Is comfrey root more than toxic pyrrolizidine alkaloids? Salvianolic acids among antioxidant polyphenols in comfrey (Symphytum officinale L.) roots


Trifan, A.; Opitz, S.E.W.; Josuran, R.; Grubelnik, A.; Esslinger, N.; Peter, S.; Bräm, S.; Meier, N.; Wolfram, E.

Food and Chemical Toxicology 112: 178-187

2017


Comfrey root preparations are used for the external treatment of joint distortions and myalgia, due to its analgesic and anti-inflammatory properties. Up to date, key activity-determining constituents of comfrey root extracts have not been completely elucidated. Therefore, we applied different approaches to further characterize a comfrey root extract (65% ethanol). The phenolic profile of comfrey root sample was characterized by HPLC-DAD-QTOF-MS/MS. Rosmarinic acid was identified as main phenolic constituent (7.55 mg/g extract). Moreover, trimers and tetramers of caffeic acid (isomers of salvianolic acid A, B and C) were identified and quantified for the first time in comfrey root. In addition, pyrrolizidine alkaloids were evaluated by HPLC-QQQ-MS/MS and acetylintermedine, acetyllycopsamine and their N-oxides were determined as major pyrrolizidine alkaloids in the comfrey root sample. Lastly, the antioxidant activity was determined using four assays: DPPH and ABTS radicals scavenging assays, reducing power assay and 15-lipoxygenase inhibition assay. Comfrey root extract exhibited significant antioxidant activities when compared to known antioxidants. Thus, comfrey root is an important source of phenolic compounds endowed with antioxidant activity which may contribute to the overall bioactivity of Symphytum preparations.

Food
and
Chemical
Toxicology
112
(2018)
178-187
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Food
and
Chemical
Toxicology
Food
and
Chemical
Toxicology
ELSEVIER
journal
homepage:
www.elsevier.com/locate/foodchemtox
Is
comfrey
root
more
than
toxic
pyrrolizidine
alkaloids?
Salvianolic
acids
among
antioxidant
polyphenols
in
comfrey
(Symphytum
officinale
L.)
roots
Adriana
Trifan
,
Sebastian
E.W.
Opitz
,
Roland
Josuran
c
,
Andreas
Grubelnik
d
,
Nils
Esslinger
,
Samuel
Peter
e
,
Sarah
Bram
e
,
Nadja
Meier
,
Evelyn
Wolfram
e
'Department
of
Phcrnnacognosy,
Faculty
of
Pharmacy,
Grigore
T.
Popa
University
of
Medicine
and
Pharmacy
Iasi,
Iasi
700115,
Romania
b
Analytical
Technologies
Group,
Institute
of
Chemistry
and
Biotechnology,
Zurich
University
of
Applied
Sciences,
Weidenswil
8820,
Switzerland
Centre
for
Biochemistry
and
Biocmalytics,
Institute
of
Chemistry
and
Biotechnology,
Zurich
University
of
Applied
Sciences,
Weidenswil
8820,
Switzerland
d
Akrinamed
AG,
Freidorf
9304
Switzerland
`Phytophcrnnacy
and
Natural
Products
Research
Group,
Institute
of
Chemistry
and
Biotechnology,
Zurich
University
of
Applied
Sciences,
Weidenswil
8820,
Switzerland
Check
for
updates
ARTICLE INFO
ABSTRACT
Keywords:
Symphytum
officinale
L
Phenolic
compounds
Salvianolic
adds
Antioxidant
activity
Pyrrolizidine
alkaloids
Comfrey
root
preparations
are
used
for
the
external
treatment
of
joint
distortions
and
myalgia,
due
to
its
an-
algesic
and
anti-inflammatory
properties.
Up
to
date,
key
activity-determining
constituents
of
comfrey
root
extracts
have
not
been
completely
elucidated.
Therefore,
we
applied
different
approaches
to
further
characterize
a
comfrey
root
extract
(65%
ethanol).
The
phenolic
profile
of
comfrey
root
sample
was
characterized
by
HPLC-
DAD-QTOF-MS/MS.
Rosmarinic
acid
was
identified
as
main
phenolic
constituent
(7.55
mg/g
extract).
Moreover,
trimers
and
tetramers
of
caffeic
acid
(isomers
of
salvianolic
acid
A,
B
and
C)
were
identified
and
quantified
for
the
first
time
in
comfrey
root.
In
addition,
pyrrolizidine
alkaloids
were
evaluated
by
HPLC-QQQ-MS/MS
and
acetylintermedine,
acetyllycopsamine
and
their
N-oxides
were
determined
as
major
pyrrolizidine
alkaloids
in
the
comfrey
root
sample.
Lastly,
the
antioxidant
activity
was
determined
using
four
assays:
DPPH
and
ABTS
radicals
scavenging
assays,
reducing
power
assay
and
15-lipoxygenase
inhibition
assay.
Comfrey
root
extract
exhibited
significant
antioxidant
activities
when
compared
to
known
antioxidants.
Thus,
comfrey
root
is
an
important
source
of
phenolic
compounds
endowed
with
antioxidant
activity
which
may
contribute
to
the
overall
bioactivity
of
Symphytum
preparations.
1.
Introduction
Symphytum
officinale
L.
(comfrey)
is
a
perennial
herbaceous
plant
very
common
in
Europe
and
Asia,
that
has
been
naturalised
throughout
North
America
(Brunton,
1999).
Dioscorides
praised
the
therapeutic
uses
of
comfrey
in
de
Materia
Medica,
the
name
of
the
genus
symphytwn
being
derived
from
Greek
symphuo
"to
make
to
grow
together"
(Cupp,
2000).
Throughout
the
Middle
ages
comfrey
was
used
as
an
external
poultice
for
healing
fractures,
bruises,
and
bums;
additional
traditional
use
implied
treatment
of
respiratory
problems,
gastrointestinal
dis-
eases,
metrorrhagia,
phlebitis,
and
tonsillitis
(Barnes
et
al.,
2007;
Cupp,
2000).
Comfrey
root
contains
carbohydrates
(mucilage
29%),
allantoin
(0.6-4.7%),
triterpenes
(phytosterols;
saponins:
symphytoxide
A,
leontoside
A,
B,
D),
tannins
(2.4%,
pyrocathechol-type),
phenolic
acids
(rosmarinic
acid
0.2%,
caffeic
acid,
chlorogenic
acid),
and
pyrrolizidine
alkaloids
(0.013-1.2%)
(Barnes
et
al.,
2007;
Staiger,
2012).
However,
the
therapeutic
application
of
comfrey
is
overshadowed
by
the
well-recognized
toxicity
of
pyrrolizidine
alkaloids.
There
are
numerous
reports
on
the
acute/chronic
ingestion
of
pyrrolizidine
al-
kaloids-containing
plant
material
and
its
toxicity
(hepatotoxicity,
car-
cinogenicity
and
mutagenicity)
(HMPC,
2014).
Bioactivation
occurs
in
the
liver
by
the
action
of
oxidases,
with
transformation
to
pyrrole
in-
termediates
that
bind
to
DNA,
proteins,
and
glutathion,
thus
causing
cell
disfunction
and
cell
death
(Wiedenfeld
and
Edgar,
2011).
Even
in
case
of
preparations
for
cutaneous
use
only,
the
main
concern
for
the
clinical
safety
of
comfrey
preparations
is
related
to
the
content
of
pyrrolizidine
alkaloids,
with
the
potential
of
absorption
through
intact
skin
followed
by
metabolic
activation
(Frost
et
al.,
2014).
In
the
as-
sessment
report
on
Symphytum
officinale
L.,
radix,
the
Committee
on
Herbal
Medicinal
Products
of
the
European
Medicines
Agency
refers
to
the
acceptable
daily
intake
of
pyrrolizidine alkaloids
as
maximum
0.007
µg/kg/day
for
cutaneous
preparations,
the
use
being
restricted
to
intact
skin
(HMPC,
2015).
Nowadays,
medicinal
products
from
comfrey
root
that
are
com-
mercialized
on
the
European
market
contain
only
extracts
from
*
Corresponding
author.
Faculty
of
Pharmacy,
Grigore
T.
Popa
University
of
Medicine
and
Pharmacy
Iasi,
Universitatii
Str.
16,
Iasi
700115,
Romania.
E-mail
address
(A.
Trifan).
https://doLorg/10.1016/j.fct.2017.12.051
Received
3
November
2017;
Received
in
revised
form
21
December
2017;
Accepted
22
December
2017
Available
online
28
December
2017
0278-6915/
©
2017
Elsevier
Ltd.
All
rights
reserved.
A
Trifan
et
al.
Food
and
Chemical
Toxicology
112
(2018)
178-187
pyrrolizidine-depleted
plant
material
or
obtained
from
special
cultivars
that
do
not
synthesize
pyrrolizidine
alkaloids
(Staiger,
2012).
Root
preparations
are
utilized
for
the
external
treatment
of
joint
complaints,
painful
muscle,
bone
fractures,
distortions
and
haematomas
(Brunton,
1999;
Frost
et
al.,
2014).
Therapeutic
properties
of
comfrey
root
are
based
on
its
anti-inflammatory
and
analgesic
effects,
mainly
due
to
allantoin
and
rosmarinic
acid
(Awang,
1987;
Barnes
et
al.,
2007).
Up
to
date,
key
activity-determining
constituents
of
comfrey
root
extracts
and
their
molecular
mechanisms
of
action
have
not
been
completely
eluci-
dated.
To
the
best
of
our
knowledge,
the
profile
of
its
phenolic
com-
pounds
has
only
been
reported
based
on
studies
using
HPLC-DAD
and
HPLC-ED
(Sowa
et
al.,
2017;
Tahirovic
et
al.,
2010;
Neagu
et
al.,
2010).
Therefore,
the
aim
of
the
present
study
was
to
assess
the
phenolic
profile
of
a
hydroalcoholic
(65%
ethanol)
extract
from
roots
of
Sym-
phytum
officinale
L.
with
higher
sensitivity
and
accurate
mass
determi-
nation
and
fragmentation
using
HPLC-DAD-QTOF-MS/MS.
Also,
major
phenolic
compounds
were
quantified
by
HPLC-QQQ-MS/MS
and
the
in
vitro
antioxidant
activity
of
the
sample
was
determined.
In
addition,
pyrrolizidine
alkaloids
were
evaluated
by
HPLC-QQQ-MS/MS.
2.
Materials
and
methods
2.1.
Chemicals
All
reagents
used
for
LC-MS
analysis
(acetonitrile,
ethanol,
formic
acid)
were
purchased
from
Sigma-Aldrich
(Steinheim,
Germany).
Gallic
acid,
butylhydroxyanisole,
sodium
carbonate,
Folin-Ciocalteu's
phenol
reagent,
2,2-diphenyl-1-picrylhydrazyl
radical
(DPPH),
2,2'-azinobis(3-
ethylbenzothiazoline-6-sulfonic
acid)
diammonium
salt
(ABTS),
po-
tassium
ferricyanide,
iron
(III)
chloride,
disodium
phosphate
dodeca-
hydrate,
boric
acid,
linoleic
acid,
lipoxidase
from
soybean,
sodium
hydroxide
were
purchased
from
Sigma-Aldrich
(Steinheim,
Germany).
Monopotassium
phosphate
and
dimethyl
sulfoxide
(DMSO)
were
pur-
chased
from
Merck
(Darmstadt,
Germany).
Trichloroacetic
acid
and
potassium
persulfate
were
purchased
from
Riedel-de
Haen
(Seelze,
Germany).
Standards
of
caffeic
acid,
rosmarinic
acid,
lithospermic
acid,
salvianolic
acid
A,
B
and
C
were
purchased
from
Phytolab
(Vestenbergsgreuth, Germany).
Standards
of
intermedine,
lycopsamine,
intermedine
N-oxide,
lycopsamine
N-oxide,
acetylintermedine,
acet-
yllycopsamine,
acetylintermedine
N-oxide,
acetyllycopsamine
N-oxide
were
purchased
from
Phytoplan
(Heidelberg,
Germany).
All
other
sol-
vents
and
reagents
were
of
analytical
grade.
2.2.
Preparation
of
comfrey
root
extract
Raw
plant
material
(root
of
Symphytum
officinale
L.)
was
purchased
from
a
local
pharmacy
(Berg-Apotheke,
Zurich,
Switzerland).
A
voucher
specimen
was
deposited
in
the
Phytopharmacy
and
Natural
Products
Research
Group
of
the
Institute
of
Chemistry
and
Biotechnology,
under
number
m20170216.
The
roots
were
powdered
and
extracted
three
times
with
65%
(v/v)
ethanol
for
30
min
(according
to
the
method
described
in
HMPC,
2015),
under
reflux
at
60
°C.
The
obtained
solution
was
concentrated
under
reduced
pressure
at
40
°C,
frozen
and
then
lyophilised.
The
extraction
yield
was
26.02%
(DER
3.84:1).
The
extract
was
kept
at
20
°C
until
analysis.
2.3.
Determination
of
total
phenolic
content
Phenolic
content
were
estimated
according
to
the
Folin-Ciocalteu
method
(Wangensteen
et
al.,
2004;
Trifan
et
al.,
2016).
Briefly,
3.16
mL
of
ultrapure
water
and
200
µL
of
Folin-Ciocalteu
reagent
were
added
to
40
µL
of
sample,
followed
by
mixing.
After
5
min,
600
µL
of
sodium
carbonate
(20%)
were
added
and
the
mixture
was
vigorously
vortexed.
After
incubation
at
room
temperature
for
2
h,
the
absorbance
was
re-
corded
at
765
nm.
Caffeic
acid
was
used
as
reference
standard.
The
results
from
triplicate
determination
were
expressed
as
mean
±
standard
deviation
mg
of
caffeic
acid
equivalents/g
of
comfrey
root
extract.
2.4.
Phenolic
compounds
analysis
Phytochemical
analyses
were
performed
using
two
complementary
LC—MS
systems.
Qualitative
analysis
of
phenolic
compounds
was
achieved
on
a
system
consisting
of
an
Agilent
1200
HPLC
with
a
diode-
array
detector
(DAD)
coupled
with
an
Agilent
6530
high-resolution
accurate
mass
quadrupole
time-of-flight
(Q-TOF)
mass
spectrometer
(Agilent
Technologies,
USA).
Chromatographic
separations
by
HPLC
were
carried
out
at
40
°C
on
a
Zorbax
Eclipse
Plus
C18
column
(2.1
x
50
min,
1.8
µm
particles;
Agilent
Technologies,
USA).
Injection
volume
was
1
µL.
The
mobile
phases
were
0.1%
formic
acid
in
water
(solvent
A)
and
acetonitrile
(solvent
B),
with
a
flow
of
0.5
mL/min
and
the
following
gradient:
0-3
min,
4%
B;
3-7
min,
4-15%
B;
7-16.5
min,
15-30%
B;
16.5-18
min,
30-95%
B;
18-21
min,
95%
B;
then
back
to
initial
conditions
in
4
min.
The
MS
operated
in
negative
ion
mode
upon
following
settings:
capillary
voltage
2.5
kV;
drying
gas
flow
rate
811
min;
sheath
gas
flow
rate
10
L/min;
temperature
of
drying
gas
and
sheath
gas
300
°C;
pressure
of
nebulizer
35
psi;
skimmer
65
V;
frag-
mentor
voltage
120
V.
The
spectra were
scanned
in
the
range
of
80-1000
m/z.
Collision
energy
for
the
MS
2
experiments
was
20
eV.
The
UV
spectra
was
recorded
between
200
and
600
nm,
and
the
wavelength
of
280
nm
was
chosen
for
determination.
Data
were
analyzed
with
Agilent
MassHunter
software
(Version
B.08.00,
Agilent
Technologies,
USA,
2016).
The
individual
compounds
were
identified
by
comparison
of
the
exact
molecular
masses,
UV
spectra,
mass
spectra,
fragmentation
pattern
and
retention
times
to
those
of
authentical
standards,
online
available
databases
and
literature
data
(Banos
et
al.,
2013;
Chen
et
al.,
2011a;
Liu
et
al.,
2007;
Yang
et
al.,
2015).
Quantification
of
main
phenolics
was
conducted
using
an
Agilent
1260
HPLC
system
(Agilent
Technologies,
USA)
equipped
with
a
binary
gradient
solvent
pump,
degasser,
autosampler
and
column
oven,
hy-
phenated
with
a
AB
Sciex
4500
QTRAP
mass
spectrometer
(AB
Sciex,
USA)
equipped
with
an
electrospray
ionisation
source
(ESI)
and
a
triple
quadrupole
(QQQ)
mass
analyser
with
a
trapping
function.
The
se-
paration
of
phenolics
was
carried
out
at
40
°C
on
an
InfinityLab
Poroshell
120
EC-C18
column
(4.6
x
50
mm,
2.7
µm
particles;
Agilent
Technologies,
USA)
with
a
mobile
phase
of
0.1%
formic
acid
in
water
and
acetonitrile,
using
5µL
injections.
The
flow
rate
was
0.5
mL/min
and
the
gradient
was
similar
to
the
one
used
for
phenolics
qualitative
analysis.
ESI
was
operated
in
the
negative
ion
mode:
capillary
tem-
perature
500
°C,
curtain
gas
40
psi,
nebulizer
gas
40
psi,
ionspray
vol-
tage
4.5
kV.
Multiple
reaction
monitoring
(MRM)
was
used
for
quan-
titative
analysis
of
main
phenolic
compounds
based
on
their
peak
areas
and
comparison
with
a
calibration
curve
for
the
corresponding
stan-
dards
at
six
concentrations
(0.2-10
µg/mL).
The
data
were
acquired
and
processed
using
Analyst
1.6.2
software
(AB
Sciex,
USA).
Triplicate
in-
jections
were
made
for
each
standard
and
for
the
sample.
The
results
were
expressed
in
mg
per
g
of
comfrey
root
extract.
2.5.
Antioxidant
activity
assays
2.5.1.
DPPH
radical
scavenging
assay
DPPH
radical
scavenging
activity
was
determined
using
the
method
described
by
Malterud
et
al.
(1993).
50
µL
of
comfrey
root
extract
(1.25-20
mg/mL
in
DMSO)
were
mixed
with
2.95
inL
of
0.1
inM
DPPH
in
methanol
(A
517
„„,
=
1.00
±
0.05).
The
absorbance
of
DPPH
radical
solution
was
measured
at
517
nm
before
(A
stan
)
and
5
min
after
adding
the
extract/positive
controls
(Aend).
The
DPPH
radical
scavenging
ac-
tivity
(%)
was
calculated
using
the
following
formula:
DPPH
radical
scavenging
activity
(%)
=
100
x
(Astart
Aend)/(Astart).
2.5.2.
ABTS
radical
cation
scavenging
assay
The
assay
was
based
on
the
method
of
ice
et
al.
(1999).
ABTS
radical
179
8
2
5
3
1
4
6
5
B
:
5.5
SS-
:
:
75
:
55.
C
55.
C
5
C
C
a.
:5.
C3•
0.25-
02-
0.15
C.1
Cos.
S
3
6
Food
and
Chemical
Toxicology
112
(2018)
178-187
A
Trifan
et
al.
x10
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1,15
1,1
1,05
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0,9
0,85
0,8
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0,7
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0,6
0,55
0
'
5
1
1,5
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2:5
3
35
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4,5
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5,5
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14,5
1
'
5
15,5
16
16,5
Response
(%)
vs.
Acquisition
Time
(min)
.5
1
4
.
5. 5.
5
.
5
5
.
5
-;
7
.
5
5
.
5
9
.
5
125
I ,
.
11
.
5
1
.
2
5
13
11.5
16
165
16
16
5
Counts
(%)
vs.
Acquisition
Time
(min)
Fig.
1.
Phenolic
compounds
profile
of
comfrey
root
sample:
A.
UV
chromatogram
recorded
at
280
nm;
B.
Total
ion
chromatogram.
Numbers
on
the
chromatograms
correspond
to
peaks
identified
in
Table
1.
cation
was
generated
by
incubation
of
equal
volumes
of
7
mM
ABTS
and
2.45
mM
potassium
persulfate
solutions
in
the
dark
at
room
tem-
perature,
for
12-16
h.
The
stock
ABTS
radical
cation
solution
was
then
diluted
with
ethanol
to
get
an
absorbance
of
0.70
±
0.02
at
734 nm.
Free
radical
scavenging
activity
was
determined
by
mixing
20
µL
of
comfrey
root
extract
(1.25-10
mg/mL
in
DMSO)
with
ABTS
radical
solution
in
a
total
volume
of
2
mL;
the
decrease
in
absorbance
was
measured
after
6
min.
The
scavenging
activity
was
estimated
by
the
following
formula:
ABTS
radical
cation
scavenging
activity
(%)
=
100
X
(Acontrol
Asample)/(Acontrol),
where
A
con
t
ro
i
is
the
absor-
bance
of
the
control
and
A
sam
pi
e
is
the
absorbance
in
the
presence
of
extract/positive
controls.
2.5.3.
Reducing
power
assay
The
assay
was
carried
out
using
the
method
of
Berker
et
al.
(2007).
Different
concentrations
(1.25-20
mg/mL
in
DMSO)
of
comfrey
root
extract
were
mixed
with
potassium
phosphate
buffer
(1.2
mL,
0.2
M,
pH
6.6)
and
1%
potassium
ferricyanide
(1.25
mL),
followed
by
incubation
at
50
°C
for
20
min.
Then,
10%
trichloroacetic
acid
(1.25
mL)
was
added
and
the
reaction
mixture
was
centrifuged
at
5000
rpm
for
10
min.
An
aliquot
of
1.25
mL
of
the
upper
layer
was
mixed
with
ultrapure
water
(1.25
mL)
and
0.1%
ferric
chloride
(0.25
mL).
After
90
s
the
absorbance
was
measured
at
700
nm.
Higher
absorbance
of
the
reaction
mixture
indicated
a
higher
reducing
power.
2.5.4.
15-Lipazygenase
inhibition
assay
The
assay
was
performed
using
a
previously
described
method
(Wangensteen
et
al.,
2004).
50
µL
of
comfrey
root
extract
(1.25-20
mg/
mL
in
DMSO)
were
mixed
with
the
same
volume
of
15-
lipoxygenase
(10,000
U/mL)
and
0.9
mL
of
borate
buffer
(0.2
M,
pH
9.0).
After
in-
cubation
at
25
°C
for
10
min
in
the
dark,
the
reaction
was
initiated
by
adding
the
substrate
-
linoleic
acid
(0.2
mM
in
borate
buffer;
2
mL).
The
absorbance
was
measured
at
234
nm,
after
30
and
90
s
reaction
time.
The
15-lipoxygenase
(15-LO)
inhibitory
activity
was
estimated
using
the
formula:
Inhibition
of
15-LO
(%)
=
100
x
[(C90
C30)
(T90
T
30)/(C90
C30)],
where
C30,
C90
and
T30,
T90
180
A
Trifan
et
al.
Food
and
Chemical
Toxicology
112
(2018)
178-187
are
the
absorbances
of
the
control
and
extract/positive
controls
after
30
and
90
s
reaction
time,
respectively.
In
all
antioxidant
assays,
butylhydroxyanisole,
gallic
acid,
and
caf-
feic
acid
were
used
as
positive
controls.
The
EC
50
values
were
calcu-
lated
by
linear
interpolation
between
values
above
and
below
50%
activity,
except
for
reducing
power
assay,
where
the
EC
50
values
are
the
effective
concentrations
at
which
the
absorbance
is
0.5
(Ferreira
et
al.,
2007).
2.6.
Pyrrolizidine
alkaloids
analysis
Analysis
was
performed
using
reversed-phase
high
performance
li-
quid
chromatography
with
electrospray
ionisation
mass
spectrometry
(RP-HPLC-ESI-MS/MS),
using
an
Agilent
1260
HPLC
system
(Agilent
Technologies,
USA)
equipped
with
a
binary
gradient
solvent
pump,
degasser,
autosampler
and
column
oven,
hyphenated
with
a
AB
Sciex
4500
QTRAP
mass
spectrometer
(AB
Sciex,
USA)
equipped
with
an
electrospray
ionisation
source
(ESI)
and
a
triple
quadrupole
mass
ana-
lyser
with
a
trapping
function.
Pyrrolizidine
alkaloids
separation
was
carried
out
at
40
°C
on
an
InfinityLab
Poroshell
120
EC-C18
column
(4.6
x
50
mm,
2.7
um
parti-
cles;
Agilent
Technologies,
USA)
with
a
mobile
phase
of
0.1%
formic
acid
in
water
(solvent
A)
and
acetonitrile
(solvent
B),
using
5µL
in-
jections.
The
flow
rate
was
1
mL
min
-1
and
the
gradient
was
0-1
min,
5%
B;
1-8
min,
5-20%
B;
8-12
min,
20-40%
B;
12-18
min,
40-95%
B;
18-21
min,
95%
B;
then
back
to
initial
conditions
in
4
min.
ESI
was
operated
in
the
positive
ion
mode:
capillary
temperature
500
°C,
curtain
gas
40
psi,
nebulizer
gas
40
psi,
ionspray
voltage
4500
V.
Nitrogen
was
used
as
curtain
and
collision
gas.
MRM
was
used
for
quantitative
ana-
lysis
of
pyrrolizidine
alkaloids.
The
data
was
acquired
and
processed
using
Analyst
1.6.2
software
(AB
Sciex,
USA).
Triplicate
injections
were
made
for
each
standard
and
for
the
sample.
Analytes
were
identified
by
comparing
retention
time
and
m/z
values
obtained
by
MS
and
MS
2
with
those
of
standards
obtained
under
the
same
conditions
and
when
not
available
by
comparison
with
literature
data
(Avula
et
al.,
2015;
Wuilloud
et
al.,
2004).
The
calibration
curves
obtained
in
MRM
mode
were
used
for
quantitation;
peak
areas
were
compared
with
calibration
curves
generated
by
three
repeated
injections
of
known
standards
at
six
concentrations
(0.2-20
ng/mL).
The
results
were
expressed
in
mg
per
g
of
comfrey
root
extract.
3.
Results
and
discussion
3.1.
Identification
and
quantitation
of
phenolic
compounds
in
comfrey
root
sample
Fig.
1
shows
the
phenolic
compounds
profile
of
comfrey
root
sample
as
typical
UV
chromatogram
at
280
nm
(A),
as
well
as
the
total
ion
chromatogram
(B).
Data
of
the
identified
compounds,
numbered
ac-
cording
to
their
elution time,
with
retention
time,
molecular
formulas
(neutral
form),
mass
of
the
molecular
ion,
MS/MS
main
fragment
ions
as
obtained
by
HPLC-DAD-QTOF-MS/MS
analysis
are
summarized
in
Table
1.
Rosmarinic
acid
(caffeic
acid
dimer)
was
identified
as
the
main
compound
in
the
comfrey
root
sample,
alongside
caffeic
acid
and
other
caffeic
acid
derivatives,
such
as
caffeic
acid
timers
(isomers
of
lithos-
permic
acid,
salvianolic
acid
A
and
C)
and
tetramers
(isomer
of
sal-
vianolic
acid
B)
(Fig.
1;
Table
1).
The
identification
of
phenolic
con-
stituents
was
achieved
by
comparison
of
their
UV,
mass
spectra,
fragmentation
pattern
and
retention
times
to
those
of
authentical
standards,
online
databases
and
literature
data
available
for
these
compounds
(Banos
et
al.,
2013;
Chen
et
al.,
2011a;
Liu
et
al.,
2007;
Yang
et
al.,
2015).
Peak
1
was
identified
as
caffeic
acid,
with
a
pseudo
molecular
ion
[M-H]
-
at
m/z
179
and
a
product
ion
at
m/z
135
(by
loss
of
a
carboxyl
group
-
44
mu)
(Oiarowski
et
al.,
2017).
Its
identity
was
confirmed
by
comparison
with
a
commercial
standard.
In
our
sample,
this
compound
was
found
in
trace
amounts,
in
contrast
to
other
reports
where
it
was
the
main
phenolic
constituent
of
Symphytum
officinale
roots
(Grabias
and
Swiatek,
1998;
Tahirovic
et
al.,
2010).
Peak
2
showed
a
pseudo
molecular
ion
[M-H]
-
at
m/z
537
with
a
similar
UV
spectra
and
fragmentation
pattern
as
lithospermic
acid
(Fig.
2a).
Compared
with
the
authentical
standard,
we
concluded
that
compound
2
is
a
lithospermic
acid
isomer,
namely
salvianolic
acid
I,
as
it
presents
a
proeminent
fragment
at
m/z
339,
which
shows
that
this
compound
easily
loses
a
danshensu
molecule
(3-(3,4-dihydroxyphenyl)-
lactic
acid)
(198
mu)
and
infirms
the
existence
of
the
benzofuran
ring
(Chen
et
al.,
2011a;
Liu
et
al.,
2007).
Lithospermic
acid
was
not
de-
tected
in
our
sample,
even
though
literature
reported
the
presence
of
this
caffeic
acid
timer
in
comfrey
root
extracts
(Barnes
et
al.,
2007;
Wagner
et
al.,
1970).
Two
peaks
showing
pseudo
molecular
ions
at
m/z
717
were
de-
tected
at
retention
times
of
10.35
min
(3)
and
11.97
min
(7)
(Table
1).
Similar
mass
spectra
and
fragments
were
reported
for
tetramers
of
caffeic
acid
such
as
salvianolic
acid
B
and
its
isomers
(Chen
et
al.,
2011a,
2011b;
Yang
et
al.,
2015).
The
MS
2
fragmentation
of
compounds
3
and
7
revealed
the
presence
of
a
characteristic
fragment
at
m/z
519
corresponding
to
the
loss
of
a
danshensu
moiety
(198
mu)
(Fig.
2b)
and
thus,
it
can
be
presumed
that
compounds
3
and
7
do
not
possess
a
benzofuran
structure
(Liu
et
al.,
2007).
Salvianolic
acid
B
was
not
present
in
our
sample,
confirmed
by
using
a
commercial
standard.
Therefore,
we
assigned
the
identity
of
compounds
3
and
7
as
corre-
sponding
to
isomers
of
salvianolic
acid
B.
Moreover,
their
elution
re-
lative
to
rosmarinic
acid
could
be
used
to
tentatively
assign
these
two
isomers.
Thus,
peak
3
eluting
before
rosmarinic
acid
could
be
salvia-
nolic
acid
E
and
peak
7
eluting
after
rosmarinic
acid
could
be
assigned
to
salvianolic
acid
L
(Liu
et
al.,
2007).
Peak
5
gave
a
pseudo
molecular
ion
[M-H]
-
at
m/z
359
and
yielded
characteristic
fragment
ions
at
m/z
197,
m/z
179
and
m/z
161
(Fig.
2c).
Its
identity
was
assigned
to
rosmarinic
acid,
a
dimer
of
caffeic
acid
(Oiarowski
et
al.,
2017),
confirmed
by
analysing
an
authentical
stan-
dard.
Rosmarinic
acid
was
also
reported
as
main
phenolic
compound
in
comfrey
roots
by
Grabias
and
Swiatek
(1998),
Sowa
et
al.
(2017),
and
Tahirovic
et
al.
(2010).
Peak
4
([M-H]
-
with
m/z
at
439)
showed
a
fragment
at
m/z
359
Table
1
Characterization
of
phenolic
compounds
in
comfrey
root
sample
by
HPLC-DAD-QTOF-MS/MS.
No.
RT
(min)
Assigned
identity
Chemical
formula
Masses
of
[M-1-1]
-
MS
2
Reference
(m/z)
(m/z)
1
3.64
Caffeic
add
C9H704
179.0345
135
Std.
2
9.18
Salvianolic
add
I
C27
14
21
0
12
537.1118
493,339,197,179,135
Chen
et
al.,
2011a
3
10.35
Salvianolic
add
B
isomer
C36
14
30016
717.1497
537,519,475,339,197
Liu
et
al.,
2007
4
10.56
Rosmarinic
acid
derivative
C19
14
20
0
12
439.0949
359,179,135
Barros
et
al.,
2013
5
11.10
Rosmarinic
acid
C18
14
1608
359.0797
197,179,161
Std.
6
11.45
Salvianolic
add
A
isomer
C261122010
493.0985
265,185
Chen
et
al.,
2011a
7
11.94
Salvianolic
add
B
isomer
C36
14
30016
717.1442
519,475,339
Liu
et
al.,
2007
8
13.44
Salvianolic
add
C
isomer
C261
-
120010
491.1018
311,267,197,179,135
Liu
et
al.,
2007
181
A
Trifan
et
al.
Food
and
Chemical
Toxicology
112
(2018)
178-187
(rosmarinic
acid),
resulting
from
the
loss
of
a
sulphate
moiety
(80
mu).
In
addition,
the
presence
of
fragments
at
m/z
197
and
m/z
179
for
this
peak,
which
were
also
observed
for
peak
5,
allowed
its
putative
iden-
tification
as
a
sulphated
derivative
of
rosmarinic
acid
(Banos
et
al.,
2013).
Peak
6
gave
a
pseudo
molecular
ion
[M-Hr
at
m/z
493
and
yielded
prominent
fragment
ions
at
m/z
295
and
m/z
185,
with
the
successive
loss
of
danshensu
(198
mu)
and
3,4-dihydroxyphenyl
moiety
(110
mu),
respectively
(Fig.
2d).
By
comparison
with
an
authentical
standard
of
salvianolic
acid
A,
peak
6
was
tentatively
identified
as
its
isomer
(Chen
et
al.,
2011a;
Liu
et
al.,
2007).
Peak
8
showed
a
pseudo
molecular
ion
[M-HF
at
m/z
491
and
X10
5
4,6-
4,4-
4,2-
4-
3,8-
3,6-
3,4-
3,2-
3-
2,8-
2,6-
2,4-
2,2-
2-
1,8-
1,6-
1,4-
1,2-
1-
0,8-
0,6-
0,4-
0,2-
0-
a)
OH
339.05
OH
HOOC
197.05
OH
COOH
295.06
HO
135.05
OH
493.12
72.99
229.01
383.08
t
50
100
150
200
250
300 350
400
450
500
550
600
650
700 750
800
850
900
950
Counts
vs.
Mass-to-Charge
(m/z)
b)
OH
519.1
OH
HOOC
475.1
HOOC
OH
OH
HO
OH
339.0
717.1
365.1
109.0
197
.
0
243.0
295
.
1
431.1
L
L
X10
5
3,6-
3,4-
3,2-
3-
2,8-
2,6-
2,4-
2,2-
2-
1,8-
1,6-
1,4-
1,2-
1-
0,8-
0,6-
0,4-
0,2-
0-
50
100 150
200
250
300
350
400
450
500
550
600 650
700 750
800
850
900
950
1000
Counts
vs.
Mass-to-Charge
(m/z)
Fig.
2.
MS
2
spectra
and
proposed
fragmentation
pattern
for
compounds
2
(a),
3
(b),
5
(c),
6
(d),
8
(e).
182
A.
Trifan
et
al.
Food
and
Chemical
Toxicology
112
(2018)
178-187
c)
161.0
OH
OH
OH
-180
HO
-162
-198
OH
197.0
73.0
135.0
L.
50
100 150
200 250
300
350
400 450
500 550
600
650
700
750
800
850
900
950
1000
Counts
vs.
Mass-to-Charge
(m/z)
d)
HO
295.1
HO
OH
185.0
OH
OH
OH
OH
109.0
135.0
73.0
269.1
493.1
50
100 150
200
250
300
350
400 450
500
550
600
650
700
750
800
850
900
950
1000
Counts
vs.
Mass-to-Charge
(m/z)
Fig.
2.
(continued)
x10
5
4,6
-
4,4-
4,2-
4
-
3,8-
3,6-
3,4-
3,2-
3
-
2,8-
2,6-
2,4-
2,2-
2
-
1,8-
1,6-
1,4-
1,2-
1
-
0,8
-
0,6-
0,4-
0,2-
0-
x10
4
3,6-
3,4-
3,2-
3
-
2,8-
2,6-
2,4-
2,2-
2-
1,8-
1,6-
1,4-
1,2-
1-
0,8-
0,6-
0,4
-
0,2
-
0
-
183
A
Trifan
et
al.
Food
and
Chemical
Toxicology
112
(2018)
178-187
e)
311.1
HO HO
OH
OH
OH
OH
491.1
135.0
197.0
267.1
x10
5
2,6-
2,4-
2,2-
2-
1,8-
1,6-
1,4-
1,2-
0,8-
0,6-
0,4-
0,2-
0
-
50
100
150
200 250
300
350
400
450
500
550
600 650
700
750
800
850
900
950
Counts
vs.
Mass-to-Charge
(m/z)
Fig.
2.
(continued)
Table
2
Major
phenolic
compounds
quantification
(mg/g
extract)
in
comfrey
root
sample.
Peak
Compound
Content
(mg/g)
2
Salvianolic
acid
1'
6.182
±
0.003
3
Salvianolic
acid
B
isomer'
4.796
±
0.002
5
Rosmarinic
add
7.557
±
0.005
6
Salvianolic
acid
A
isomer'
0.649
±
0.000
8
Salvianolic
acid
C
isomer
d
6.750
±
0.000
Expressed
as
lithospermic
acid
equivalents.
b
Expressed
as
salvianolic
add
B
equivalents.
Expressed
as
salvianolic
acid
A
equivalents.
d
Expressed
as
salvianolic
acid
C
equivalents.
fragmented
into
a
characteristic
fragment
ion
at
m/z
311,
corre-
sponding
to
the
loss
of
a
caffeic
acid
moiety
(180
mu)
(Fig.
2e).
The
mass
spectra
and
the
predominant
fragment
were
consistent
with
known
trimers
of
caffeic
acid,
namely
salvianolic
acid
C
and
its
isomers
(Liu
et
al.,
2007;
Yang
et
al.,
2015).
Salvianolic
acid
C
was
not
found
in
our
sample,
as
assayed
by
using
a
commercial
standard;
therefore,
we
could
tentatively
identify
peak
8
as
an
isomer
of
salvianolic
acid
C.
Total
phenolic
content
in
comfrey
sample
was
determined
by
Folin-
Ciocalteu
method,
and
revealed
a
content
of
73.69
±
0.32
mg
caffeic
acid
equivalents
per
g
of
comfrey
root
extract.
Main
phenolic
acids
identified
in
comfrey
root
sample
were
further
quantified
by
HPLC-
QQQ-MS/MS
(Table
2).
Rosmarinic
acid
(peak
5)
content
was
calcu-
lated
based
on
the
calibration
curve
obtained
for
its
respective
com-
mercial
standard.
The
other
four
compounds
were
tentatively
quanti-
fied
on
the
basis
of
calibration
curves
from
other
compounds
with
structural
similarities.
The
lithospermic
acid
standard
curve
was
used
for
the
quantification
of
salvianolic
acid
I
(2).
Compound
3,
isomer
of
salvianolic
acid
B,
was
quantified
using
a
salvianolic
acid
B
calibration
curve.
Compound
6,
isomer
of
salvianolic
acid
A,
was
expressed
as
salvianolic
acid
A
equivalents.
Salvianolic
acid
C
was
used
for
the
quantification
of
compound
8
tentatively
identified
as
a
salvianolic
acid
C
isomer.
The
quantitative
results
showed
that
the
most
abundant
compound
in
the
comfrey
root
sample
was
rosmarinic
acid
(7.557
±
0.005
mg/g
extract).
If
we
consider
the
amount
of
rosmarinic
acid
reported
to
root
dry
weight,
the
obtained
value
-1.77
mg/g
root
dry
weight
-
is
similar
to
that
previously
reported
by
Sowa
et
al.
(2017)
(1.85
mg/g
root
dry
weight);
however,
Tahirovic
et
al.
(2010)
determined
lower
values
for
rosmarinic
acid
(0.85
mg/g
root
dry
weight)
in
a
water
extract
obtained
from
roots
of
S.
officinale.
This
variation
might
be
assigned
to
different
extraction
conditions
but
also
to
spatial
and
temporal
environmental
variability
(sunlight,
temperature,
soil
type,
nutrients,
climate),
and
plant
developmental
state
which
influence
the
synthesis
of
specific
compounds.
Regarding
the
content
in
salvianolic
acids,
salvianolic
acid
I
and
C
showed
similar
amounts,
followed
by
salvianolic
acid
B
isomer,
and
to
a
lesser
extent,
by
the
isomer
of
salvianolic
acid
A
(Table
2).
The
main
phenolic
compound
rosmarinic
acid
an
ester
of
caffeic
acid
and
3,4-dihydroxyphenyllactic
acid,
commonly
found
in
species
of
the
Lamiaceae
(Nepetoideae
subfamily)
and
the
Boraginaceae
(Kim
et
al.,
2015).
This
dimer
of
caffeic
acid
is
known
for
its
numerous
biological
activities
proven
in
cultured
cells
and
animal
models,
such
as
anti-inflammatory
effects
via
inhibition
of
expression
of
interleuldn
(IL)-6,
IL-1j3,
tumor
necrosis
factor
(TNF)-a
and
nuclear
factor
kappa
B
(NF-KB)
activation
(Sotnikova
et
al.,
2013);
antioxidant
activity
by
up-
regulation
of
glutathione
and
superoxide
dismutase
(Kim
et
al.,
2005);
antitumor
and
anti-metastasis
effects
via
down-regulation
of
matrix
metalloproteinase
(MMP)-2,
MMP-9
expression
and
inhibition
of
ad-
hesion,
migration
and
invasion
in
cancer
cells
(Xu
et
al.,
2010a,
2010b);
antimicrobial
(active
against
different
pathogenic
prokaryotic
micro-
organisms,
both
gram-positive
and
gram-negative)
(Abedini
et
al.,
2013)
and
anti-allergic
activity
via
inhibition
of
immunoglobulin
E
and
histamine
production
(Oh
et
al.,
2011).
Herein,
we
report
for
the
first
time
the
presence
of
salvianolic
acids
A,
B
and
C
isomers
in
S.
offwinale
root.
Salvianolic
acids
are
poly-
phenolic
acids,
conjugates
of
3,4-dihydroxyphenyllactic
acid
and
184
Comfrey
root
extract
Butylhydroxyanisole
Gallic
acid
Caffeic
add
80.25
±
0.42
12.66
±
0.11
1.64
±
0.05
3.63
±
0.02
±
0.13
32.75
±
0.59
63.68
±
1.55
±
0.05
4.30
±
0.06
78.66
±
0.45
±
0.07
1.58
±
0.00
39.06
±
0.75
±
0.03
2.01
±
0.00
25.54
±
0.28
20.14
1.76
0.52
1.67
A
Trifan
et
al.
Food
and
Chemical
Toxicology
112
(2018)
178-187
caffeic
acid,
mostly
known
as
major
hydrophilic
constituents
of
roots
and
rhizome
of
danshen
(Salvia
milthiorrhiza
Bunge,
Lamiaceae)
(Wang,
2010).
Danshen
is
widely
used
in
traditional
Chinese
medicine
for
the
treatment
of
coronary
artery/cerebrovascular
disease
and
inflammatory
conditions
(Ho
and
Hong,
2011).
Recently,
several
reports
pointed
out
danshen
as
a
source
of
lead
compounds
for
developing
new
drugs
against
cardio-cerebrovascular
diseases
and
Alzheimer's
disease
(Chen
et
al.,
2011a;
Liu
et
al.,
2007;
Wang,
2010;
Zhang
et
al.,
2016).
Sal-
vianolic
acid
B
was
intensively
studied
in
animal
models;
thus,
it
proved
cardioprotective
activity
via
anti-apoptotic
and
anti-inflammatory
ef-
fects
by
silent
information
regulator
(SIRT)-1
activation
in
experimental
stroke
rats
(Lv
et
al.,
2015);
anti-hyperalgesic
activity
was
shown
in
an
animal
model
of
neuropathic
pain
using
liposomal
formulations
of
salvianolic
acid
B
(Isacchi
et
al.,
2011);
neuroprotective
effects
via
anti-
inflammatory
activity
in
microglia
(inhibition
of
nitric
oxide,
TNF-a,
IL-
IA
and
reactive
oxygen
species
induced
by
lipopolysaccharide)
was
determined
in
rats
(Wang
et
al.,
2010b);
anti-steatotic
and
anti-in-
flammatory
effects
were
shown
in
rats
(inhibition
of
TNF-a,
IL-6)
by
inducing
SIRT-1
mediated
inhibition
of
C
reactive
protein
and
lipo-
protein
carbohydrate
response
element
binding
protein
expression
(Zhang
et
al.,
2017).
Also,
salvianolic
acid
A
possesses
protective
effects
against
acute
ischemic
stroke
by
imparing
the
NF-KB
signal
in
rats
(Chien
et
al.,
2016);
also,
it
proved
cardiovascular
protection
by
mod-
ulation
of
CD36
receptors
of
oxidized
low
density
lipoproteins
(oxLDL),
with
a
lesser
uptake
of
oxLDL
by
the
macrophage
(Wang
et
al.,
2010a).
Thus,
comfrey
root
can
be
considered
an
important
source
of
phe-
nolic
compounds
such
as
rosmarinic
acid
and
salvianolic
acids,
espe-
cially
exhibiting
antioxidant
and
anti-inflammatory
effects;
this
could
translate
into
a
significant
contribution
of
phenolic
constituents
to
the
overall
bioactivity
of
Symphytum
derived
preparations.
3.2.
Antioxidant
activity
In
vitro
antioxidant
testing
was
done
in
order
to
estimate
the
puta-
tive
contribution
of
phenolic
compounds
to
the
biological
activities
of
comfrey
roots.
It
is
known
that
antioxidant
activity
exhibited
by
phe-
nolic
compounds
is
related
to
various
beneficial
health
effects
espe-
cially
in
the
case
of
oxidative
stress-related
diseases
(Pandey
and
Rizvi,
2009);
therefore
screening
of
such
effects
is
of
great
importance
in
our
attempt
to
identify
bioactive
components
of
comfrey
root
extract.
Antioxidant
activities
are
attributed
to
the
multifunctional
proper-
ties
of
phenolics
which
may
act
as
radical
scavenging,
reducing
agents
and
transition
metal
ions
chelators
(Prior
et
al.,
2005).
The
antioxidant
activity
of
comfrey
root
sample
was
assessed
by
four
different
in
vitro
antioxidant
assays
and
compared
with
the
activity
of
commonly
used
positive
controls,
such
as
butylhydroxyanisole,
gallic
acid,
and
caffeic
acid.
Effects
of
comfrey
root
extract
in
these
antioxidant
tests
are
shown
in
Table
3.
DPPH
and
ABTS
assays
are
standard
colorimetric
methods,
used
for
the
assessment
of
free
radical
scavenging
ability
of
antioxidants.
Due
to
their
hydrogen
donating
ability,
phenolics
reduce
the
free
radical
DPPH
to
DPPH
2
,
with
a
decrease
in
absorbance
at
517
nm
(Malterud
et
al.,
1993).
Comfrey
root
extract
exhibited
good
DPPH
scavenging
activity
(EC
50
=
80.25
±
0.42
vg/mL)
when
compared
with
positive
controls,
which
increased
in
a
concentration-dependent
manner
(data
not
shown).
The
ABTS
cation
radical
scavenging
assay
is
based
on
the
ability
of
antioxidants
to
reduce
the
preformed
ABTS•
+
,
a
blue-green
chromophore
with
characteristic
absorption
at
734
nm,
to
ABTS,
causing
radical
decolourization
(Re
et
al.,
1999).
The
EC
50
value
(EC
50
=
20.14
±
0.13
vg/mL)
demonstrated
significant
ABTS
radical
scavenging
activity
for
comfrey
extract.
The
reducing
power
assay
was
used
to
evaluate
the
ability
of
phe-
nolics
in
comfrey
root
extract
to
donate
an
electron,
thus
reducing
the
Fe
3+
/ferricyanide
complex
to
the
ferrous
form
(Perl's
Prussian
blue),
spectrophotometrically
quantified
at
700
nm
(Berker
et
al.,
2007).
Si-
milar
to
results
obtained
in
the
radical
scavenging
assays,
comfrey
ex-
tract
showed
noticeable
reducing
power,
with
EC
50
values
(32.75
±
0.59
vg/mL)
approximately
eight-fold
higher
than
positive
control
butylhydroxyanisole
(EC
50
=
4.30
±
0.06
vg/mL).
The
lipoxygenases
catalyze
the
hydroxyperoxidation
of
poly-
unsaturated
fatty
acids
and
are
involved
in
the
etiology
and
progress
of
several
inflammatory
diseases
(Brash,
1999).
Among
them,
15-LO
is
strongly
related
to
the
pathogenesis
of
asthma
and
chronic
bronchitis,
glomerular
inflammation
and
atherosclerosis
(Conrad,
1999).
Comfrey
extract
showed
significant
15-LO
inhibitory
activity
(EC
50
=
63.68
±
1.55
vg/mL),
even
higher
than
that
of
the
known
antioxidant
butylhydroxyanisole
(EC
50
=
78.66
±
0.45
vg/mL).
Thus,
phenolics
from
comfrey
could
be
regarded
as
potential
15-LO
in-
hibitors,
which
may
contribute
to
the
anti-inflammatory
effects
of
root
extracts.
A
direct
comparison
of
our
results
obtained
in
the
four
antioxidant
assays
with
those
reported
in
other
studies
for
comfrey
root
(Neagu
et
al.,
2010;
Paun
et
al.,
2012;
Sowa
et
al.,
2017)
is
not
feasible
due
to
the
fact
that
different
methods
and
different
ways
of
expressing
anti-
oxidant
results
are
used.
Nevertheless,
major
phenolic
compounds
identified
in
comfrey
root
extract,
such
as
rosmarinic
acid
and
salvia-
nolic
acids,
are
known
as
potent
antioxidants
which
possess
the
ability
to
scavenge
free
radicals
and
chelate
metal
ions
(Erkan
et
al.,
2008;
Zhao
et
al.,
2008).
To
summarize,
comfrey
root
sample
exhibited
significant
in
vitro
antioxidant
activity,
as
assayed
by
DPPH
radical
and
ABTS
cation
ra-
dical
scavenging
assays,
reducing
power
and
15-LO
inhibition,
sug-
gesting
that
phenolic
compounds
may
play
a
major
role
in
antioxidant
activities
of
comfrey
root
extract.
3.3.
Identification
and
quantification
of
pyrrolizidine
alkaloids
in
comfrey
root
sample
Although
our
research
focused
mainly
on
phenolic
compounds
from
comfrey
root,
pyrrolizidine
alkaloids
profile
and
their
amount
in
the
sample
were
also
investigated
by
means
of
HPLC-QQQ-MS/MS.
This
technique
allows
a
good
separation,
fragmentation
and
quantification
of
specific
masses,
thus
providing
a
high
degree
of
selectivity
and
structural
information
of
the
analyzed
molecules
(Wuilloud
et
al.,
2004).
Thereby,
analytes
were
identified
by
comparing
retention
time
and
m/z
values
obtained
by
MS
and
MS
2
with
those
of
standards
ob-
tained
under
the
same
conditions
or
when
not
available
by
comparison
with
literature
data
(Avula
et
al.,
2015;
Wuilloud
et
al.,
2004).
Major
Table
3
Effects
of
comfrey
root
sample
in
different
antioxidant
assays.
Extract/
DPPH
radical
scavenging
assay
ABTS
cation
radical
Reducing
power
assay
15-LO
inhibition
assay
Positive
control
EC
50
scavenging
assay
EC
5O
'
EC
50
EC
5O
'
pg/mL
extract.
185
A
Trifan
et
al.
Table
4
Major
pyrrolizidine
alkaloids
identified
in
comfrey
root
sample.
Food
and
Chemical
Toxicology
112
(2018)
178-187
RT
(min)
Assigned
identity
[M+H]
+
MS
2
References
(m/z)
(m/e)
3.38
Intermedine
300
156,138,120
Std.
3.59
Lycopsamine
300
156,138,120
Std.
4.37
Intermedine
N-oxide
316
172,156,138
Std.
4.55
Lycopsamine
N-oxide
316
172,156,138
Std.
6.40
Acetylintermedine
342
198,180,120
Std.
6.46
Acetyllycopsamine
342
198,180,120
Std.
6.49
Acetylintermedine
N-oxide
358
340,214,180,154,138,120
Std.
6.65
Acetyllycopsamine
N-oxide
358
340,214,180,154,138,120
Std.
10.17
Symphytine
isomer
382
238,220,154,138,120
Wuilloud
et
al.,
2004
10.55
Symphytine
N-oxide
isomer
398
380,254,236,154,138,120
Wuilloud
et
al.,
2004
Table
5
Pyrrolizidine
alkaloids
quantification
(mg/g
extract)
in
comfrey
root
sample.
RT
(min)
Alkaloid
Content
(mg/g)
3.38
Intermedine
0.142
0.003
3.59
Lycopsamine
0.146
0.009
4.37
Intermedine
N-oxide
0.027
0.001
4.55
Lycopsamine
N-oxide
0.031
0.002
6.40
Acetylintermedine
3.667
0.088
6.46
Acetyllycopsamine
3.049
0.097
6.49
Acetylintermedine
N-oxide
0.979
0.074
6.65
Acetyllycopsamine
N-oxide
1.040
0.069
10.17
Symphytine
isomers'
0.008
0.000
10.55
Symphytine
N-oxide
isomers"
0.146 0.006
Total
content
9.235
Expressed
as
acetyllycopsamine
equivalents.
b
Expressed
as
acetyllycopsamine
N-oxide
equivalents.
pyrrolizidine
alkaloids
identified
in
comfrey
root
sample
are
presented
as
the
pseudo
molecular
ion
[M
+
H]
+
and
characteristic
MS
2
fragments
in
Table
4.
Thus,
intermedine,
lycopsamine
and
their
N-oxides,
acet-
ylintermedine,
acetyllycopsamine
and
their
N-oxides
were
identified
by
comparison
with
commercially
available
standards.
Isomers
of
sym-
phytine
and
symphytine
N-oxide
were
identified
by
comparison
of
their
fragmentation
pattern
with
literature
data
(Wuilloud
et
al.,
2004).
Quantification
of
pyrrolizidine
alkaloids
was
made
using
a
targeted
approach
(MRM),
in
order
to
minimize
the
matrix
effects,
but
also
to
overcome
the
drawbacks
due
to
the
presence
of
isomers
and
their
poor
separation.
Contents
of
intermedine,
lycopsamine
and
their
N-oxides,
acetylintermedine,
acetyllycopsamine
and
their
N-oxides
were
calcu-
lated
based
on
the
calibration
curve
obtained
for
their
respective
commercial
standards.
Isomers
of
symphytine
and
symphytine
N-oxide
were
tentatively
quantified
on
the
basis
of
calibration
curves
from
other
compounds
with
structural
similarities.
Therefore,
the
acet-
yllycopsamine
standard
calibration
curve
was
used
for
the
quantifica-
tion
of
symphytine
isomer
and
the
acetyllycopsamine
N-oxide
calibra-
tion
curve
was
used
for
the
quantification
of
symphytine
N-oxide
isomer.
Acetylintermedine
and
acetyllycopsamine
were
found
as
main
components
in
comfrey
root,
followed
by
their
N-oxides
(Table
5).
Brauchli
et
al.
(1982)
also
showed
that
these
compounds
are
the
major
constituents
in
comfrey
root.
Total
alkaloid
content
was
9.235
mg/g
extract
(meaning
0.24%
root
dry
weight),
which
is
in
accordance
with
literature
data
(0.013-1.2%)
(Staiger,
2012).
4.
Conclusions
Symphytum
officinale
root
is
still
widely
used
as
a
phytoremedy
and
its
therapeutic
application
should
not
be
overshadowed
by
the
toxicity
concerns
related
to
its
pyrrolizidine
alkaloids
content.
Nowadays
only
pyrrolizidine-depleted
extracts
are
used
in
topical
medicinal
products,
which
is
why
the
health
benefits
of
this
phytotherapeutic
product
should
be
considered.
Herein,
we
report
for
the
first
time
the
presence
of
salvianolic
acids
A,
B
and
C
isomers
in
S.
officinale
root.
More,
we
intend
to
isolate
these
major
phenolic
compounds
that
have
been
only
tentatively
identified
by
HPLC-QTOF-MS/MS
and
to
determine
their
chemical
structure
by
nuclear
magnetic
resonance
spectroscopy.
To
summarize,
comfrey
root
is
an
important
source
of
phenolic
compounds
such
as
rosmarinic
acid
and
salvianolic
acids
endowed
with
antioxidant
activity
which
may
contribute
to
the
overall
bioactivity
of
Symphytum
derived
preparations.
Conflicts
of
interest
The
authors
declare
no
conflict
of
interest.
The
funding
sponsors
had
no
role
in
the
design
of
the
study;
in
the
collection,
analysis,
or
interpretation
of
data;
in
the
writing
of
the
manuscript,
or
in
the
de-
cision
to
publish
the
results.
Acknowledgements
We
thank
Alpinamed
AG
and
Zurich
University
of
Applied
Sciences
for
financial
support.
Transparency
document
Transparency
document
related
to
this
article
can
be
found
online
at
http://dx.doi.org/10.1016/j.fct.2017.12.051.
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