. [CrossRef] [PubMed]
35. Perveen, S.; Anis, M.; Aref, I.M. In vitro morphogenic response and metal accumulation in Albizia lebbeck (L.) cultures grown
under metal stress. Eur. J. For. Res. 2012, 131, 669–681. [CrossRef]
36. Atabayeva, S.D.; Minocha, R.; Minocha, S.C.; Rakhymgozhina, A.; Nabieva, A.M.; Nurmahanova, A.C.; Kenzhebayeva, S.S.;
Alybayeva, R.A.; Asrandina, S.S. Response of plants to cadmium stress. Int. J. Biol. Chem. 2020, 13, 109. [CrossRef]
37. Gugale, G.S.; Bhusare, B.P.; Ambawade, M.S.; Kshatriya, A.S.; Barwant, M.M.; Mhaske, A.K. Effect of cadmium chloride on seed
germination, seedling growth parameters, and proline content in maize (Zea mays). Int. J. Res. Educ. Sci. Meth. 2021, 9, 1818–1822.
38. Bora, G.; Gogoi, H.K.; Handique, P.J. Influence of silver nitrate and glutamine on in vitro organogenesis of Lota Bhot (Capsicum
chinense Jacq.), an indigenous pungent pepper variety of Assam. J. Appl. Biol. Biotechnol. 2019, 7, 21–28.
39. Zhao, X.C.; Qu, X.; Mathwes, D.E.; Schaller, G.E. Effect of ethylene-pathway mutations upon expression of the ethylene receptor
ETR1 from Arabidopsis. Plant Physiol. 2002, 130, 1983–1991. [CrossRef]
40. Ali, A.; Mohammad, S.; Khan, M.A.; Raja, N.I.; Arif, M.; Kamil, A.; Mashwani, Z.U.R. Silver nanoparticles elicited in vitro callus
cultures for accumulation of biomass and secondary metabolites in Caralluma tuberculata. Artif. Cells Nanomed. Biotechnol. 2019, 47,
715–724. [CrossRef]
41. Hall, J.L. Cellular mechanisms for heavy metal detoxication and tolerance. J. Exp. Bot. 2002, 53, 1–11. [CrossRef]
42. Kisa, D.; Elmastaş, M.; Öztürk, L.; Kayır, Ö. Responses of the phenolic compounds of Zea mays under heavy metal stress. Appl.
Biol. Chem. 2016, 59, 813–820. [CrossRef]
43. Zhang, S.; Zhang, H.; Qin, R.; Jiang, W.; Liu, D. Cadmium induction of lipid peroxidation and effects on root tip cells and
antioxidant enzyme activities in Vicia faba L. Ecotoxicology 2009, 18, 814–823. [CrossRef] [PubMed]
Molecules 2024, 29, 5263 14 of 14

44. Martins, L.L.; Mourato, M.P. Effect of excess copper on tomato plants: Growth parameters, enzyme activities, chlorophyll, and
mineral content. J. Plant Nutr. 2006, 29, 2179–2198. [CrossRef]
45. Irfan, M.; Hayats, S.; Ahmed, A.; Alyemeni, M.N. Soil cadmium enrichment: Allocation and plant physiological manifestations.
Saudi J. Biol. Sci. 2013, 20, 1–10. [CrossRef]
46. Hassan, M.J.; Raza, M.A.; Rehman, S.U.; Ansar, M.; Gitari, H.; Khan, I.; Wajid, M.; Ahmed, M.; Shah, G.A.; Peng, Y.; et al. Effect of
cadmium toxicity on growth, oxidative damage, antioxidant defense system and cadmium accumulation in two orghum cultivars.
Plants 2020, 9, 1575. [CrossRef]
47. Carmen, E.M.; Souza, V.; Bucio, L.; Hernández, E.; Damián-Matsumura, P.; Zaga, V.; Gutiérrez-Ruiz, M.C. Cadmium induces
alpha(1)collagen (I) and metallothionein II gene and alters the antioxidant system in rat hepatic stellate cells. Toxicology 2002, 170,
63–73. [CrossRef]
48. Anjitha, K.S.; Sameena, P.P.; Puthur, J.T. Functional aspects of plant secondary metabolites in metal stress tolerance and their
importance in pharmacology. Plant Stress 2021, 2, 100038. [CrossRef]
49. Bhaduri, A.M.; Fulekar, M.H. Antioxidant enzyme responses of plants to heavy metal stress. Rev. Environ. Sci. Biotechnol. 2012, 11,
55–69. [CrossRef]
50. Jiao, Z.; Shi, Y.; Wang, J.; Wang, Z.; Zhang, X.; Jia, X.; Du, Q.; Niu, J.; Liu, B.; Du, R.; et al. Integration of transcriptome and
metabolome analyses reveals sorghum roots responding to cadmium stress through regulation of the flavonoid biosynthesis
pathway. Front. Plant Sci. 2023, 14, 1144265. [CrossRef]
51. Manquián-Cerda, K.; Escudey, M.; Zúñiga, G.; Arancibia-Miranda, N.; Molina, M.; Cruces, E. Effect of cadmium on phenolic
compounds, antioxidant enzyme activity and oxidative stress in blueberry (Vaccinium corymbosum L.) plantlets grown in vitro.
Ecotoxicol. Environ. Saf. 2016, 133, 316–326. [CrossRef]
52. Lam, V.P.; Beomseon, L.; Anh, V.K.; Loi, D.N.; Kim, S.; Kwang-Ya, L.; Park, J. Effectiveness of silver nitrate application on plant
growth and bioactive compounds in Agastache rugosa (Fisch. & CA Mey.) Kuntze. Heliyon 2023, 9, e20205l.
53. Gonçalves, S.; Mansinhos, I.; Rodríguez-Solana, R.; Pérez-Santín, E.; Coelho, N.; Romano, A. Elicitation improves rosmarinic acid
content and antioxidant activity in Thymus lotocephalus shoot cultures. Ind. Crop. Prod. 2019, 137, 214–220. [CrossRef]
54. Zhang, J.I.; Yan, R.J.; Yu, N.; Zhang, X.; Chen, D.J.; Wu, T.; Xin, J.G. A new caffeic acid tetramer from the Dracocephalum moldavica
L. Nat. Prod. Res. 2018, 32, 370–373. [CrossRef] [PubMed]
55. Selenge, E.; Murata, T.; Tanaka, S.; Sasaki, K.; Batkhuu, J.; Yoshizaki, F. Monoterpene glycosides, phenylpropanoids, and acacetin
glycosides from Dracocephalum foetidum. Phytochemistry 2014, 101, 91–100. [CrossRef] [PubMed]
56. Dang, J.; Wang, S.; Shao, Y. Preparative isolation of antioxidative compounds from Dracocephalum heterophyllum using off-line two-
dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography guided by on-line HPLC-DPPH
assay. J. Chromatogr. B 2018, 1095, 267–274. [CrossRef]
57. Li, S.M.; Yang, X.W.; Li, Y.L.; Shen, Y.H.; Feng, L.; Wang, Y.H.; Zeng, H.W.; Liu, X.H.; Zhang, C.S.; Long, C.L.; et al. Chemical
constituents of Dracocephalum forestii. Planta Med. 2004, 75, 1591–1596. [CrossRef]
58. Darki, B.S.; Shabani, L.; Pourvaez, R.; Ghannadian, S.M. Effects of CuSO4 and AgNO3 on artemisinin and phenolic compound in
shoot cultures of Artemisia annua L. J. Plant Process Funct. 2019, 8, 31.
59. Grzegorczyk-Karolak, I.; Rytczak, P.; Bielecki, S.; Wysokińska, H. The influence of liquid systems for shoot multiplication,
secondary metabolite production and plant regeneration of Scutellaria alpina. Plant. Cell Tiss. Organ Cult. 2017, 128, 479–486.
[CrossRef]
60. Wellburn, A.R. The spectra determination of chlorophylls a and b as well as total carotenoids, using various solvents with
spectrophotometers of different resolution. J. Plant Physiol. 1994, 144, 307–313. [CrossRef]
61. Grzegorczyk-Karolak, I.; Ejsmont, W.; Kiss, A.K.; Tabaka, P.; Starbała, W.; Krzemińska, M. Improvement of bioactive polyphenol
accumulation in callus of Salvia atropatana Bunge. Molecules 2024, 29, 2626. [CrossRef]
62. Grzegorczyk-Karolak, I.; Kuźma, Ł.; Wysokińska, H. The effect of cytokinins on shoot proliferation, secondary metabolite
production and antioxidant potential in shoot cultures of Scutellaria alpina. Plant Cell Tiss. Organ Cult. 2015, 122, 699–708.
[CrossRef]
63. Grzegorczyk-Karolak, I.; Kiss, A. Determination of the phenolic profile and antioxidant properties of Salvia viridis L. shoots: A
comparison of aqueous and hydroethanolic extracts. Molecules 2018, 23, 1468. [CrossRef] [PubMed]
64. Van Rossun, M.W.P.C.; Alberda, M.; Van Der Plas, L.H.W. Role of oxidative damage in tulip bulb scale micropropagation. Plant
Sci. 1997, 130, 207–216. [CrossRef]
65. Hemeda, H.M.; Klein, B.P. Effects of naturally occurring antioxidants on peroxidase activity of vegetable extracts. J. Food Sci. 1990,
55, 184–185. [CrossRef]

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