Indus ial
C ops
and
P oduc s
45 (2013) 170–
181
Con en s
lis s
a ailable
a
SciVe se
ScienceDi ec
Indus ial
C ops
and
P oduc s
jou na
l
h
o
me
pag
e:
www.else ie .com/loca e/indc op
A emisia
annua
L.:
Essen ial
oil
and
ace one
ex ac
composi ion
and
an ioxidan
capaci y
Sand a
C.
Gou eia∗,
Paula
C.
Cas ilho
Cen o
de
Química
da
Madei a,
CCCEE,
Uni e sidade
da
Madei a,
Campus
Uni e si á io
da
Pen eada,
piso
0,
9000-390
Funchal,
Po ugal
a
i
c
l
e
i
n
o
A icle
his o y:
Recei ed
26
May
2012
Recei ed
in
e ised
o m
21
Sep embe
2012
Accep ed
17
Decembe
2012
Keywo ds:
A emisia
annua
Ca eoylquinic
acids
Phenolic
Essen ial
oil
An ioxidan
a
b
s
a
c
Ae ial
pa s
o
A emisia
annua
g ow h
in
h ee
di e en
loca ions
o
Madei a
A chipelago
we e
s udied.
The
essen ial
oil
composi ion
was
es ablished
by
GC-MS
and
he
main
componen s
we e
mono-
and
sesqui e penes;
a emisia
ke one
was
no
de ec ed.
The
p esence
o
phenolic
compounds
in
he
ace one
ex ac s
was
in es iga ed
by
HPLC-DAD-ESI/MSn
and
a
di e sified
phenolic
p ofile
o
40
hyd ocinnamic
acid
de i a i es
and
glycosyla ed
fla onoids
was
ound.
A
ew
compounds
we e
epo ed
o
he
fi s
ime
in
A emisia
annua.
The
an ioxidan
capaci y
o
essen ial
oils
and
ex ac s
we e
measu ed
by
h ee
di e en
in
i o
assays.
Fo
he
essen ial
oils,
a
e y
good
an ioxidan
esponse
was
ound
and
he
ex ac s
also
showed
a
good
an ioxidan
capaci y,
in
pa icula
as
an i adical
sca enge s.
© 2012 Else ie B.V. All igh s ese ed.
1.
In oduc ion
The
genus
A emisia
( amily
o
As e aceae)
includes
mo e
han
300
species,
mainly
small
he bs
and
sh ubs
(Yoon
e
al.,
2011).
The e
a e
se e al
epo s
(Ca alho
e
al.,
2011)
desc ibing
A emisia
plan s
as
die a y
oods
and
as
adi ional
he bal
medicines
agains
inflamma o y
diseases,
in ec ions
by
ungi,
bac e ia
and
i uses,
gas ic
ulce ,
cance
among
o he s.
One
o
he
mos
s udied
A emisia
plan s
is
A emisia
annua
L.,
commonly
known
as
“qinghao”
o
“annual
wo mwood”.
I
is
a
plan
used
o
many
cen u ies
in
Chinese
olk
medicine
o
he
ea men
o
mala ia
and
e e
and
se e al
bioac i e
me abo-
li es
ha e
been
epo ed,
he
mos
impo an
o
hem
being
a emisinin,
an
endope oxide
sesqui e pene
lac one
(Cas ilho
e
al.,
2008).
A emisia
annua
lea es
ha e
a
high
con en
o
essen ial
oil
(EO)
con aining
cineole,
␣-pinene,
camphene,
bo neol,
campho ,
ge mac ene-D
and
a emisia
ke one
(Bo a
and
Sha ma,
2011).
The
essen ial
oil
o
A emisia
annua
is
e e enced
as
ha ing
an i ungal
and
an imic obial
ac i i y
(Ju eau
e
al.,
2002;
Ve ma
e
al.,
2011).
In
addi ion
o
hese,
o he
biological
p ope ies
a e
associa ed
o
mo e
pola
ex ac s
o
A emisia
annua
such
∗Co esponding
au ho
a :
Kemihuse ,
Linnaeus
äg
10,
KB6A14,
Umeå
uni e -
si e ,
SE-901
87
Umeå,
Sweden.
Tel.:
+46
907865182.
E-mail
add ess:
[email p o ec ed] (S.C.
Gou eia).
as
an imala ial,
an ibac e ial,
an i-inflamma o y,
an i umo
and
an iulce ogenic
(Bhakuni
e
al.,
2001; ´
Ca a
e
al.,
2012).
The
an ioxidan
capaci y
e e ed
in
he
li e a u e
is
associa ed
o
he
high
fla onoid
con en
and
di e sified
ypes
o
compounds
(B isibe
e
al.,
2009;
Fe ei a
e
al.,
2010).
Also,
in
i o
an i-
mala ial
and
an icance
ac i i y
o
a emisinin
and
i s
de i a i es
is
enhanced
by
he
p esence
ce ain
fla onoids
(Fe ei a
e
al.,
2010).
The
phenolic
composi ion
o
A emisia
annua
has
been
desc ibed
using
HPLC-MS/MS
echniques
by
(Lai
e
al.,
2007),
(Han
e
al.,
2008)
and
(Ca bona a
e
al.,
2012);
Ca alho
and
co-wo ke s
(Ca alho
e
al.,
2011)
epo ed
he
HPLC-DAD
quan ifica ion
o
se e al
phe-
nolic
compounds.
The
main
phenolic
compounds
epo ed
we e
fla onoids
and
hyd oxycinnama es.
Based
on
li e a u e
su ey,
he
chemical
composi ion
and
biological
p ope ies
o
A emisia
annua
can
ex en-
si ely
di e
acco ding
o
hei
geog aphical
o igin
and
how
he
plan
ma e ial
is
p ocessed.
The
in e es
in
he
po en ial
applica-
ions
o
his
plan
is
s ill
inc easing
and
in
he
p esen
s udy
we
analysed
A emisia
annua
plan s
g own
in
Madei a
A chipelago,
p e iously
epo ed
as
ha ing
ca.
1%
o
a emisin
(Cas ilho
e
al.,
2008).
The
main
goals
we e
o
es ablish
he
phenolic
composi ion
o
c ude
ace one
and
me hanol
ex ac s
by
high-pe o mance
liquid
ch oma og aphy
wi h
diode
a ay
de ec ion
coupled
wi h
mass
spec ome y
(HPLC-DAD-ESI/MSn);
he
EO
composi ion
by
GC-
MS
and
he
an ioxidan
capaci y
o
he
EO
and
he
ex ac s
we e
e alua ed
by
h ee
di e en
assays
(DPPH,
ABTS
and
FRAP).
0926-6690/$
–
see
on
ma e ©
2012 Else ie B.V. All igh s ese ed.
h p://dx.doi.o g/10.1016/j.indc op.2012.12.022
S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181 171
2.
Ma e ials
and
me hods
2.1.
Chemicals
and
s anda ds
The
ollowing
eagen s
we e
pu chased
om
Me ck
(Da ms ad ,
Ge many):
disodium
phospha e
dodecahyd a ed
(99%),
po assium
pe sul a e
(99%),
e ous
sul a e
hep ahyd a e
(99%),
glacial
ace ic
acid
(100%),
sodium
ca bona e
(p.a.)
and
sodium
chlo ide
(99.5%).
2,2-diphenyl-1-pic ylhyd azyl
(DPPH)
(>95%),
T olox
(≥99.8%,
HPLC),
2,2azinobis-(3-e hylbenz hiazoline-6-sul onic
acid)
(ABTS)
(≥99%,
HPLC),
2,4,6-T i(2-Py idyl)-s- iazine
(TPTZ)
(≥99.0%,
TLC)
and
Folin-Ciocal eu’s
phenol
eagen
we e
pu chased
om
Fluka
(Lisbon,
Po ugal).
Po assium
chlo ide
(>99.5%),
gallic
acid
(99%,
HPLC),
po assium
ace a e
(p.a.),
u in
(≥98%,
HPLC)
and
e ic
chlo ide
hexahyd a e
(97–100%)
we e
pu chased
om
Pan eac
(Ba celona,
Spain);
po assium
dihyd ogen
phospha e
(99.5%),
alu-
minium
chlo ide
(98%)
and
sodium
ace a e
ihyd a e
(pu e)
we e
pu chased
om
Riedel-de
Haën
(Hano e ,
Ge many).
All
sol en s
used
o
plan
ex ac ion
we e
AR
g ade,
pu chased
om
Fishe
(Lisbon,
Po ugal).
HPLC-MS
g ade
ace-
oni ile
(99.9%,
LabScan,
Gliwice,
Poland)
and
ul a-pu e
wa e
(Milli-Q
Wa e s
pu ifica ion
sys em,
EUA)
we e
used
o
HPLC
analysis.
S anda ds
p epa ed
in
e hanol
(100
␮g/mL):
apigenin
(>99%),
lu eolin
(>99%),
que ce in
(>99%),
p-couma ic
acid
(>99%)
and
ca -
eic
acid
(>99%)
om
Ex asyn hese
(Lyon,
F ance),
kaemp e ol
(>99%)
and
5-O-ca eoylquinic
acid
(99%)
om
Ac os
O ganics
(Geel,
Belgium).
1,3-O-dica eoylquinic
acid,
1,5-O-dica eoylquinic
acid,
3,4-O-dica eoylquinic
acid,
3,5-O-dica eoylquinic
acid,
4,5-
O-dica eoylquinic
acid
and
3,4,5-O- ica eoylquinic
acid
(>98%
by
HPLC
o
all)
we e
ob ained
om
Chengdo
Biopu i y
Phy ochemi-
cals,
L d
China
(Sichuan,
China).
2.2.
Plan
ma e ial
and
sample
p epa a ion
Samples
o
A emisia
annua
we e
ob ained
om
seeds
o
a emisinin
ich
hyb ids
(CPQBA
×
POP)
kindly
o e ed
by
UNI-
CAMP,
B azil
and
cul i a ed
in
h ee
di e en
loca ions
o
Madei a
A chipelago
(P eces,
Ja dim
Bo ânico
and
Po o
San o).
Be o e
flowe ing,
he
whole
plan s
we e
cu
and
d ied.
Plan s
g own
in
Madei a
Bo anical
Ga den
(Ja dim
Bo ânico)
expe imen-
al
field
eached
abou
3.5
m
high.
These
plan s
we e
di ided
in o
wo
lo s
and
one
o
hem
was
d ied
unde
di ec
sunligh
in
o de
o
maximize
a emisinin
p oduc ion
(Cas ilho
e
al.,
2008).
Lea es
om
he
op
hi d
o
plan s
we e
p ocessed
sepa a ely
om
hose
o
he
es
o
he
plan .
Vouche s
we e
deposi ed
in
he
Madei a
Bo anical
Ga den
He ba ium
collec ion.
The
d ied
plan
ma e ial
was
g ound
o
fine
powde
in
a
mechanic
g inde .
Po ions
o
his
powde ed
plan
we e
sepa a ely
ex ac ed
by
solid
liquid
ex ac ion,
using
wo
sol en s
(ace one
and
me hanol):
The
plan
ma e ial
(50
g)
was
mace a ed
wi h
500
mL
o
sol en ,
a
oom
empe a u e,
o
24
h.
In
all
cases,
he
solu ions
we e
fil e ed
and
concen a ed
unde
educed
p essu e
in
a
o a y
e apo a o
(40 ◦C)
and
kep
in
he
da k
a
−20 ◦C
un il
es ed.
Essen ial
oils
we e
ob ained
by
hyd odis illa ion
in
a
Cle enge
ype
appa a us.
2.3.
Essen ial
oil
analysis
by
GC-MS
analysis
Quali a i e
and
quan i a i e
composi ion
o
essen ial
oils
was
pe o med
by
GC
and
GC-MS
analysis,
using
a
HP
5889
se ies
II
and
a
Va ian
Sa u n
3
ion
ap
sys em,
espec i ely
in
bo h
cases,
columns
DB-5
(30
m
×
0.15
mm
×
0.25
␮m;
J.
W.
Scien ific)
we e
used;
helium
N60
was
used
as
ca ie
gas.
The
analysis
condi ions
we e:
ini ial
empe a u e
40 ◦C
o
14
min,
g adien
o
1.5 ◦C/min
up
o
250 ◦C,
iso he m
o
10
min.
Injec o
(and
ans e
line)
empe a u e
was
270 ◦C.
1
␮L
was
injec ed
in
spli less
mode.
GC-MS
analysis
was
pe o med
by
ESI
in
a
mass
ange
24
o
400
m/z
wi h
a
delay
ime
o
2
min.
Iden ifica ion
o
componen s
was
pe o med
by
compa ing
he
mass
spec a
o
hose
o
he
a ailable
NIST
da abase
(GC-MS)
and
by
hei
ela i e
e en ion
index
(Ko acs
Index,
KI)
owa ds
a
mix u e
o
alkanes
C4 o
C26,
injec ed
in
he
same
expe imen al
condi ions
(GC-FID).
2.4.
HPLC-DAD-ESI/MSnanalysis
2.4.1.
Liquid
ch oma og aphy
S ock
solu ions
wi h
concen a ions
(m/ )
o
5
mg/mL
we e
p epa ed
by
dissol ing
each
d ied
ex ac
in
ini ial
HPLC
mobile
phase
(Ace oni ile/Wa e
(20/80,
/ )).
These
solu ions
we e
fil-
e ed
h ough
0.45
mm
Nylon
mic opo e
memb anes
p io
o
use
and
10
␮L
we e
injec ed
o
HPLC-DAD-ESI/MSnanalysis.
Th ee
independen
assays
we e
pe o med
o
each
sample.
The
HPLC
sepa a ion
was
ca ied
ou
on
a
Dionex
ul ima e
3000
se ies
ins umen
(Cali o nia,
EUA)
coupled
o
a
bina y
pump,
a
diode-a ay
de ec o
(DAD),
an
au osample
and
a
column
com-
pa men .
Samples
we e
sepa a ed
on
a
Phenomenex
Gemini
C18 col-
umn
(5
␮m,
250
mm
×
3.0
mm
i.d.;
Phenomenex)
wi h
a
sample
injec ion
olume
o
10
␮L.
The
mobile
phase
was
composed
o
ace-
oni ile
(A)
and
wa e / o mic
acid
(100/0.1,
/ )
(B).
A
g adien
p og am
was
used
as
ollows:
20%
A
(0
min),
25%
A
(10
min),
25%
A
(20
min),
50%
A
(40
min),
100%
A
(42–47
min),
20%
A
(49–55
min).
The
mobile
phase
flow
a e
was
0.4
mL/min;
he
ch oma og am
was
eco ded
a
280
nm
and
350
nm
and
spec al
da a
o
all
peaks
we e
accumula ed
in
he
ange
o
190–400
nm.
Column
empe a u e
was
con olled
a
30 ◦C.
2.4.2.
Mass
spec ome y
Fo
HPLC-ESI/MSnanalysis,
a
model
6000
ion
ap
mass
spec-
ome e
(B uke
Esqui e,
B emen,
Ge many)
fi ed
wi h
an
ESI
sou ce
ope a ing
in
he
nega i e
mode
was
used.
Da a
acquisi ion
and
p ocessing
we e
pe o med
using
Esqui e
con ol
so wa e.
Nega i e
ion
mass
spec a
o
he
column
elua e
we e
eco ded
in
he
ange
m/z
100–1000
a
a
scan
speed
o
13,000
Da/s.
High
pu i y
ni ogen
(N2)
was
used
bo h
as
d ying
gas
a
a
flow
o
10.0
mL/min
and
as
a
nebulizing
gas
a
a
p essu e
o
50
psi.
The
nebulize
em-
pe a u e
was
se
a
365 ◦C
and
a
po en ial
o
+400
V
was
used
on
he
capilla y.
Ul a-high-pu i y
helium
(He)
was
used
as
collision
gas
a
a
p essu e
o
1
×
10−5mba
and
he
collision
ene gy
was
se
a
40
V.
The
acquisi ion
o
MSnda a
was
made
in
au o
MSnmode,
wi h
an
isola ion
wid h
o
4.0
m/z.
Fo
MSnanalysis,
he
mass
spec ome e
was
scanned
om
10
o
1000
m/z
wi h
agmen a ion
ampli ude
o
1.0
V
(MSnup
o
MS4)
and
wo
p ecu so
ions.
2.5.
To al
phenolic
con en
(TPC)
The
o al
phenolic
con en
he
ex ac s
was
de e mined
ollow-
ing
he
Folin-Ciocal eu
me hod
(Zheng
and
Wang,
2001)
wi h
some
modifica ions
(Gou eia
and
Cas ilho).
Each
sample
aliquo
o
50
␮L
(10
mg/mL,
w/ )
was
mixed
wi h
1.25
mL
o
Folin-Ciocal eu
eagen
(dilu ed
1:10
old)
and
1
mL
o
7.5%
sodium
ca bona e
solu ion.
The
mix u e
was
incuba ed
o
30
min
a
oom
empe a u e
and
hen
abso bance
was
measu ed
a

=
765
nm.
The
final
esul s
we e
exp essed
as
millig ams
o
gallic
acid
equi alen s
pe
100
g
o
d ied
plan
(mg
GAE/100
g).
172 S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181
2.6.
DPPH
adical
sca enging
ac i i y
Fo
he
DPPH
assay,
100
␮L
o
he
sample
solu ions
(10
mg/mL)
we e
added
o
3.5
mL
o
a
0.06
mM
me hanol
DPPH
adical
solu-
ion
(Gou eia
and
Cas ilho,
2012a,
b).
The
dec ease
in
abso bance
a

=
516
nm
was
measu ed
du ing
30
min.
The
DPPH
adical
sca enging
e ec
o
he
ex ac s
was
exp essed
as
␮mol
T olox
equi alen
pe
100
g am
o
d ied
plan
(␮mol
eq.
T olox/100
g
d ied
plan )
o
plan
ex ac s
and
as
␮mol
T olox
equi alen
pe
mL
o
essen ial
oil
(␮mol
eq.
T olox/mL
EO)
o
essen ial
oils.
2.7.
ABTS•+ adical
sca enging
ac i i y
The
ABTS•+ adical
wo king
solu ion
was
p epa ed
by
mixing
50
mL
o
2
mM
ABTS•+solu ion
wi h
200
␮L
o
70
mM
po assium
pe sul a e
solu ion
(Gou eia
and
Cas ilho,
2012a,b).
This
mix u e
was
kep
in
he
da k
o
16
h
a
oom
empe a u e.
Fo
each
analysis,
he
ABTS•+solu ion
was
dilu ed
wi h
pH
7.4
phospha e
bu e ed
saline
(PBS)
solu ion
o
an
ini ial
abso bance
o
0.700
±
0.021
a
734
nm.
This
solu ion
was
eshly
p epa ed
o
each
analysis.
Fo
he
assessmen
o
he
adical
sca enging
ac i i y,
an
aliquo
o
100
␮L
(10
mg/mL,
w/ )
was
added
o
1.8
mL
o
ABTS•+wo king
solu ion
and
he
abso bance
dec ease,
a
a

=
734
nm,
was
eco ded
du ing
6
min.
Resul s
we e
exp essed
as
␮mol
T olox
equi alen
pe
100
g
o
d ied
plan
(␮mol
eq.
T olox/100
g
d ied
plan )
o
plan
ex ac s
and
as
␮mol
T olox
equi alen
pe
mL
o
essen ial
oil
(␮mol
eq.
T olox/mL
EO)
o
essen ial
oils.
2.8.
Fe ic
educing
ac i i y
(FRAP
assay)
The
e ic
educing
abili y
o
he
ex ac s
was
measu ed
based
on
he
FRAP
assay
(Benzie
and
S ain,
1996).
FRAP
eagen
was
p epa ed
daily
by
mixing
2.5
mL
o
solu ion
e ic
ichlo ide
hexa-
hyd a e
(20
mM),
2.5
mL
o
solu ion
TPTZ
(10
mM
in
40
mM
o
hyd ochlo ic
acid)
and
25
mL
o
ace a e
bu e
0.3
M
(pH
3.6)
and
incuba ing
a
37 ◦C.
Fo
each
analysis,
30
␮L
o
me hanolic
solu-
ion
(1
mg/mL,
w/ )
we e
added
o
180
␮L
o
dis illed
wa e
and
1.8
mL
o
FRAP
solu ion.
The
inc ease
o
abso bance
was
eco ded
a

=
593
nm
in
15
s
in e als,
du ing
30
min
a
37 ◦C.
The
FRAP
esul s
we e
exp essed
as
mmol
I on(II)
sul a e
hep ahyd a e
pe
mg
o
d ied
plan
(mmol
Fe(II)/mg)
o
plan
ex ac s
and
as
mmol
I on(II)
sul a e
hep ahyd a e
pe
mL
o
essen ial
oil
(mmol
Fe(II)/mL
EO)
o
essen ial
oils.
3.
Resul s
and
discussion
3.1.
Essen ial
oil
composi ion
The
chemical
composi ion
o
essen ial
oils
ob ained
om
d ied
lea es
is
p esen ed
in
Table
1.
Simila
esul s
ha e
been
published
by
Magalhães
e
al.
(2004),
who
p oduced
his
hyb id
and
cul i a ed
i
in
B azil.
Compa ing
hese
se s
o
esul s
i
was
clea
ha
he
composi ion
o
essen ial
oil
was
no
subs an ially
a ec ed
by
clima e,
loca ion
o
cul i a -
ing
condi ion
bu
i
is
in insic
o
he
plan .
E en
d ying
in
di ec
sunshine
o
shade
did
no
a ec
much
he
essen ial
oil
composi ion,
wi h
a
li le
loss
o
he
mo e
ola ile
componen s
being
obse ed
o
he
plan
d ied
unde
sunligh .
The
CPQBA
×
POP
hyb id
has
no
hu-
jone
de i a i es
o
a emisia
ke one,
ha m ul
compounds
no mally
p esen
in
wild- ype
A emisia
annua
L.
(´
Ca a
e
al.,
2012;
Reale
e
al.,
2011).
The
only
possible
oublesome,
in
e ms
o
sa e y,
com-
ponen
could
be
campho
(ca.
40%).
The
pai
1,8-cineole/campho
as
he
wo
majo
componen s
in
essen ial
oils
has
been
ound
no
only
in
A emisia
annua
(´
Ca a
e
al.,
2012;
Holm
e
al.,
1997;
M
R,
2009)
bu
in
se e al
o he
A emisia
subspecies
(Ko dali
e
al.,
2005;
Lopes-Lu z
e
al.,
2008;
Shang
e
al.,
2012).
3.2.
Phenolic
composi ion
by
HPLC-DAD-ESI/MSn
Compa ison
be ween
he
HPLC-DAD-ESI/MSnsc eening
o
me hanolic
and
ace one
ex ac s
showed
ha ,
o
he
me hanolic
ex ac ,
he
numbe
o
peaks
de ec ed
was
lowe
and
wi h
educed
ch oma og aphic
esolu ion
leading
o
a
poo
agmen a ion
o
he
de ec ed
compounds.
This
can
be
associa ed
o
ma ix
e ec s
and
high
molecula
weigh
compounds
common
in
mo e
pola
sol-
en s
ex ac ions.
Se e al
au ho s
(Kalli h aka
e
al.,
1995;
Ko eka
e
al.,
2011)
ha e
e alua ed
me hanol
e sus
ace one
as
ex ac ion
sol en s
o
phenolic
compounds
wi h
clea
ad an age
o
ace-
one.
Ou
choice
o
his
sol en
esul ed
om
ou
p e ious
wo k
whe e
i
was
he
bes
o
ex ac
a emisinin.
The
ace one
ex ac s
om
plan s
cul i a ed
in
h ee
di e en
loca ions
we e
e y
sim-
ila
be ween
hem,
hus
we
only
p esen
he
ch oma og am
and
agmen a ion
da a
o
one
o
hem
(P eces).
The
HPLC-DAD-ESI/MSnbase
peak
ch oma og am
p ofile
o
ace one
ex ac
is
shown
in
Fig.
1.
The
iden ifica ion
and
cha ac e iza ion
o
he
de ec ed
com-
pounds
was
made
by
compa ison
o
he
HPLC
e en ion,
UV
and
mass
spec a
wi h
hose
o
s anda d
compounds.
Since
only
a
limi ed
numbe
o
s anda d
compounds
was
a ailable,
s uc u es
o
unknown
compounds
we e
es ablished
la gely
based
on
hei
andem
MSn agmen a ion
beha iou .
Among
he
de ec ed
compounds,
we
ound
ypical
hyd oxycin-
namic
acid
UV
abso p ions
(max 230–240,
300
sh.
320–340
nm)
and
fla onoids
cha ac e is ic
UV
abso p ions:
fla onols
de i a-
i es
exhibi ed
wo
maximum
abso p ions
a
250–270
nm
and
320–360
nm,
de i ed
om
he
aglycone
A
and
B
ings,
espec i ely.
Peaks
co esponding
o
fla ones
conjuga es
showed
h ee
abso p-
ions
a
210–230
nm,
250–280
nm
and
330–350
nm
(Mab y
e
al.,
1970).
In
ou
s udy,
38
compounds
we e
en a i ely
iden ified
and
some
o
hem
a e
epo ed
o
he
fi s
ime
o
A emisia
annua.
Fig.
2
ep esen s
he
chemical
s uc u es
o
main
classes
o
compounds
de ec ed
in
A emisia
annua.
Table
2
shows
he
expe imen al
analy ical
da a:
e en ion
ime
( R),
wa eleng h
o
maximum
abso bance
(max),
dep o ona ed
molecula
ions
[M–H]−,
and
mos
impo an
MSn agmen
ions
o
each
peak.
Usually,
he
base
peak
in
he
MS1spec um
was
assigned
as
he
dep o ona ed
molecula
ion
[M–H]−.
When
isome s
we e
obse ed,
hei
iden ifica ion
was
pe o med
based
on
li e a u e
da a.
3.2.1.
Iden ifica ion
o
fla onoids
The
occu ence
o
fla onoids,
in
pa icula
hose
conjuga ed
wi h
one
o
mo e
suga
moie ies,
in
A emisia
species
has
been
p e iously
epo ed
(Fe ei a
e
al.,
2010,
Gou eia
and
Cas ilho,
Han
e
al.,
2008;
Lai
e
al.,
2007).
In
HPLC-ESI/MSnsc eening
o
A emisia
annua,
mainly
apigenin
and
que ce in
conjuga es
ha e
been
epo ed.
In
he
p esen
wo k,
in
addi ion
o
hese
wo
ypes
o
aglycones,
lu eolin,
iso hamne in
and
kaemp e ol
de i a i es
compounds
we e
also
iden ified.
The
andem
MS/MS
expe imen s
o
he
fla onoids
esul ed
on
he
dep o ona ed
molecula
ion
([M–H]−)
and
he
dep o ona ed
aglycone
ion
(Y−
0)
as
a
esul
o
he
loss
o
he
suga
uni .
Mos
o
he
de ec ed
fla onoids
we e
in
hei
glycosyla ed
o m
and/o
es e ified
wi h
acyl
g oups.
Bu
wo
aglycones
we e
de ec ed
on
i s
ee
o m
–
lu eolin
and
apigenin.
3.2.1.1.
F ee
aglycones
(36
and
40).
Compound
36
( R=
27.7
min)
was
iden ified
as
lu eolin
by
compa ison
wi h
a
e e ence
s anda d
(da a
no
shown).
In
he
MSnexpe imen s
he
[M–H]−ion
a
m/z
285
ga e
se e al
ypical
lu eolin
agmen
ions
a
m/z
199,
217
([M-
H-C3O2]−),
175
([M-H-C3O2-C2H2O]−)
and
241
([M-H-CO2]−).
Compound
40
( R=
33.9
min)
exhibi ed
a
[M–H]−ion
a
m/z
269
and
he
i s
MSn agmen a ion
p esen ed
main
agmen
ions
a
S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181 173
Table
1
Composi ion
o
he
essen ial
oil
(EO)
om
lea es
o
A emisia
annuag ow h
in
P eces,
Po o
San o
and
Ja dimBo ânico
(d ied
unde
di ec
sunligh
and
in
he
shade).
KI
e *KI
Compound
P eces
Po o
San o
Ja dimBo ânico
Shade
Ja dimBo ânico
Sunshine
937
937
␣-pinene
3.58
1.55
3.19
2.79
953 954 camphene 0.28
0.01
0.23
0.31
990 902
␤-pinene
0.60
0.27
1.35
0.88
1018
1017.0
␣- e pinene
0.68
0.68
0.77
0.78
1026 1027.0
p-cymene
3.32
1.23
2.10
3.31
1030
1031.0
limonene
0.23
0.05
0.39
0.24
1030
1035.0
1,8-cineole
11.6
6.54
9.87
7.92
1059
1061.0
␥- e pinene
1.27
1.22
1.35
1.28
1141 1141.0
ans-pinoca eol 0.51
0.88
0.86
0.64
1143
1149.0
campho
42.8
45.5
42.0
44.33
1165 1173.0
bo neol
2.50
8.96
2.58
4.74
1177
1180.0
e pinen-4-ol
2.80
2.59
2.45
2.51
1194
1196.0
my enol
1.23
1.21
1.00
1.08
1217
1222.0
ans-ca eol
1.77
1.76
1.79
1.55
1242 1247.0
l-ca one
1.03
0.52
0.30
0.78
1345 1346.0
␣-cubebene
0.10
0.10
0.08
0.13
1351
1353.0
eugenol
1.13
0.66
1.23
1.09
1376 1373.0
␣-copaene
1.75
0.17
0.82
1.15
1389
1388.0
␤-cubebene
1.93
1.31
1.02
1.66
1394 1392.0
cis-jasmone
0.24
0.72
0.24
0.48
1428
1417.0
␤-ca yophyllene
7.77
10.21
6.44
8.65
1444
1452.0
␣-humulene
0.46
0.94
6.70
0.94
1480
1479.0
ge mac ene-D
7.15
8.41
8.61
7.77
1485
1486.0
␤-selinene
1.93
3.09
2.78
3.04
1494 1493.0
bicycloge mac ene 3.29
1.4
1.86
1.95
*See
h p://www.phe obase.com/da abase/ko a s/ko a s-index.php.
m/z
225
([M-.H-CO2]−),
151
(1,3A−)
and
149
(1,4B−+
2H).
Compa ing
hese
esul s
o
hose
ob ained
o
a
s anda d
solu ion
o
apigenin,
40
was
iden ified
as
ee
apigenin.
Two
ypes
o
fla onoid
glycosila ion
we e
obse ed:
O-
glycosila ion
and
C-glycosila ion.
The
iden ifica ion
o
hese
wo
g oups
was
based
on
he
andem
MSn agmen a ion
expe imen s.
Fo
C-glycosyla ed
compounds
he
suga
uni
is
linked
o
he
aglycone
wi h
a
ca bon-ca bon
bond
ha
is
esis an
o
up u e.
The
ep esen a i e
MSn agmen s
o
C-glycosyla ed
fla onoids
a e
ela ed
o
he
suga
pa
and
occu s
a
m/z 0,2 X−[M-H-120]−,0,3
X−[M-H-90]−and 0,2 X−[M-H-60]−(Cuyckens
and
Claeys,
2004).
The
non-obse a ion
o
dep o ona ed
aglycone
ion,
Y−
0,
makes
iden-
ifica ion
p ocess
ha de .
Fo
O-glycosyla ed
compounds
he
suga
moie ies
a e
los
as
neu al
agmen s
and
he
fla onoid
s uc u e
is
mo e
easily
iden-
ified
due
o
aglycone
bonds
clea ages.
3.2.1.2.
C-glycosides
(7,
8,
9,
12
and
13).
Fo
hese
compounds
UV
spec a
showed
maximum
abso p ions
bands
a

=
225,
272
and
314
nm
iden ical
o
cha ac e is ics
abso p ions
bands
o
fla ones
(Mab y
e
al.,
1970).
Howe e ,
he
andem
MSnexpe imen s
e ealed
a
di e en
pa e n
o
ha
known
o
O-glycosyla ed
fla ones.
Compound
7
( R=
5.4
min)
and
8
( R=
5.8
min)
showed
a
[M-
H]−ions
a
m/z
563.
The
MS2 agmen a ion
e ealed
a
beha iou
ypical
o
he
asymme ical
di-C-glycosides
wi h
agmen
ions
a
[M-H-210]−,
[M-H-90]−and
[M-H-60]−.
The
neu al
loss
o
60
Da
(0,3X−)
indica es
he
p esence
o
a
pen ose
moie y.
Compound
8
has
been
p e iously
desc ibed
by
ou
g oup
in
A emisia
a gen ea
(Gou eia
and
Cas ilho,
2011a,b)
and
was
cha -
ac e ized
as
apigenin-6-C-hexoside-8-C-pen oside.
Compound
7
simila
o
8
had
MS3 agmen
ions
a
m/z
353
[0,2Xi0,2Xj]−and
383
[0,2Xi0,3Xj]−sugges ing
apigenin
as
he
aglycone
(Vukics
e
al.,
2008).
Howe e ,
con a ily
o
compound
8,
7
did
no
p esen
an
in ense
[M-H-60]−ion
a
m/z
503
(2.9%
o
base
peak).
Based
on
hese
e idences
and
knowing
ha
6-C-pen oside-8-C-hexoside
elu es
be o e
he
iso-
me
6-C-hexoside-8-C-pen oside,
compound
7
was
iden ified
as
apigenin-6-C-pen oside-8-C-hexoside.
Fig.
1.
HPLC-DAD-ESI/MSnanalysis
o
he
ace one
ex ac s
o
A emisia
annua
o al
ae ial
pa s
–
HPLC-MS
nega i e
ion
ESI/MSnbase
peak
ch oma og am
(BPC).
174 S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181
Table
2
Cha ac e iza ion
o
phenolic
componen s
o
he
ace one
ex ac
om
A emisia
annua
by
HPLC-DAD-ESI/MSn.
No.
R(min)
Iden ifica ion
max (nm)
[M-H]−(m/z)
HPLC-DAD-ESI/MSnm/z
(%
base
peak)
1
2.8
Ca eic
acid
hexoside
de i a i e
275,
304
473
MS2[473]:
342
(14.4),
341
(100),
179
(13.8),
161
(13.8),
131
(29.2)
MS3[473
→
341]:
179
(100),
161
(43.7),
143
(10.8),
119
(44.2),
101
(11.3)
MS4[473
→
341
→
179]:
119
(92.0),
113
(54.0),
101
(63.2),
89
(100)
2
3.1
Quinic
acid
-
191
MS2[191]:
173
(74.9),
127
(100),
111
(47.0),
109
(69.7),
85
(70.7)
MS3[191
→
127]:
163
(30.8),
109
(69.7),
99
(100)
3
4.0
Kaemp e ol-3,7-di-O-
hexoside
-
609
MS2[609]:
449
(18.2),
448
(11.6),
447
(100),
285
(34.9)
MS3[609
→
447]:
327
(7.6),
284
(73.0),
285
(100),
255
(62.3)
MS4[609
→
447
→
284]:
256
(31.1),
255
(100),
151
(34.2)
4
4.4
3-O-Ca eoylquinic
acid
241,
300,
324
353
MS2[353]:
191
(100),
179
(51.4),
135
(15.9)
MS3[353
→
191]:
173
(100),
171
(16.9),
127
(56.8),
111
(30.2),
109
(39.2),
85
(37.4)
MS4[353
→
191
→
127]:
109
(100),
99
(90.9),
85
(12.9)
5*5.0
5-O-Ca eoylquinic
acid
242,
300,
325
353
MS2[707]:
353
(100)
MS2[353]:
191
(100)
MS3[707
→
353]:
191
(100)
MS3[353
→
191]:
173
(57.1),
127
(100),
109
(55.9),
85
(97.7)
MS4[707
→
353
→
191]:
173
(100),
127
(63.7),
135
(21.8),
111
(17.4)
MS4[353
→
191
→
127]:
109
(100)
6
5.2
4-O-Ca eoylquinic
acid
240,
300,
323
353
MS2[353]:
191
(18.4),
179
(58.3),
173
(100),
135
(8)
MS3[353
→
173]:
111
(30.8),
93
(100)
7 5.4 Apigenin-6-C-
pen oside-8-C-
hexoside.
224,
273,
314 563
MS2[563]:
503
(2.9),
473
(98.0),
443
(78.2),
383
(83.5),
353
(100)
MS3[563
→
353]:
326
(13.4),
325
(100),
297
(78.2)
MS4[563
→
352
→
325]:
298
(5.1),
297
(100)
8
5.9
Apigenin-6-C-
hexoside-8-C-
pen oside.
224,
273,
314
563
MS2[563]:
503
(59.3),
473
(68.6),
443
(100),
383
(63.0),
353
(63.2)
MS3[563
→
443]:
383
(50.0),
354
(12.0),
353
(100)
MS4[563
→
443
→
353]:
326
(35.1),
325
(100),
297
(56.2)
9
6.5
Apigenin-6-C-
hamnoside-8-C-
hexoside
224,
272,
313
577
MS2[577]:
559
(21.9),
503
(2.8),
487
(28.3),
473
(40.0),
457
(53.1),
353
(100)
MS3[577
→
353]:
326
(18.3),
325
(100),
297
(71.2)
MS4[577
→
353
→
325]:
297
(100)
10
6.9
3-O-Fe uloylquinic
acid
326
367
MS2[367]:
193
(100),
191
(1.5),
173
(3.7)
MS3[367
→
193]:
149
(31.9),
134
(100),
109
(10.3)
11
7.4
5-O-Fe uloylquinic
acid
327
367
MS2[367]:
191
(100)
MS3[367
→
191]:
179
(100),
134
(53.0),
111
(68.6),
127
(55.5),
109
(12.7),
85
(93.0)
12
7.9
Apigenin-6-C-
hexoside-8-C-
hamnoside
224,
272,
313
577
MS2[577]:
487
(41.2),
473
(2.7),
457
(100),
353
(36.9)
MS3[577
→
457]:
383
(18.5),
354
(33.0),
353
(100)
MS4[577
→
457
→
353]:
326
(55.5),
325
(100),
298
(22.0)
13
8.3
Apigenin-8-C-hexoside
224,
272,
314
431
MS2[431]:
341
(23.1),
311
(100)
MS3[431
→
311]:
284
(38.1),
283
(100)
MS4[431
→
311
→
283]:
283
(100),
224
(77.3),
163
(23.7)
14
8.8
Que ce in-O-
dihexoside
–
625
MS2[625]:
302
(17.1),
301
(100),
300
(19.1)
MS3[625
→
301]:
271
(22.9),
212
(10.8),
179
(82.9),
151
(100)
MS4[625
→
301
→
151]:
169
(100),
107
(20.2)
15
9.1
Lu eolin-7-O-hexoside
–
447
MS2[447]:
286
(14.0),
285
(100)
MS3
[447
→
285]:
285
(100),
241
(24.9),
176
(19.0),
175
(80.1),
149
(117.5)
16 9.3
Mea nse in-O-
hexoside
257,
300,
342
493
MS2[493]:
332
(19.0),
331
(100),
330
(10.3),
316
(11.7)
MS3[493
→
331]:
317
(12.8),
316
(100),
315
(16.3)
MS4[493
→
331
→
316]:
287
(49.6),
271
(46.1),
229
(38.2),
166
(100)
17*9.5
Que ce in-3-O-
glucoside
258,
353
463
MS2[463]:
302
(17.1),
301
(100),
300
(19.1)
MS3[463
→
301]:
271
(22.9),
212
(10.8),
179
(82.9),
151
(100)
MS4[463
→
301
→
151]:
169
(100),
107
(20.2)
18
9.8
Iso hamne in-O-
hexoside
256,
270,
344
477
MS2[477]:
316
(17.1),
315
(100),
300
(29.3)

S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181 175
Table
2
(Con inued)
No.
R(min)
Iden ifica ion
max (nm)
[M-H]−(m/z)
HPLC-DAD-ESI/MSnm/z
(%
base
peak)
MS3[477
→
315]:
301
(11.4),
300
(100)
MS4[477
→
315
→
300]:
284
(62.1),
271
(67.5),
245
(63.1),
229
(100),
213
(61.9)
19*11.8
3,4-O-Dica eoylquinic
acid
246,
299,
325
515
MS2[515]:
354
(17.5),
353
(100),
335
(12.3),
299
(12.2),
173
(21.8)
MS3[515
→
353]:
191
(24.8),
179
(60.9),
173
(100),
135
(14.8)
MS4[515
→
353
→
173]:
155
(20.8),
111
(100),
93
(61.1)
20*12.5
1,5-O-Dica eoylquinic
acid
243,
300,
328
515
MS2[515]:
353
(100)
MS3[515
→
353]:
191
(100)
MS4[515
→
353
→
191]:
173
(43.8),
127
(100),
109
(22.1)
21*12.9
3,5-O-Dica eoylquinic
acid
242,
300,
328
515
MS2[515]:
354
(10.7),
353
(100)
MS3[515
→
353]:
191
(100),
179
(53.9)
MS4[515
→
353
→
191]:
173
(100),
127
(90.9),
93
(34.5)
22
13.1
Dica eoylquinic
acid
isome
240,
326
515
MS2[515]:
354
(15.8),
353
(100)
MS3[515
→
353]:
191
(100),
179
(52.1),
135
(15.2)
MS4[515
→
353
→
191]:
85
(100),
173
(76.6),
127
(66.8),
109
(76.6)
23*14.2
4,5-O-dica eoylquinic
acid
243,
300,
327
515
MS2[515]:
354
(14.5),
353
(100)
MS3[515
→
353]:
191
(31.5),
179
(53.9),
173
(100),
MS4[515
→
353
→
179]:
155
(33.6),
111
(57.0),
93
(100)
24 14.6 Lu eolin-7-O-pen oside 417
MS2[417]:
285
(100),
284
(22.4)
MS3[417
→
285]:
257
(5.1),
243
(72.1),
241
(54.1),
217
(93.1),
199
(98.2),
175
(100),
151
(39.2)
25
15.3
Dihyd oxy-
dime hoxyl-O-
hexoside
fla one
491
MS2[491]:
371
(12.1),
330
(12.5),
329
(100),
314
(10.3)
MS3[491
→
329]:
315
(21.1),
314
(100)
MS4[491
→
329
→
314]:
300
(22.8),
299
(100)
26
16.8
3-p-O-Couma oyl-5-O-
ca eoylquinic
acid
499
MS2[499]:
337
(100),
221
(11.4),
179
(),
163
(14.2)
MS3[499
→
337]:
179
(28.2),
163
(100),
135
(15.8)
MS4[499
→
337
→
163]:
119
(100)
27
18.1
Unknown
423
MS2[423]:
262
(10.6),
261
(100),
173
(100)
MS3[423
→
261]:
175
(11.6),
173
(100)
MS4[423
→
261
→
173]:
93
(100)
28
18.9
1-O-Ca eoyl-5-O-
e uloylquinic
acid
328
529
MS2[529]:
368
(12.9),
367
(100),
353
(30.0),
191
(8.5)
MS3[529
→
367]:
191
(100)
MS4[529
→
367
→
191]:
173
(69.0),
127
(100),
109
(52.9)
29 20.2
1
o
5-O-ca eoyl-4-O-
e uloylquinic
acid
–
529
MS2[529]:
368
(42.4),
367
(100),
173
(23.3)
MS3[529
→
367]:
193
(81.5),
191
(28.5),
173
(100)
MS4[529
→
367
→
173]:
137
(21.5),
111
(14.6),
93
(100)
30
20.6
3-O- e uloyl-5-O-
ca eoylquinic
acid
529
MS2[529]:
368
(2.8),
367
(100),
353
(18.4),
193
(21.4)
MS3[529
→
367]:
194
(8.4),
193
(100),
191
(42.2),
135
(2.4)
31
22.2
3,4-O-di e uloylquinic
acid
543
MS2[543]:
367
(19.5),
349
(100),
193
(3.8),
173
(12.4)
MS3[543
→
349]:
287
(38.6),
193
(28.4),
175
(100),
155
(34.2)
32
23.2
3,5-O-di e uloylquinic
acid
543
MS2[543]:
367
(100),
349
(38.2),
191
(18.28)
MS3[543
→
367]:
193
(100),
173
(2.4)
33 23.8
Kaemp e ol-3-O-
ca eoylhexoside
–
609
MS2[609]:
447
(20.1),
323
(17.1),
286
(11.5),
284
(100),
285
(98.2),
179
(29.4)
MS3[609
→
285]:
213
(55.2),
151
(89.4),
107
(100)
34 26.5
Que ce in-O-
ca eoylhexoside
253,
330
625
MS2[625]:
463
(57.1),
445
(24.2),
323
(14.3),
301
(100),
300
(5.1)
MS3[625
→
301]:
273
(4.1),
271
(5.9),
255
(19.6),
176 S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181
Table
2
(Con inued)
No.
R(min)
Iden ifica ion
max (nm)
[M-H]−(m/z)
HPLC-DAD-ESI/MSnm/z
(%
base
peak)
179
(45.9),
151
(100),
107
(7.0)
MS4[625
→
301
→
151]:
107
(100)
35 27.2 Unknown
579
MS2[579]:
418
(24.7),
417
(100)
MS3[579
→
417]:
243
(100),
179
(26.5)
MS4[579
→
417
→
243]:
225
(16.7),
199
(100),
183
(31.0)
36*27.7
Lu eolin
–
285
MS2[285]:
243
(38.2),
241
(92.1),
217
(12.5),
199
(69.5),
175
(100),
151
(40.2)
MS3[285
→
175]:
147
(100)
37 28.5 Ca eoylcouma oyl a a ic
acid
232,
300,
311 457 MS2[457]:
296
(10.5),
295
(100),
173
(12.9)
MS3[457
→
295]:
163
(100),
121
(36.2)
MS4[457
→
295
→
163]:
111
(38.8),
93
(100)
38*29.2
3,4,5-O-
T ica eoylquinic
acid
677
MS2[457]:
296
(10.5),
295
(100),
173
(12.9)
MS3[457
→
295]:
163
(100),
121
(36.2)
MS4[457
→
295
→
163]:
111
(38.8),
93
(100)
39 30.4 E iodic yol-7-O-
hexoside
247,
329 449 MS2[449]:
287
(100),
173
(14.3)
MS3[449
→
287]:
173
(100)
MS4[449
→
287
→
173]:
111
(100),
83
(73.6)
40*33.9
Apigenin
260,
331
269
MS2[269]:
227
(33.4),
225
(100),
201
(59.2),
151
(32.6),
149
(80.3)
MS3[269
→
225]:
198
(23.1),
183
(66.9),
181
(100)
(–)
Thei
UV
spec a
ha e
no
been
p ope ly
obse ed
due
o
low
in ensi y.
*Compa ison
wi h
e e ence
s anda ds.
Fig.
2.
Chemical
s uc u es
o
phenolic
compounds
cha ac e ized.
S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181 177
Compounds
9
( R=
6.5
min)
and
12
( R=
7.9
min)
displayed
a
[M-
H]−ion
a
m/z
577.
MSnexpe imen s
o
hese
wo
peaks
did
no
e ealed
he
agmen
co esponding
o
he
neu al
loss
o
60
Da
and
he
p esence
o
a
agmen
ion
a
m/z
353
indica es
ha
he
suga
g oups
mus
be
a
hexoside
o
a
me hylpen ose.
Compound
9
p esen ed
MS2ions
a
m/z
559
(21.9%
o
base
peak)
[M-H-H2O]−and
m/z
503
[M-H-74]−sugges s
ha
he
hamnoside
g oup
should
be
linked
a
6-C-posi ion.
Thus,
9
was
iden ified
as
apigenin-6-C- hamnoside-8-C-hexoside.
Compound
12
has
been
epo ed
be o e
and
was
iden ified
as
apigenin-6-C-hexoside-8-C- hamnoside.
Compound
13
( R=
8.3
min)
showed
a
[M-H]−ion
a
m/z
431.
The
MS2spec um
p esen ed
main
agmen
ions
a
m/z
311
[M-
H-120]−
0,2X−(base
peak)
and
341 0,3X−(23.1%
o
base
peak).
The
loss
o
wa e
molecules
is
indica i e
o
C-6
isome s.
This
ype
o
clea age
was
no
obse ed
o
compound
13
and
based
on
li e a u e
epo s
(Gou eia
S.
and
Cas ilho)
his
compound
was
iden ified
as
apigenin-8-C-hexoside.
3.2.1.3.
O-glycosides
(3,
14,
15,
16,
17,
18,
24,
25,
33
and
34).
The
agmen s
esul ed
om
he
o
O-glycosila ed
fla onoids
we e
labelled
as
p oposed
by
Cuyckens
and
Claeys
(2004).
The i,jA−and
i,jB−labels
co espond
o
ions
con aining
in ac
A-
and
B- ings,
espec i ely,
and
i
and
j
speci y
he
C- ing
bonds
ha
ha e
been
b oken.
Compound
3
( R=
4.0
min)
displayed
a
[M-H]−ion
a
m/z
609.
I s
analysis
by
MS2 agmen a ion
esul ed
in
a
neu al
loss
o
162
Da
o ming
a
agmen
ion
a
m/z
447
and
an
in ense
agmen
a
m/z
285
(34.9%
o
base
peak).
MS3spec um
displayed
a
agmen
ion
a
m/z
285
(loss
o
162
Da),
as
base
peak,
and
i s
subsequen
agmen a ion
ga e
he
ypical
agmen s
o
kaemp e ol
a
m/z
255
[Y0−-CH2OH]−
and
151
(1,3A−)
(compa ison
made
wi h
a
s anda d
solu ion
o
kaemp e ol).
The
absence
o
a
agmen
ion
a
m/z
323
excluded
he
hypo hesis
o
he
wo
162
Da
esidues
being
ca eoylhexo-
sides
and
we e
cha ac e ized
as
being
wo
hexosides
esidues.
Based
on
he
p e ious
epo s
(Gou eia
and
Cas ilho,
2010)
and
acco ding
o
he
ules
desc ibed
by
Ablajan
and
co-wo ke s
(Ablajan
e
al.,
2006)
3
was
iden ified
as
kaemp e ol-3,7-O-
dihexoside.
Compound
33
( R=
23.8
min)
also
displayed
a
[M-H]−ion
a
m/z
609.
Howe e ,
MSn agmen a ion
was
qui e
di e en
o
ha
ound
o
compound
4.
The
MS2spec a
showed
he
adical
aglycone
ion
[Y0-H]−a
m/z
284,
as
base
peak,
and
also
an
in ense
agmen
a
m/z
285
(98.2%
o
base
peak).
The
loss
o
a
324
Da
esidue
was
a ibu ed
o
a
combined
loss
o
wo
162
Da
g oups
and
was
confi med
by
he
p esence
o
a
agmen
ion
a
m/z
447
(20.1%
o
base
peak)
o med
by
he
loss
o
162
Da.
The
agmen
ion
a
m/z
323
(17.1%
o
base
peak)
assigned
as
[ca eoylhexoside-H]−poin
ou
o
a
hexoside
g oup
es e ified
wi h
a
ca eoyl
g oup
a he
han
wo
hexosides
moi-
e ies.
The
aglycone
was
iden ified
as
being
kaemp e ol
based
on
he
MSn agmen s
and
compa ison
wi h
a
kaemp e ol
e e ence
solu ion.
The
a ou ed
glycosila ion
posi ions
o
fla onols,
such
as
kaemp e ol
a e
3-OH
and
7-OH.
When
he
aglycone
adical
is
mo e
abundan
han
he
dep o ona ed
aglycone
ion,
indica es
an
agly-
cone
subs i u ed
a
posi ion
3-OH
(Cuyckens
and
Claeys,
2005).
Fo
compound
33,
he
MS2spec um
base
peak
was
he
aglycone
adical
ion,
a
m/z
285,
and
he e o e
i
was
iden ified
as
being
kaemp e ol-3-O-ca eoylhexoside.
Compound
17
( R=
9.5
min)
exhibi ed
a
[M-H]−ion
a
m/z
463
which
easily
los
a
162
Da
moie y,
in
MS2 agmen a ion,
esul ing
in
a
agmen
ion
a
m/z
301.
Fu he
agmen a ion
o
his
ion
ga e
he
ep esen a i e
agmen s
o
que ce in
a
m/z
151
(1,2 A−-CO),
179
([1,2A−-H]−)
and
271
[M-H-CH2O]−.
The e o e,
compound
17
was
unequi ocally
iden ified
as
que ce in-3-O-glucoside
by
compa ison
wi h
a
e e ence
s anda d
solu ion.
Compounds
14
( R=
8.8
min)
and
34
( R=
26.5
min)
ga e
he
same
[M-H]−ion
a
m/z
625
and
hei
MSn agmen a ion
was
simila .
In
he
MS2spec a,
he
same
base
peak
a
m/z
301
was
obse ed
(loss
o
324
Da).
The
occu ence
o
a
MS1ion
a
m/z
463
(loss
o
162
Da)
sugges s
ha
he
esidue
o
324
Da
is
composed
o
wo
uni s
o
162
Da
linked.
Howe e ,
he
na u e
o
hese
wo
g oups
appea s
o
be
dis inc
o
each
compound.
Fo
34,
a
MS1
ion
a
m/z
323
(14.3%
o
base
peak)
was
obse ed
indica ing
a
ca -
eoylhexoside
g oup
which
is
in
good
ag eemen
wi h
he
long
e en ion
ime
o
his
compound.
The
lowe
e en ion
ime
o
14
and
he
absence
o
a
agmen
ion
a
m/z
323
indica e
a
dihexoside
esidue.
F agmen a ion
o
he
dep o ona ed
aglycone
ion,
Y−
0a
m/z
301
allowed
o
iden i y
common
agmen
ions
o
que ce in,
as
desc ibed
o
compound
17.
Since
he
Y−
0ion
is
he
MS2base
peak,
he
subs i u ion
g oups
mus
be
linked
o
only
one
OH
g oup
o
aglycone
s uc u e.
The
aglycone
adical
ion
was
no
obse ed,
so
he
3-OH
posi-
ion
is
excluded
bu
no
o he
agmen s
we e
ound
o
suppo
in
which
posi ion
he
subs i u ion
occu s.
Thus,
14
was
en a i ely
iden ified
as
que ce in-O-dihexoside
and
34
was
as
que ce in-O-
ca eoylhexoside.
Compound
15
( R=
9.1
min)
displayed
a
[M-H]−in
a
m/z
447
and
i s
MS2 agmen a ion
e ealed
he
loss
o
162
Da
o ming
a
ag-
men
ion
a
m/z
285,
Y−
0.
This
compound
was
al eady
desc ibed
o
A emisia
a gen ea
(Gou eia
and
Cas ilho,
2011a,
b)
and
iden ified
as
lu eolin-7-O-hexoside.
Ano he
lu eolin
de i a i e
was
de ec ed
a
a
e en ion
ime
o
14.6
min
(compound
24).
I
ga e
a
[M-H]−ion
a
m/z
417
easily
los
a
neu al
g oup
o
132
Da
(pen oside)
ising
he
dep o ona ed
aglycone
ion
a
m/z
285.
F agmen a ion
o
he
ion
a
m/z
285
ga e
he
cha ac e is ics
agmen s
o
lu eolin
a
m/z
175
([M-H-C3O2-
C2H2O]−),
217
([M-H-C3O2]−)
and
241
([M-H-CO2]−)
(Fig.
3).
The
a ou ed
subs i u ion
posi ion
o
fla ones
such
as
lu eolin
is
he
7-
OH
posi ion
(Cuyckens
and
Claeys,
2004).
Consequen ly,
compound
24
was
cha ac e ized
as
lu eolin
7-O-pen oside.
To
ou
knowledge
i
is
he
fi s
ime
ha
his
compound
is
epo ed
o
A emisia
species.
Compound
16
( R=
9.3
min)
showed
a
[M-H]−ion
a
m/z
493.
I s
MSn agmen a ion
esul ed
in
he
aglycone
ion
a
m/z
331
due
o
he
loss
o
162
Da.
The
MS3 adical
ion
a
m/z
316
is
simila
o
he
agmen a ion
beha iou
desc ibed
o
mea nse in-O-hexoside,
also
de ec ed
in
A emisia
a gen ea
and
A emisia
annua
(Gou eia
and
Cas ilho,
2011a,b;
Han
e
al.,
2008).
Compound
18
( R=
9.8
min)
displayed
a
[M-H]−ion
a
m/z
477
and
was
cha ac e ized
as
iso hamne in-O-hexoside
by
compa ison
wi h
li e a u e
da a
(Gou eia
and
Cas ilho,
2009).
This
compound
was
no
epo ed
be o e
o
A emisia
annua
bu
was
de ec ed
in
A emisia
species
(Gou eia
and
Cas ilho,
2011a,b).
Compound
25
( R=
15.3
min)
showed
a
[M-H]−ion
a
m/z
491.
A
loss
o
162
Da
was
obse ed
in
he
MS2 agmen-
a ion,
o ming
a
agmen
ion
a
m/z
329.
In
he
u he
MSn agmen a ions,
wo
losses
o
15
Da
each
we e
obse ed
and
associa ed
o
wo
me hoxyl
g oups.
So,
25
was
cha -
ac e ized
as
dihyd oxy-dime hoxyl-O-hexoside
fla ones.
This
compound
was
also
epo ed
o
A emisia
a gen ea
(Gou eia
and
Cas ilho,
2011a,b)
bu
i
was
no
epo ed
o
A emisia
annua
be o e.
One
compound
(39)
om
he
g oup
o
fla anones
is
epo ed
o
A emisia
annua
o
he
fi s
ime.
I
was
ound
a
a
e en ion
ime
o
30.4
min
and
iden ified
as
a
e iodic yol-7-O-hexoside.
The
[M-
H]−ion
appea ed
a
m/z
449
and
he
hexoside
esidue
was
easily
expelled
in
he
MS2 agmen a ion
esul ing
in
he
dep o ona ed
aglycone
ion,
Y−
0,
a
m/z
287.
178 S.C.
Gou eia,
P.C.
Cas ilho
/
Indus ial
C ops
and
P oduc s
45 (2013) 170–
181
Fig.
3.
P oposed
agmen a ion
pa hway
o
compound
24–lu eolin-O-pen oside.
3.2.2.
Iden ifica ion
o
hyd oxycinnamic
acids
(2,
4,
5,
6,
10,
11,
19,
20,
21,
22,
23,
26,
28,
29,
30,
31,
32
and
38)
Compound
2
( R=
3.1
min)
displayed
a
[M-H]−ion
a
m/z
191
and
was
iden ified
as
quinic
acid
(Gou eia
and
Cas ilho,
2011a,b).
Despi e
ha
his
compound
was
ound
as
a
ela i e
in ense
com-
ponen
o
he
A emisia
annua
ex ac ,
i
was
no
epo ed
be o e
o
his
plan .
A
o al
o
19
quinic
acid
de i a i es
we e
de ec ed
and
iden ified
in
his
s udy,
mos
o
hem
quinic
acid
es e ified
wi h
acyl
g oups.
The
iden ifica ion
o
each
compound
was
made
based
on
he
main
agmen
ions
ob ained
in
he
MSnexpe imen s.
The
hie a chical
key
o
he
iden ifica ion
by
LC-MSno
quinic
acid
de i a i es
p o-
posed
by
Cli o d
e
al.
(Cli o d
e
al.,
2003,
2005)
was
used
o
iden i y
his
class
o
compounds.
Mono-,
di-
and
ica eoylquinic
acids
we e
iden ified
by
com-
pa ison
o
he
e en ion
ime,
MSn agmen a ion
beha iou
and
UV
spec a
wi h
hose
o
s anda d
compounds.
I
was
he
case
o
com-
pounds
5
( R=
4.4
min)
as
5-O-ca eoylquinic
acid;
19
( R=
11.8
min)
as
3,4-O-dica eoylquinic
acid;
20
( R=
12.5
min)
as
1,5-O-
dica eoylquinic
acid;
21
( R=
12.9
min)
as
3,5-O-dica eoylquinic
acid;
23
( R=
14.2
min)
as
4,5-O-dica eoylquinic
acid
and
38
( R=
29.2
min)
as
3,4,5-O- ica eoylquinic
acid.
Addi ionally,
wo
monoca eoylquinic
acids,
compounds
4
and
6,
we e
iden ified
based
on
MSn agmen a ion
o
he
[M-H]−ion
a
m/z
353.
Compound
4
( R=
4.4
min)
ga e
a
MS2ion
a
m/z
191
(base
peak)
and
an
in ense
agmen
ion
a
m/z
179
(51.4%
o
base
peak)
which
is
ep esen a i e
o
a
ca eoyl
g oup
linked
o
he
3-OH
posi ion
o
quinic
acid.
Thus,
4
was
iden ified
as
3-O-ca eoyquinic
acid.
Compound
6
( R=
5.2
min)
showed
a
MS2ion
a
m/z
173
(base
peak)
poin ing
o
a
quinic
acid
es e ified
a
posi ion
4-OH
(Fig.
4).
The e o e,
6
was
classified
as
4-O-ca eoylquinic
acid.
Rema kably,
his
compound
ga e
a
highe
in ensi y
han
he
isome
5-O-CQA
commonly
ound
in
o he
As e aceae
plan s.
Compound
22
( R=
13.1
min)
ga e
a
[M-H]−ion
a
m/z
515
and
u he
MSn agmen a ion
ga e
he
cha ac e is ic
agmen
ions
o
dica eoylquinic
acid
isome s
a
m/z
191,
173
and
353.
This
should
be
a
cis
isome
o
a
dica eoylquinic
acid
wi h
no
subs i u ion
posi ion
4-OH
(Jaiswal
e
al.,
2011;
Ma
e
al.,
2008).
The
unequi -
ocally
iden ifica ion
o
his
compound
can
only
be
es ablished
by
NRM
s udies
o
compa ison
wi h
a
e e ence
s anda d
since
e en
UV-i adia ion
s udies
would
no
cla i y
i
i
is
a
mono-cis
o
a
di-cis
compound.
Compounds
10
( R=
6.9
min)
and
11
( R=
7.4
min)
exhibi ed
he
same
[M-H]−ion
a
m/z
367
bu
a
di e en
MSn agmen a-
ion
beha iou .
Fo
10,
he
MS2spec um
showed
a
agmen
ion
a
m/z
193
(base
peak).
Based
on
he
ules
epo ed
by
Cli o d
e
al.
(Cli o d
e
al.,
2003)
his
compound
was
iden ified
as
3-O-
e uloylquinic
acid.
Compound
11
ga e,
as
base
peak
o
he
MS2
spec um,
a
agmen
ion
a
m/z
191
[quinic
acid-H]−.
This
pa e n
is
consis en
o
ha
epo ed
o
5-O- e uloylquinic
acid
(Cli o d
e
al.,
2005).
Compound
26
( R=
16.8
min)
displayed
a
[M-H]−ion
a
m/z
499.
The
MS2spec um
showed
a
agmen
ion
a
m/z
337,
as
base
peak,
indica ing
he
loss
o
162
Da
and
sugges ing
a
couma oylquinic
acid
de i a i e.
MS3 agmen a ion
o
he
ion
a
m/z
337
esul ed
in
a
agmen
ion
a
m/z
163
and
a
MS4ion
a
m/z
119,
as
base
peaks,
poin ing
o
a
3-p-O-couma oylquinic
acid
s uc u e,
acco ding
o
li e a u e
epo s
(Cli o d
e
al.,
2003).
The
absence
o
a
s ong
agmen a ion
a
m/z
173
sugges s
a
3,5-O-p-couma oyl-ca eoylquinic
acid.
Since
he
ca eoyl
g oup
is
he
fi s
o
be
los
i
should
be
linked
o
he
5-OH
posi ion.
Taking
in o
accoun
hese
da a,
26
was
iden ified
as
3-p-O-couma oyl-5-O-ca eoylquinic
acid.
To
ou
knowledge,
quinic
acids
es e ified
wi h
couma oyl
g oups
ha e
no
been
epo ed
be o e
in
phenolic
sc eenings
o
A emisia
annua.
Th ee
compounds
wi h
[M-H]−ions
a
m/z
529
we e
obse ed
(compound
28
( R=
18.9
min),
29
( R=
20.2
min)
and
30
( R=
20.7
min)).
In
he
MS2 agmen a ion
all
compounds
ga e
MS2
base
peaks
a
m/z
367
[ e uloylquinic
acid-H]−,
due
o
he
loss
o
a
ca eoyl
esidue,
and
a
seconda y
agmen
ion
a
m/z
353
(ca.
20%
o
base
peak).
The
dis inc ion
o
hese
h ee
isome s
was
based
on
he
MS3 agmen a ion.
Compound
28
ga e
a
MS3ion
a
m/z
191,
as
base
peak,
which
is
consis en
wi h
a
5-O- e uloylquinic
acid
s uc u e.
Acco ding
o
he
agmen a ion
da a,
a
agmen
ion
a
m/z
179
was
no
obse ed,
hus
he
ca eoyl
g oup
mus
be
linked
o
he
1-OH
posi ion
o
quinic
acid.
So,
compound
28
was
iden ified
as
1-O-ca eoyl-5-O-
e uloylquinic
acid.
Compound
29
( R=
20.2
min)
exhibi ed
a
MS3base
peak
a
m/z
173
which
indica es
a
4-O- e uloylquinic
acid
s uc u e.
The
exac