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Flavonol glycosides found in hydroethanolic extracts from Tilia cordata, a species utilized as anxiolytics

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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 flavonoids reported as anxiolytics. The major flavonoids 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.
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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 cromatograa 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
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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 identied 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 identied 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 certicates. 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 acidied 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 basied 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 prole, the
extract was initially analyzed via TLC (Stahl, 1969;
Wagner & Bladt, 1996; Jayaprakasha et al., 2006).
Dried TLC plates were sprayed with specic 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. Identication of the kaempferol
and quercetin aglycones after hydrolysis reactions
were conrmed 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 sufcient 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-glycosylavonol. 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-glycosylavonol. 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. Identication
of compounds 6 was also conrmed 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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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 identied 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-
inammatory 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 interavonoid 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 purication of an ethanol extract of
Tilia petiolaris DC. inorescences resulted in the
isolation and identication 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 Passiora 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
modications 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 identied as promising
phytochemical class (Psterer 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).
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The flavonoid baicalin, isolated from the dried root of Scutellaria baicalensis. G. (Labiatae), is widely used in traditional Chinese herbal medicine. In the present study, baicalin, at doses of 20, 40, and 80 mg/kg (p.o.), reduced immobility time in tail suspension test (TST) and the forced swimming test (FST) in mice. Baicalin also decreased immobility time at 12.5, 25, and 50 mg/kg (p.o.) in FST in rats. Furthermore, baicalin (25 mg/kg), as well as fluoxetine (FLU; 20 mg/kg), showed a significant recovery in sucrose intake compared with the vehicle-treated stressed animals for 5 weeks treatment in a chronic mild stress (CMS) model in rats. The effect of baicalin at the dose of 25 mg was as potent as that of reference antidepressant FLU (20 mg/kg) in the CMS model. With the monoamine oxidase (MAO A and B) assay, oral administration of baicalin at the doses of 12.5, 25, and 50 mg/kg significantly inhibited MAO A activity in a dose-dependent manner in rats. However, only baicalin at the doses of 25 and 50 mg/kg markedly inhibited MAO B activity. Neither baicalin nor FLU, at the doses tested, produced a significant effect on locomotor activity in mice. These results suggest that baicalin had a specific antidepressant-like effect in vivo.. The antidepressant activity of baicalin may be mediated in part through MAO A and B inhibition in rat brain.
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A qualitative and quantitative characterisation of the main non-anthocyanin phenolic compounds from two different colored oca (Oxalis tuberosa Mol.) genotypes with potential antioxidant capacity was carried out by high performance liquid chromatography with photodiode array detection (HPLC-DAD). Phenolic compounds were fractionated in two main fractions: an aqueous (Faq) and a ethyl acetate fraction (Fea). In addition, the contribution of these phenolic fractions to the antioxidant capacity was evaluated. The Faq revealed the presence of caffeic, vanillic and cinnamic acid derivatives, flavan-3-ols and flavones derivatives, as the main non-anthocyanin phenolic compounds for both genotypes. Anthocyanins for the purple genotype were significantly present in this fraction. Acid hydrolysis revealed the presence of vanillic, caffeic and cinnamic acids and malvidin in Faq. The Fea was composed mainly of caffeic and cinnamic acid derivatives as well as flavan-3-ols, flavones and flavanone derivatives. Based on their UV–Vis spectral data the flavan-3-ols, flavones and flavanones detected in both fractions seem to correspond to bound forms of catechin, luteolin and apigenin and naringenin, respectively. The Faq fractions were the major contributors to the ABTS antioxidant capacity (77–82%). The results obtained in the present study suggest that oca tubers could potentially be considered beneficial for human health and for potential industrial applications.
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The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar range of non-acylated flavonoids.
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To compare the anxiolytic activities and flavonoid compositions of the two populations of the species Passiflora edulis, Passiflora edulis 'edulis' with purple fruit and Passiflora edulis 'flavicarpa' with yellow fruit. Four samples for each population of Passiflora edulis were collected from different districts of China. Swiss albino mice were used as experimental animals in elevated plus-maze (EPM) test to assay the anxiolytic effects of ethanol extracts of the samples. The conventional parameters and ethological items of the behavior of the mice were recorded and analyzed. Flavonoid compositions of the samples were analyzed by RP-HPLC monitored with diode array detection and the chromatograms were compared. The ethanol extracts of the samples of Passiflora edulis 'flavicarpa' displayed anxiolytic activity at 400 mg/kg, while those of Passiflora edulis 'edulis' exhibited sedative effect at 400 mg/kg. The chromatograms of the samples belonging to similar population of Passiflora edulis were identical, but those belonging to different population were distinct from each other. The series of peaks between 16 and 24 min in the chromatograms of Passiflora edulis 'flavicarpa' did not appear in those of Passiflora edulis 'edulis', either did the peaks between 54 and 90 min in chromatograms of Passiflora edulis 'edulis' not appear in those of Passiflora edulis 'flavicarpa'. The six major flavonoid compounds isolated from the leaves of Passiflora edulis 'flavicarpa', lucenin-2, vicenin-2, isoorientin, isovitexin, luteolin-6-C-chinovoside, and luteolin-6-C-fucoside, had not been detected in Passiflora edulis 'edulis'. Passiflora edulis 'flavicarpa' is extremely different from Passiflora edulis 'edulis' and they should be distinguished when pharmacological studies are performed on them. The aerial part of Passiflora edulis 'flavicarpa' is possible to be utilized as the resource of Passionflower Extract.
Article
Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-α-l-(2″,4″-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC₅₀ 10.4 μM). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.