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217
Recebido para publicação em 09/06/2011
Aceito para publicação em 09/05/2012
Flavonol glycosides found in hydroethanolic extracts from Tilia cordata, a species
utilized as anxiolytics
NEGRI, G.*, SANTI, D.; TABACH, R.
Department of Psychobiology (CEBRID), Federal University of São Paulo, UNIFESP/EPM. São Paulo, Brazil.
Rua Botucatu, 862 – Ed. Ciências Biomédicas – 1º andar, CEP - 04023-062, São Paulo, SP, Brazil. Phone: 55
(2111) 2149-0161 / Fax: 55 (2111) 5084-2793, e-mail: gnegri@terra.com.br
ABSTRACT: Tilia species, among which is Tilia cordata Mill. (Tiliaceae), have been used
in folk medicine as anxiolytic. The hydroethanolic extract was analyzed by using liquid
chromatography with mass spectrometry HPLC-DAD-ESI-MS/MS in negative ion mode, and its
chemical composition was compared to avonoids reported as anxiolytics. The major avonoids
found were: quercetin-3,7-di-O-rhamnoside, kaempferol-3,7-di-O-rhamnoside and kaempferol
3-O-(6”-p-coumaroyl glucoside) or tiliroside. The anxiolytic activity of the genus Tilia has been
attributed to the presence of quercetin and kaempferol derivatives, while the anxiolytic activity
of T. americana var. Mexicana was attributed to tiliroside, which was also found among the
major constituents of this species.
Key words: Tilia cordata, HPLC-DAD-ESI-MS/MS, quercetin and kaempferol glycosides,
tiliroside.
RESUMO: Flavonóides glicosídeos encontrados no extrato hidroalcoólico de Tilia cordata,
espécie usada como ansiolítico. As espécies de Tilia, entre elas, a Tilia cordata Mill. (Tiliaceae)
são utilizadas como ansiolíticas na medicina popular. O extrato hidroalcoólico foi analisado
usando cromatograa líquida acoplada à espectrometria de massas HPLC-DAD-ESI/MS/
MS no modo negativo e a sua composição química foi comparada com os avonóides já
reportados como ansiolíticos. Os principais avonóides encontrados foram: quercetina-3,7-di-
O-rhamnosideo, canferol-3,7-di-O-rhamnosideo, e canferol 3-O-(6”-p-cumaroil glucosideo) ou
tilirosideo. A atividade ansiolítica do gênero Tília tem sido atribuída à presença de derivados
de canferol e quercetina, enquanto que a atividade ansiolítica da T. americana var. Mexicana
foi atribuída ao tilirosideo, o qual também foi encontrado entre os principais constituintes desta
espécie.
Palavras-chave: Tilia cordata, HPLC-DAD-ESI-MS/MS, glicosídeos de quercetina e canferol,
tilirosideo
Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
INTRODUCTION
Tilia cordata Mill. (Tiliaceae) has been used
in folk medicine, primarily as a non-narcotic sedative
for sleep disorders or anxiety. The anxiolytic effect of
Tilia species, such as T. americana var. Mexicana,
has been attributed to the presence of tiliroside
(Anesini et al., 1999; Perez-Ortega et al., 2008).
Phytochemical studies have demonstrated that Tilia
species possess hydrocarbons, esters, aliphatic
acids (Fitsiou et al., 2007), terpenoids, quercetin
and kaempferol derivatives (Pietta et al., 1994),
phenolic compounds, condensed tannins (Behrens
et al., 2003) and a coumarin scopoletin (Arcos et al.,
2006). Tilia americana var. Mexicana has several
avonoids such as rutin, hyperoside, quercitrin and
tiliroside (Aguirre-Hernandez et al., 2010).
The combination of liquid chromatography
with electrospray ionization mass spectrometry (LC-
ESI-MS) provides the molecular weight and a partial
characterization of avonoid glycosides present in
complex mixtures. The structural characterization
of avonoid glycosides has been carried out with
mass spectral methods based on collision-induced
dissociation (CID) of molecular species, such
as protonated molecules [M + H]+, deprotonated
molecules [M – H]- and sodiated molecules [M
+ Na]+, generated by a ionization technique like
ESI (Kachlicki et al., 2008; Shahat et al., 2005).
Thus, MS/MS spectra of protonated, deprotonated
218
Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
and sodiated molecules can be used to obtain
information about several structural features, such
as the aglycone and the types of carbohydrates that
are present (mono-, di-, tri- or tetrasaccharides and
hexoses, deoxyhexoses or pentoses) (Kachlicki et
al., 2008; Shahat et al., 2005).
Type-A α-aminobutyric acid (GABAA)
receptors are the major inhibitory neurotransmitter
receptors in the human central nervous system,
which are involved in epilepsy, sedation and
anxiolysis, producing these effects by binding
to GABAA receptors. The GABAergic system
has á-aminobutyric acid as neurotransmitter.
Anxiolytics facilitate the coupling of GABAergic
receptors to GABAA (Mewett et al. 2009) and
produce their pharmacological effect by binding to
a benzodiazepine recognition site on the GABAA
receptor complex. GABAA receptors are heteromeric
GABA-gated chloride channels. GABA released from
GABAergic interneurons exerts inhibition by acting on
GABA receptors at pre-synaptic terminals and post-
synaptic neurons to reduce pre-synaptic glutamate
release and produce inhibitory postsynaptic currents,
or membrane hyperpolarization, in post-synaptic
neurons (Mewett et al. 2009). Benzodiazepines
(BDZ) are the most common anxiolytic drugs
used today. Benzodiazepines bind to the so-called
benzodiazepine site, where they modulate the
receptor to be more sensitive to GABA, thereby
yielding an anticonvulsant, sedative or anxiolytic
effect (Huen et al., 2003).
Flavonoids have recently increased in
importance because they have been identied as a
new type of ligand with in vivo anxiolytic properties
(Jager & Saaby, 2011). Flavonoids are present in
food and medicinal plants and are thus consumed
by humans. The flavones apigenin and luteolin
derivatives have shown an anxiolytic effect in
rodents exposed to behavioral tests (Coleta et al.,
2008). In the Central Nervous System (CNS) several
avonoids bind to the benzodiazepine site on the
GABAA receptor and modulate the á-aminobutyric
acid (GABA)ergic system to produce the biological
effect of sedation, anxiolytic or anti-convulsive
(Aguirre-Hernandez et al., 2010). Flavonoids of
several classes are also inhibitors of monoamine
oxidase A or B (Zhu et al., 2006). Various in vivo
studies have shown that avonoids can be absorbed
after oral administration, pass the blood-brain barrier
and produce various effects on the CNS (Jager &
Saaby, 2011).
In this study, the major avonoids present
in the hydroethanolic extract of Tilia cordata were
characterized by using HPLC–DAD–ESI-MS/
MS in negative ion mode and a correlation was
established between them and the flavonoids
reported as anxiolytics. Quercetin and kaempferol
derivatives were found in Tilia species. But there is
no literature report regarding the determination of
phenolic compounds from hydroethanolic extract
of T. cordata. Tilia species have been used in
folk medicine due to their anxiolytic and sedative
activities, which have been attributed to avonoids,
such as tiliroside. Therefore, the aim of this study was
to compare the avonoids identied in this species
with the avonoids reported as anxiolytics. Tiliroside
is one of the main avonoids found in this species.
MATERIAL AND METHOD
Plant Material
The leaves of T. cordata were purchased
from “Quimer Ervas & Especiarias S. A.” and came
with their respective certicates. Quercetin (Q4951, >
95%), apigenin (A3145, > 95%), kaempferol (K0133,
> 96%) and quercetrin (Q3001, > 90%) standards
were purchased from Sigma-Aldrich Chemical Co.
(St. Louis, MO, USA); their purities were above
97%, as determined by HPLC/DAD analysis. Stock
solutions of these standards (100µg / mL) were
prepared in methanol. HPLC grade methanol was
purchased from Merck (Darmstadt, Germany). HPLC
grade water was prepared from distilled water using
a Milli-Q system (Millipore, Waters, Milford, MA,
USA).
Sample preparation
The leaves were air-dried in the shade at
room temperature to a constant weight, ground
to pass through a 30 mesh screen, and stored in
sealed glass vials. For preparation of lyophilized
extracts, 100 g of the powder were extracted
with 1 L of hydroethanolic solution at 50% (V/V)
by maceration (Mendes et al., 2002). The crude
preparation was ltered through Whatman paper
no. 1 and concentrated under reduced pressure in
a rotaevaporator to produce a crude extract, which
was placed in a lyophilizer (4 atm of pressure and
temperature of - 40o C) for 48 hours. The lyophilized
extracts were stored in amber asks at 5o C (freezer).
Phytochemical Assays
The lyophilized hydroethanolic extract
was screened via thin layer chromatography (TLC)
for alkaloids, phenolic acids, steroids, terpenoids,
cardioactive glycosides, flavonoids, coumarins,
saponins, lignans, tannins and iridoids (Stahl, 1969;
Wagner & Bladt, 1996). The extract was dissolved
in methanol PA (10 mg/mL) and applied to silica-
gel 60 F254 plates (Merck). For alkaloid analyses,
lyophilized samples (50 mg) were dissolved in 2 mL
of water to form a suspension that was acidied with
a solution of 20% of sulfuric acid (H2SO4) to pH 4.
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Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
The acidic suspension was rst partitioned with ethyl
acetate (EtOAc) to remove neutral compounds, and
the aqueous phase was then basied with sodium
carbonate (Na2CO3) to pH 10, followed by extraction
with chloroform (Xu et al., 2006).
Hydrolysis Assays
The free avonoid aglycones of avonoid-
O-glycosides were released by acidic hydrolysis,
as follows: 60mg of samples from T. cordata were
dissolved in 4mL of 10% (V/V) H2SO4 solution, and
heated in boiling water for 1 h (Chirinos et al., 2009).
After cooling, the reaction mixture was neutralized
with saturated aqueous sodium carbonate and
ltered under reduced pressure. The ltrate obtained
after the hydrolysis reactions was concentrated
to approximately 1mL and analyzed by HPLC/
DAD using the same chromatographic conditions
that were utilized for the analyzes of standards of
quercetin and kaempferol.
Reversed Phase HPLC-DAD-ESI-MS/MS
analysis
For reversed phase high performance liquid
chromatography (RPHPLC) analysis, lyophilized
extract was dissolved in water : methanol (80:20)
v/v (10mg / 3mL) and ltered with a 0.45 µm lter,
prior to injection of 30.0µL into the HPLC system.
Spectral UV data from all peaks were collected in
the range 240 – 400nm, and chromatograms were
recorded at 370 and 260nm for phenolic compounds.
A DADSPD-M10AVP Shimadzu equipped with a
photodiode array detector was coupled to Esquire
3000 Plus, Bruker Daltonics mass spectrometer with
electrospray ionization (ESI) source and ion-trap
analyser. All the operations, acquisition and data
analysis were controlled by the system controller
Shimadzu VP series HPLC system (SCL-10A VP).
The mobile phases consisted of eluent A (0.1% aq.
formic acid) and eluent B (methanol). A reverse
phase, C18, Zorbax – 5B - RP-18 (Hewlett Packard)
column (4.6×250mm, 5µm), connected to a guard
column and a gradient of 20–90% B (V/V) over 50
min were utilized for separations, as follows: 0min
–20% B in A; 10min – 30% B in A; 20min – 50% B in
A; 30 min – 70% B in A; 40min– 90% B in A; 45min –
40% B in A and nally returned to the initial conditions
(20%B) to re-equilibrate the column prior to another
run. The ow rate was kept constant at 0.5mL min”1,
and the temperature of the column was maintained
at 30°C. The ionization conditions were adjusted as
follows: electrospray ionization was performed using
an ion source voltage of - 39 V and a capillary offset
voltage of 4400V. Collision-induced dissociation
(CID) spectra were performed in the ion trap using
helium as collision gas, with voltage ramping cycles
from 0.5 up to 1.3V. Ultrahigh pure Helium (He) was
used as the collision gas and high-purity nitrogen
(N2) as the nebulizing gas. Nebulization was aided
with a coaxial nitrogen sheath gas provided at a
pressure of 26 psi. Desolvation was facilitated by
using a counter current nitrogen ow set at a ux
of 7.0 liter / minute and a capillary temperature
of 325°C. The full scan mass acquisition both in
negative and positive ion mode were performed
by scanning from m/z 100 up to 900. Due to the
unavailability of commercial standards of avonoids
glycosides, these compounds were characterized by
the interpretation of their UV absorbance band, the
mass spectra (ESI/MS and ESI/MS/MS) obtained,
including their respective aglycone, which are
determined after hydrolysis reaction using standards
of quercetin, kaempferol and also taking into account
the ESI-MS and ESI-MS/MS data provided by the
literature.
RESULTS AND DISCUSSION
RPHPLC-DAD-ESI-MS/MS analyses
The hydroethanolic extract of T. cordata
has an acidic pH (5.5). Flavonoids are acidic due
to phenolic hydroxyl groups that interface with the
neighboring benzene ring, causing a conjugation
effect. The yield of the hydroethanolic extract of T.
cordata was 9.2 g per 100 g of crude plant material.
To characterize the qualitative chemical prole, the
extract was initially analyzed via TLC (Stahl, 1969;
Wagner & Bladt, 1996; Jayaprakasha et al., 2006).
Dried TLC plates were sprayed with specic reagent
(methanolic hydrochloric acid 2 M; ferric chloride;
dragendorff reagent; Liebermann-Burchard; Carr-
Price reagent and antimony pentachloride) and
heated to observe the color reaction characteristic
for each chemical class. The spots of procyanidins
exhibited a pink color upon heating with methanolic
hydrochloric acid 2 M. The hydroethanolic extracts
reacted positively with ferric chloride, indicating the
presence of phenolic hydroxyl groups. This extract
showed the presence of avonoids and procyanidins
(condensed tannins). Alkaloids were not detected.
Formic acid is a common modifier for
RPHPLC, and its volatility also makes it highly
suitable for mass spectrometry. Table 1 summarizes
the following information on peaks observed during
RPHPLC-DAD-ESI-MS/MS analyses: (1) peak
labels, (2) retention times (RT) (min), (3) proposed
structure, (4) wavelengths of absorbance maxima
(ëmax), (5) m/z ratios for the protonated [M + H]+
molecules, (6) m/z ratios for the deprotonated [M –
H]- molecules and (7) the major MS/MS fragments
obtained in negative mode. The retention time (RT)
on the column is governed not only by the polarity of
the molecules but also by their size. Besides, RT of
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Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
the separated substances depend on their solubility
in water and/ or their hydrophobicity, and RT having
been demonstrated to increase as the hydrophobicity
of the compound on the reverse phase columns
increase. The sugar position is more important for
the retention time than the nature of the sugar (Abad-
Garcia et al., 2009).
The susceptibility of the sugar aglycone in
acidic hydrolysis depends on the attachment position
of the sugar. The loss of the glycan substituent at the
3’ or 5’ positions occur more readily in comparison
with 7’ and 3’ positions (Abad-Garcia et al., 2009).
Compounds 2 - 8 (Figure 1) were easily hydrolyzed
in an acidic medium. Identication of the kaempferol
and quercetin aglycones after hydrolysis reactions
were conrmed by comparing retention time, UV,
MS and co-injection with standards. In addition,
inspection of the UV spectra of compounds 2 - 7
showed absorptions typical of avonol derivatives,
with maximum absorption at band I (347–370nm)
and band II (250–267nm) (Abad-Garcia et al., 2009).
Most of the authentic compounds exhibited
[M – H]- ions of sufcient abundance that could be
subjected to MS/MS analysis. The rst-order mass
spectra obtained during analyses after the LC
separation provided information about molecular
weight of avonoid glycosides. The number and
size of sugars (hexose, deoxyhexose or pentose)
substituted to the aglycone and also the aglycone
were established from the second-order product
ion mass spectra, which were performed in the
Collision-Induced Dissociation (CID) MS/MS
negative mode. Ions of deprotonated molecules [M
- H]- are usually more stable than their protonated
[M + H]+ counterparts; thus, higher collision energy
is necessary for the fragmentation of the precursor
ions (Kachlicki et al., 2008; Shahat et al., 2005;
Abad-Garcia et al., 2009). Protonated avonol di-
O-glycosides containing a substituent at both the
3-O and 7-O positions more easily lose the 3-O than
the 7-O glycoside (Kachlicki et al., 2008; Shahat et
al., 2005). Thus, while the loss of the 3-O glycoside
is more pronounced than that of the 7-O glycoside
in the MS/MS spectra of protonated molecules, the
opposite behavior is noted for deprotonated and
sodiated molecules (Kachlicki et al., 2008; Shahat
et al., 2005).
The peak at 25.2 min for compound 2
(Table 1, Figure 1 and 2) exhibited deprotonated
and protonated molecules at m/z 609.2 and 611.3,
and a sodiated molecule [M + Na]+ at m/z 633.1,
respectively, in the ESI/MS spectra. The MS/MS
spectrum of deprotonated molecule gave rise to
the m/z 447.0 [M – H - 162]- as the base peak,
formed after the loss of the glucose sugar attached
to the C-7 carbon atom of avonol. The fragment
at m/z 462.9 probably correspond to the loss of
rhamnose sugar at C-3 carbon atom of avonol,
and at m/z 301.0 (aglycone quercetin), being typical
of di-O-glycosylavonol. This type of fragmentation
occurring in negative ion mode, in which the loss
of a sugar unit gives the most abundant base peak
(m/z 447.0) different from the peak of the aglycone
(m/z 301.0), indicated that residues of glycosilation
are present in more than one phenolic hydroxyl
group of the aglycone (Kachlicki et al., 2008;
Shahat et al., 2005). Glucose is more common
than galactose. Compound 2 was tentatively
characterized as quercetin-3-O-rhamnoside-7-O-
glucoside or quercetrin-7-O-glucoside.
Compound 3 (Table 1, Figure 1 and 2), peak
at 27.4 min, exhibited deprotonated and protonated
molecules at m/z 593.1 and m/z 595.2, and a sodiated
molecule [M + Na]+ at m/z 617.2, respectively, in the
TABLE 1. HPLC/MS data, protonated and deprotonated molecules (m/z) for peaks, including the retention times
(RT), MS/MS experiments and maximal absorption wavelength (λmax) of the constituents found in T. cordata.
ND – not determined
Rt Proposedstructure UV λmax (M + H)+ (M - H)- MS/MS (m/z) (ESI-) (%)
(min) (nm) (m/z) (m/z)
1 16.2 procyanidin dimer B2 275 579.2 577.1 407.1 (100), 425.0 (90),
451.0 (30), 559.0 (50), 289.0 (20)
2 25.2 quercetin-3-O- rhamnoside - 260, 355 611.3 609.2 447.0 (100), 462.9 (70),
7-O- glucoside 301.0 (60)
3 27.4 quercetin-3,7-di-O-rhamnoside 263, 355 595.2 593.1 447.0 (100), 301.0 (30)
4 28.6 quercetin-3-O-glucoside 260, 355 465.1 463.1 301.0 (100)
5 29.5 kaempferol-3,7-di-O-rhamnoside 260, 350 579.2 577.1 431.0 (100), 285.0 (80)
6 30.6 quercitrin 260, 350 449.1 447.1 301.0 (100)
7 32.7 kaempferol-3-O-rhamnoside ND 433.1 431.1 285.0 (100)
8 34.2 tiliroside 260, 315 595.2 593.1 285.0 (100)447.0 (10)
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Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
ESI/MS spectra. The MS/MS spectrum in negative
ion mode showed a base peak [(M – H) - 146]- at
m/z 447.0 and a fragment at m/z 301.0 (quercetin),
typical of di-O-glycosylavonol. The most abundant
product ion obtained from deprotonated molecule
was formed at m/z 447.0, after loss of the rhamnose
residue probably attached to the C-7 carbon atom
of avonol (Kachlicki et al., 2008; Shahat et al.,
2005). Compound 3 was tentatively characterized
as quercetin-3,7-di-O-rhamnoside.
At the peak at 29.5 min, compound 5
showed deprotonated and protonated molecules at
m/z 577.1 and 579.2 and a sodiated molecule [M
+ Na]+ at m/z 601.3, respectively, and the MS/MS
spectrum in negative ion mode showed a base peak
[(M – H) - 146]- at m/z 431.0 and a fragment at m/z
285.0 (aglycone kaempferol), exhibiting the same
pattern of fragmentation of compound 3, leading to
the characterization of this compound as kaempferol-
3,7-di-O-rhamnoside.
In this species, avonoids monoglycosides,
such as quercetin-3-O-glucoside (peak at 28.6 min,
compound 4), quercitrin (quercetin-3-O-rhamnoside)
(peak at 30.6 min, compound 6) and kaempferol-
3-O-rhamnoside (peak at 32.7 min, compound 7
) were also found (Table 1, Figure 2), which were
characterized by comparing with the previously
reported MS/MS fragmentation data and UV spectra.
Compound 4 exhibited deprotonated and protonated
molecules at m/z 463.1 and m/z 465.1, respectively,
and the MS/MS spectrum of deprotonated molecule
showed a base peak at m/z 301.0 that indicated
the presence of quercetin as aglycone and hexose
(162 u) as sugar. The other quercetin derivative,
compound 6 exhibited protonated and deprotonated
molecules at m/z 449.1 and m/z 447.1, respectively.
In this case, the base peak obtained at m/z 301.0 was
formed by the loss of rhamnose (146 u). Compound 7
exhibited deprotonated and protonated molecules at
m/z 431.1 and m/z 433.1, respectively, and the MS/
MS spectrum of deprotonated molecule exhibited a
base peak at m/z 285.0, indicating kaempferol as
aglycone and rhamnose as glycoside. Identication
of compounds 6 was also conrmed by comparing
the retention times, UV and mass data with authentic
standards.
Compound 8, peak at 34.2 min (Table
1, Figure 1 and 2), exhibited deprotonated and
Compound R R1 R2
2 OH Rha Glc
3 OH Rha Rha
4 OH Glc H
5 H Rha Rha
6 OH Rha H
7 H Rha H
8 H (6”-p-coum-glc) H
FIGURE 1. Proposed structure of avonols glycosides found in T. cordata.
FIGURE 2. HPLC-ESI/MS chromatogram of hydroethanolic extract of T. cordata.
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Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
protonated molecules at m/z 593.1 and 595.2, and a
sodiated molecule [M + Na]+ at m/z 617.2, respectively.
This compound presented a UV spectrum with ëmax
at 260.0 (band II) and 315.0 nm (band I), suggesting
that this avonol is acylated because it showed an
absorption maximum for band I at 315nm. The main
characteristics of p-coumaroylglycosylated avonols
are the shift of absorption for 315nm (Shahat et
al., 2005; Abad-Garcia et al., 2009). The MS/MS
spectrum in negative ion mode produced a base
peak [(M – H) – 146 - 162]- at m/z 285.0, resulting
from the loss of the coumaroyl glucoside moiety and
revealing the presence of kaempferol and another
fragment [(M – H) - 146]- at m/z 447 (10% of the
base peak), showing the presence of coumaroyl
group. Acyl groups are predominantly located at
the 6-position of hexose moiety (Abad-Garcia et
al., 2009; Kite et al., 2011). Generally, avonoid
glycosides esterified with aromatic acids have
higher retention times on RP-HPLC columns than
diglycosides and monoglycosides (Abad-Garcia et
al., 2009). The MS/MS spectrum in positive ion mode
exhibited fragments at m/z 577.1 (10%) [(M + H) –
18]+, at m/z 309.0 (100%), resulting from glucose
more p-coumaroyl moiety and at m/z 287.1 (10%)
(kaempferol). This compound was characterized as
kaempferol 3-O-(6”-p-coumaroyl glucoside), known
as tiliroside (Abad-Garcia et al., 2009). Kaempferol
and quercetin derivatives were previously identied in
T. cordata (Pietta et al., 1994; Loscalzo et al., 2009).
Quercetin-3,7-di-O-rhamnoside and kaempferol-3,7-
di-O-rhamnoside were also found in Tilia argentea
and were shown to possess potent antinociceptive
(reduced the sensitivity to painful stimuli) and anti-
inammatory activity (Toker et al., 2004).
Type B procyanidin dimers
Condensed tannins consist of
polyhydroxyflavan subunits with interflavonoid
C-C-linkages. The fragmentation reflects the
oligomeric composition and the major fragment ions
are due to the cleavage of the interavonoid C-C
linkages with losses of catechin units (288 mass
units). The peak at 16.2min is probably procyanidin
dimer B2 (dimer of avonol catechin) (compound
1, Table 1, Figure 2) based on UV absorption
maximum at 275 nm, deprotonated molecule [M -
H]- at m/z 577.1, protonated molecule [M +H]+ at
m/z 579.2, and sodiated molecule [M + Na]+ at m/z
599.2, respectively. The MS/MS spectrum of the
deprotonated molecule gave several product ions
characteristic of procyanidins [(M - H) - 18]- at m/z
559.0, [(M - H) - 126]- at m/z 451.0, [(M - H) - 152]-
at m/z 425.0, a base peak [(M - H) - 170]- at m/z
407.1, and [(M - H) - 288]- at m/z 289.0 (catechin).
The MS/MS spectrum in positive ion mode gave a
fragment at m/z 561.9 [(M + H) - 18]+, a base peak
at m/z 427.0 and a fragment at m/z 409.0. Retro-
Diels-Alder ssion of the heterocyclic ring system
of the avan-3-ol (Hellstrom et al., 2007) subunits
gave rise to a fragment of m/z 425.0 from anion
m/z 577.1. The ion at m/z 425.0 eliminates water,
probably from ring C at position C3/C4, resulting in
a fragment ion of m/z 407.1.
Relationship among avonoids found in
this species and avonoids known as anxiolytics.
Tilia sp. (Tiliaceae) have been used around
the world due to its anxiolytic and sedative activity,
and quercetin and kaempferol flavonoids have
been shown to be responsible for its sedative effect
(Aguirre-Hernandez et al., 2010; Viola et al., 1994;
Martinez et al., 2009). Tilia extracts acted as an
agonist of the peripheral benzodiazepine receptor,
suggesting the presence of avonoids capable of
binding to the peripheral type of benzodiazepine
receptor binding sites (Aguirre-Hernandez et al.,
2010; Viola et al., 1994). The pharmacological assay
that guided a purication of an ethanol extract of
Tilia petiolaris DC. inorescences resulted in the
isolation and identication of neuroactive avonoid
glycosides, among which areisoquercitrin, quercetin-
3-O-glucoside-7-O-rhamnoside and kaempferol-3-
O-glucoside-7-O-rhamnoside (Toker et al., 2004). T.
americana var. mexicana aqueous extract produced
an antinociceptive effect in models like formalin and
arthritic pain, reinforcing its use for treating this type
of affection in folk medicine, in which an anxiolytic
compound is probably involved. The chromatographic
analyses of aqueous extracts indicate the presence
of glycosides from quercetin such as kaempferitrin,
isoquercitrin, astragalin and tiliroside as avonoids
responsible for the antinociceptive activity (Loscalzo
et al., 2009; Martinez et al., 2009). Kaempferitrin,
isoquercitrin, astragalin and tiliroside have been
reported as the major compounds in polar extracts
of various Tilia species such as: Tilia cordata, Tilia
rubra, Tilia argentea, and Tilia platyphyllos (Loscalzo
et al., 2009; Martinez et al., 2009). Phytochemical
analyses evidenced that flavonoids comprise
the principal group of compounds present in the
anxiolytic extract of T. Americana (Herrera-Ruiz et
al., 2008) and the anxiolytic activity of T. americana
var. Mexicana, widely used in Mexican traditional
medicine to relieve sleeplessness, headache and
nervous excitement, has been attributed to tiliroside
(3-O-(6”-O-(E)-p-coumaroyl)-β-glucosylkaempferol)
(Herrera-Ruiz et al., 2008), which was also found
in the current study for T. cordata hydroethanolic
extract. The anxiolytic activity of Passiora species
has been attributed to avones C-glycosides (Li et
al., 2011). Flavonols that act as monoamineoxidase
A and B (MAO A and B) inhibitors can modulate
monoamine levels in the brain (serotonine, dopamine,
223
Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.
and norepinephrine),which causes behavioral
modications in rodents, indicating an anxiolytic
effect (Chimenti et al., 2006). Beyond its antioxidant
effect, quercetin, like other avonoids, exhibits a wide
range of neuropharmacological actions including
analgesia, sleep, anticonvulsant, sedative and
anxiolytic effects (Dai et al., 2006). Acylated avonol
monorhaminoside have been identied as promising
phytochemical class (Psterer et al., 2011).
In conclusion, since T. cordata exhibited a
high content of avonol O-glycosides (mono- and di-)
quercetin and kaempferol derivatives and tiliroside,
its medicinal use as anxiolytic could be attributed to
the presence of these avonoids, in special tiliroside,
which has been reported as the anxiolytic constituent
of T. americana var. Mexicana.
ACKNOWLEDGMENTS
The authors would like thank the Dr. E. A.
Carlini and Dr. Joaquim Mauricio
Duarte Almeida and Alessandra de Carvalho
Ramalho (Central Analytical of São Paulo University
- USP). This work was supported by grants from
Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP) and Associação Fundo de Incentivo
à Psicofarmacologia (AFIP).
REFERENCE
ABAD-GARCIA, B. et al. A general analytical strategy
for the characterization of phenolic compounds in fruit
juices by high-performance liquid chromatography with
diode array detection coupled to electrospray ionization
and triple quadrupole mass spectrometry. Journal of
Chromatography A, v.1216, n.28, p.5398- 5415, 2009.
AGUIRRE-HERNANDEZ, E. et al. HPLC/MS analysis
and anxiolytic-like effect of quercetin and kaempferol
avonoids from Tilia americana var. mexicana. Journal
of Ethnopharmacology, v. 127, n.1, p. 91-97, 2010.
ANESINI, C.; WERNER, S.; BORDA, E. Effect of Tilia
cordata ower on lymphocyte proliferation: participation
of peripheral type benzodiazepine binding sites.
Fitoterapia, v.70, n.4, p.361-67, 1999.
ARCOS, M.L.B. et al. Tília cordata Mill. extracts and
scopoletin (isolated compound): Differential cell growth
effects on lymphocytes. Phytotherapy Research, v.
20, n.1, p.34-40, 2006.
BEHRENS, A.; MAIE, N.; KNICKER, H.; KOGEL-
KNABNER, I.; MALDI-TOF mass spectrometry and PSD
fragmentation as means for the analysis of condensed
tannins in plant leaves and needles. Phytochemistry,
v. 62, n.7, p. 1159-170, 2003.
CHIMENTI, F. et al. Quercetin as the active principle of
Hypericum hircinum exerts a selective inhibitory activity
against MAO-A: Extraction, biological analysis, and
computational study. Journal of Natural Products, v.
69, n. 6, p. 945-49, 2006.
CHIRINOS, R.et al. HPLC-DAD characterisation of
phenolic compounds from Andean oca (Oxalis tuberose
Mol.) tubers and their contribution to the antioxidant
capacity. Food Chemistry, v.113, n.4, p.1243-1251,
2009.
COLETA, M. et al. Assessment of luteolin
(3’,4’,5,7-tetrahydroxyavone) neuropharmacological
activity. Behavioural Brain Research, v.189, n.1,
p.75-82, 2008.
DAI, F. et al. Protective effects of avonols and their
glycosides against free radical-induced oxidative
hemolysis of red blood cells. Life Sciences, v. 78, n.
21, p.2488-493, 2006.
FITSIOU, I.; TZAKOU, O.; HANCIANU, M.; POIATA, A.;
Volatile constituents and antimicrobial activity of Tilia
tomentosa Moench and Tilia cordata Miller oils. Journal
of the Essential Oil Research, v.19, n.2, p.183-85,
2007.
HELLSTROM, J.; SINKKONEN, J.; KARONEN, M.;
MATTILA, P. Isolation and structure elucidation
of procyanidin oligomers from saskatoon berries
(Amelanchier alnifolia). Journal of Agricultural and
Food Chemistry, v.55, n. 1, p.157-164, 2007.
HERRERA-RUIZ, M. et al. Flavonoids from Tilia americana
with anxiolytic activity in plus-maze test. Journal of
Ethnopharmacology, v. 118, n.2, p.312-317, 2008.
HUEN, M.S.Y.et al. 5,7-Dihydroxy-6-methoxyavone, a
benzodiazepine site ligand isolated from Scutellaria
baicalensis Georgi, with selective antagonistic
properties. Biochemical Pharmacology, v.66, n.1,
p.125-132, 2003.
JAGER, A.K.; SAABY, L.; Flavonoids and the CNS.
Molecules, v. 16, n.2, p.1471-485, 2011.
JAYAPRAKASHA, G.K. et al. Phenolic constituents in the
fruits of Cinnamomum zeylanicum and their antioxidant
activity. Journal of Agricultural and Food Chemistry,
v. 54, n. 5, p.1672-679, 2006.
KACHLICKI, P. et al. Evaluation of glycosylation and
malonylation patterns in avonoid glycosides during
LC/MS/MS metabolite profiling. Journal of Mass
Spectrometry, v.43, n.5, p.572-586, 2008.
KITE, G.C.; ROWE, E.R.; LEWIS, G.P.; VEITCH, N.C.
Acylated avonol tri- and tetraglycosides in the avonoid
metabolome of Cladrastis kentukea (Leguminosae)
Phytochemistry, v.72, n. 4-5, p. 372-384, 2011.
LI, H.W. et al. Comparative studies on anxiolytic activities
and flavonoid compositions of Passiflora edulis
‘edulis’ and Passiora edulis ‘avicarpa’. Journal of
Ethnopharmacology, v. 133, n.3, p.1085-1090, 2011.
LOSCALZO, L.M.; WASOWSKI, C.; MARDER, M.
Neuroactive Flavonoid Glycosides from Tilia petiolaris
DC. Extracts. Phytotherapy Research, v.23, n. 10,
p.1453-1457, 2009.
MARTINEZ, A.L. et al. Antinociceptive activity of Tilia
americana var. mexicana inorescences and quercetin
in the formalin test and in an arthritic pain model in rats
Neuropharmacology, v. 56, n.2, p.564-571, 2009.
MENDES, F.R.; MATTEI, R.; CARLINI, E.A. Activity of
Hypericum brasiliense and Hypericum cordatum on the
central nervous system in rodents. Fitoterapia, v.73, n.
6, p.462-471, 2002.
MEWETT, K.N. et al. Synthesis and biological evaluation
of avan-3-ol derivatives as positive modulators of
GABA(A) receptors. Biorganic Medicinal Chemistry,
v.17, n.20, p.7156-173, 2009.
224
PEREZ-ORTEGA, G. et al. Sedative and anxiolytic efcacy
of Tilia americana var. mexicana inorescences used
traditionally by communities of State of Michoacan,
Mexico. Journal of Ethnopharmacology, v.116, n. 3,
p.461-68, 2008.
PFISTERER, P.H. et al. In silico discovery of acylated
avonol monorhamnosides from Eriobotrya japonica
as natural, small-molecular weight inhibitors of XIAP
BIR3. Bioorganic Medicinal Chemistry, v. 19, n.2,
p.1002-1009, 2011.
PIETTA, P.; FACINO, R.M.; CARINI, M.; MAURI, P.;
Thermospray liquid chromatography mass spectrometry
of avonol glycosides from medicinal plants. Journal
of Chromatography A, v. 661, n.1-2, p.121-26, 1994.
SHAHAT, A. A. et al. Structural characterization of avonol
di-O-glycosides from Farsetia aegyptia by electrospray
ionization and collision-induced dissociation mass
spectrometry. Rapid Communication in Mass
Spectrometry, v.19, n.15, p. 2172-178, 2005.
STAHL, E.; Thin Layer Chromatography. A Laboratory
Handbook. Springer Verlag, Berlin, 1969, 398p.
TOKER, G.; KUPELI, E.; MEMISOGLU, M.; YESILADA,
E. Flavonoids with antinociceptive and anti-inammatory
activities from the leaves of Tilia argentea (silver linden).
Journal of Ethnopharmacology, v. 95, n. 2-3, p. 393-
397, 2004.
VIOLA, H. et al. Isolation of pharmacologically active
benzodiazepine receptor ligands from Tilia tomentosa
(Tiliaceae). Journal of Ethnopharmacology, v. 44,
n.1, p.47-53, 1994.
WAGNER, H.; BLADT, S. Plant Drug Analysis. A thin layer
chromatography atlas, 2a. edição. Springer, Berlin,
1996, 400p.
XU, Y.K. et al. Alkaloids from Gelsemium elegans. Journal
of Natural Products, v. 69, n. 9, p.1347-350, 2006.
ZHU, W.L. et al. Antidepressant effect of baicalin extracted
from the root of Scutellaria baicalensis in mice and rats.
Pharmaceutical Biology, v.44, n.7, p.503-510, 2006.
Rev. Bras. Pl. Med., Campinas, v.15, n.2, p.217-224, 2013.