Services on DemandArticleIndicatorsRelated linksShareOn-line version ISSN J. Braz. Chem. Soc. vol.16 no.3b S?o Paulo May/June 2005 http://dx.doi.org/10.-00005
A new neolignan and antioxidant
phenols from Nectandra grandiflora
Alan B. RibeiroI; Vanderlan da
S. BolzaniI; Massayoshi YoshidaII,III; Leonardo S. SantosIV;
Marcos N. EberlinIV; Dulce H. S. SilvaI,
IInstituto de Qu&mica, Universidade
Estadual Paulista, CP 355,
Araraquara - SP, Brazil
IIInstituto de Qu&mica, Universidade de S&o Paulo,
CP 2-970 S&o Paulo - SP, Brazil
IIIInstituto de Pesquisas da Amaz&nia, CP 476,
- AM, Brazil
IVInstituto de Qu&mica, Universidade Estadual de Campinas,
CP -971 Campinas - SP, Brazil
The EtOH crude extract from the leaves of Nectandra
grandiflora collected in the Atlantic Forest, Brazil, showed antioxidant
activity towards b-carotene in a TLC assay. The bioassay-guided
fractionation led to the isolation of protocatechuic acid and two flavonol glycosides:
afzelin and quercetrin, which showed free radical scavenging activity towards
DPPH (DA 32, 16 and 73% at 50 µmol
L-1) and were compared to commercial antioxidants rutin (81% at 50
µmol L-1) and BHT (9% at 50 µmol
L-1), used as standard compounds. Additionally, three inactive neolignans,
including the new bicycle[3.2.1]octane neolignan 2'-oxo-piperol B were obtained
and characterized by spectrometric methods.
Keywords: Nectandra grandiflora,
Lauraceae, neolignan, flavonoid, radical scavenging activity, antioxidant
O extrato etan&lico das folhas de Nectandra
grandiflora coletadas na Mata Atl&ntica, Brasil, mostrou atividade
antioxidante frente ao b-caroteno em teste usando
cromatografia em camada delgada. O fracionamento biomonitorado levou ao isolamento
do &cido protocatecuico e de dois flavon&ides glicosilados: afzelina
e quercetrina, os quais exibiram atividade sequestradora de radicais livres
frente ao DPPH (DA 32, 16 e 73% a 50 µmol
L-1) e foram comparados aos antioxidantes comerciais rutina (81%
a 50 µmol L-1) e BHT (9% a 50 µmol
L-1), usados como subst&ncias de refer&ncia. Al&m
disso, tr&s neolignanas inativas, incluindo a nova neolignana biciclo[3.2.1]oct&nica
2'-oxo-piperol B, foram obtidas e caracterizadas por m&todos espectrom&tricos.
Introduction
Nectandra grandiflora Nees is a tree belonging
to Lauraceae family, which is known for economically important species such
as Aniba roseodora ("pau-rosa"), the main source of sesquiterpene
(linalool)-rich essential o and species with scented leaves
and bark such as Laurus nobilis (bay leaves) and Ocotea odorifera
(Brazilian cinnamon), widely used as condiments.1-3 Lignans and neolignans
are important classes of secondary metabolites occurring in Lauraceae species,
which include several medicinally important closely related compounds as the
anti-mitotic podophyllotoxin, extracted from Podophyllum peltatum (Berberidaceae),
used in the preparation of the antitumoral lignans etoposide and teniposide,
and the antioxidant nor-dihydroguaiaretic acid (NDGA), obtained from
Larrea tridentata (Zygophyllaceae).4
Nectandra species are extensively used
in folk medicine due to their antirrheumatic, digestive and diuretic properties.5
Some of the claimed medicinal properties have been substantiated by studies,
e.g. on Nectandra salicifolia, which showed antifungal and vascular muscle
relaxing properties,6 in addition to antimalarial activity due to
the presence of the alkaloid costaricin in its bark extract.7 Previous
work on N. grandiflora evidenced the presence of aporphine alkaloids
and antitumoral activity in its bark extract.8
A number of pathologies are known to be ultimately
associated with the imbalance of pro and antioxidant factors in living systems.
Exogenous antioxidant compounds may therefore exert beneficial actions upon
systems which have been deprived from sufficient amounts of endogenous antioxidants
as in some cardiovascular diseases, tumors, inflammation, ulcer and aging.9
As part of our ongoing projects aiming the phytochemical
investigation of S&o Paulo State flora,10-12 the EtOH crude
extract from leaves of N. grandiflora collected at Jureia State Park,
Atlantic Forest, have been subjected to bioassay-guided fractionation using
the b-carotene test on TLC for the detection of antioxidant
compounds.13 Pure compounds isolated from this extract were further
evaluated for their free radical scavenging activity towards the stable free
radical DPPH and compared to standard compounds rutin and BHT.11,14 Non-active
fractions were also investigated and afforded the benzofuran neolignan burchellin
and two bicyclooctane[3.2.1] neolignans: piperol B and the new 2'-oxo-piperol
Results and Discussion
The EtOH crude extract of N. grandiflora
leaves was successively partitioned with organic solvents affording hexane,
CHCl3, EtOAc and MeOH extracts. The TLC test using b-carotene
as revealing agent indicated the presence of antioxidant compounds in the EtOAc
and MeOH fractions. Chromatographic fractionation of these extracts led to the
isolation of protocatechuic acid (6) and flavonol glycosides afzelin
(4) and quercetrin (5), which were identified through analyses
of their MS and NMR spectra and comparison with literature data.15,16
Fractionation of the CHCl3 extract led to the isolation of neolignans
Compound 1 was obtained as a light brown
viscous oil and its 13C NMR spectrum showed 12 signals for sp2
carbons and 9 signals for sp3 carbons, including three signals
at ca. d 56, assigned to three methoxy groups
on sp2 carbons (). These data combined
with HRMS analysis in the positive ion mode [(C21H24O5,
(M+1)+? 357; expanded to C18H15O2.(OCH
3)3] suggested compound 1 is a bicarbonyl neolignan,
with 13C NMR signals for C=O groups at d
190.5 and d 203.0.
The 1H NMR spectrum of compound 1
showed three broad doublets at d 2.48, d
5.08 and d 5.14, and one multiplet at d
5.89 assigned to hydrogens of an allyl moiety. Additionally, a doublet at d
0.99 (3 H-9) and a quintuplet at d 2.08 (H-8) indicated
compound 1 is a guianin type bicyclooctane neolignan.17,18
The absence of a doublet at ca. d 5.90 for
an oxybenzylic hydrogen of the related benzofuran type neolignans, gave additional
support to this proposal. Its relative stereochemistry was deduced from the
absence of anisotropy from the aromatic ring on the deshielded H-9 (d
0.99) evidencing a trans relationship between methyl group C-9 and the
aromatic ring.17,18 Moreover, the bicycle bridge associated with
the guianin type configuration of 1 is evidenced by the absorption of
H-6' at d 5.72. Further analysis of NMR spectra and
comparison with literature data for other guianin-type compounds suggested the
presence of an a,b-unsaturated
carbonyl moiety and an additional carbonyl (C-2', n
1760 cm-1) in the bicyclic moiety.17,18 HMQC correlations
allowed the complete assignment of hydroge and HMBC correlations
of C-2' to H-7, H-3' and H-7'; and C-4' to H-7 and H-3' supported the position
of carbonyl groups at C-2' and C-4' in the bicyclic moiety, respectively (). This spectrum also showed correlations of C-5' to H-3'; C-7' to H-6';
and C-1 to H-7, H-8 and H-3', which supported the position of one methoxyl at
C-5', the allyl group at C-1' and aromatic ring at C-7. Additional cross-peaks
in the HMBC spectrum gave support to the proposed structure for compound 1
ESI(+)-MS/MS experiments for the protonated molecule
of m/z 357 with dissociation induced by collisions with argon provides
further structural characterization as dissociation forms major ionic fragments
of m/z 316 (24), 341 (8), and 301 (22) owing to the losses of an allyl,
methyl and allyl plus methyl radicals, in addition to a fragment ion of m/z
151 (9), assigned to the dimethoxytropilium ion. Additionally, the protonated
1, of m/z 357, dissociates by the loss of a neutral molecule of
1,2-dimethoxybenzene to yield the ion of m/z 219 (), which likely rearranges to form an acylium ion that subsequently loses
a neutral C3H4 allene to yield an ion of m/z 179.
Additional data from a LRMS experiment showed the base peak of m/z 179
and peaks at 357, 325, 316 and 219 (see Experimental section), which gave support
to the structure proposed for compound 1. These data are fully consistent
with the new bicyclooctane neolignan 1, which is therefore assigned as
(7S*, 8R*, 1'R*, 3'R*)-3,4,5'-trimethoxy-3,7'-cyclo-8,1'-neolig-8'-ene
-2,4'(3'H)-dione.
Compound 2 was identified as the bicycle
[3.2.1]octane neolignan piperol B and compound 3, as the benzofuran neolignan
burchellin, previously isolated from Piper betle and Aniba burchelli,
respectively.19,20 Compound 2 is aguianin type and compound
3 is a porosin type neolignan and their identification was based on extensive
analyses of NMR data as well as on comparison with literature data for guianin,
porosin and ferrearin type neolignans, which are widely distributed in Lauraceae
species.21
The antiradicalar activity of compounds 1-6
has been evaluated towards the stable free radical DPPH, which exhibits an absorption
maximum at 517 nm, evidencing prominent activities of compounds 4-6
() and poor activities for compounds 1-3.
The absence of phenolic hydroxyls in the latter renders them less effective
in promoting DPPH reduction, therefore appearing as poor radical scavengers.
The free radical scavenging activity of flavonoids and other phenols is mostly
due to their aromatic hydroxy groups, which confer great stability to the phenolic
radical as soon it is formed, after one hydrogen radical donation to DPPH. The
stronger activity of compound 5 when compared to compounds 4 and
6 is probably due to the catechol moiety in the B ring of quercetrin
along with its extended conjugation through the a,b-unsaturated
carbonyl system on the C ring, which are some of the major structural features
associated with antioxidant activity of flavonoids.22 The comparison
with standard compound rutin (DA=81% at 50 µmol
L-1) evidences the reported major roles of some structural features
of flavonoids as the catechol moiety present in the B ring and the C2-C3 double
bond for the enhancement of the free radical scavenging activity.22
On the other hand, the synthetic antioxidant BHT (DA=9%
at 50 µmol L-1) exhibited the weakest
antiradicalar activity as expected, due to its slow reaction rate with DPPH.23
Experimental
General experimental procedures
Silica gel (Merck 230-400 Mesh) and Sephadex
LH-20 (Pharmacia) were used for all column chromatography separations unless
otherwise stated. HPLC separations were performed on a Waters Prep LC 4000 System
and C-18 Luna (Phenomenex) columns (4.6 mm and 20mm i.d. for analytical and
preparative columns, respectively) and pre-columns were used. All solvents (Merck
or Malinckrodt) were of analytical or HPLC grade. 1H and 13C
NMR spectra were recorded on a Varian Unity 500 spectrometer at 500 and 125
MHz, respectively, using CDCl3 or DMSO-d6 as solvent
and TMS as reference. IR spectra were obtained on a Nicolet spectrometer. Low
resolution ESI(+)-MS were recorded on a VG Platform II spectrometer. High resolution
ESI(+)-MS analysis were performed on a hybrid double quadrupole (Qq) and orthogonal
time-of-flight (Tof) mass spectrometer (Qtof, Micromass UK) operating at 7.000
resolution and mass accuracy of 10 ppm. The temperature of the nebulizer was
50 ?C. The ESI and mass spectrometer were operated in the positive ion
mode. The cone and extractor potential were set to 40 and 0 V, respectively.
The m/z scan range was from 50 up to 1000. Tandem mass spectrometric
[ESI(+)-MS/MS] experiments were performed using the product ion scan mode via
Q1 mass selection of the desirable product ion, q2 collision-induced dissociation
(CID) with N2, and orthogonal TOF mass analysis of the CID ionic
fragments. The collision energy ranged from 15 to 25 eV, depending on the dissociation
lability of the precursor ion.
Plant material
Nectandra grandiflora leaves were collected in
Jureia-Itatins State Park, Atlantic Forest, Brazil, and identified by Dr. Ines
Cordeiro. The voucher specimens (number: Moraes 05) were deposited at Herbarium
of Instituto de Bot&nica-SEMA, SP, Brazil.
Extraction and isolation
Dried and ground leaves (2.0 kg) were extracted
with ethanol at r.t. and afforded a dark green EtOH extract (5.0 g) after solvent
evaporation under vacuum. Partition of the EtOH extract with organic solvents
gave the hexane (0.6g), CHCl3 (1.6 g), EtOAc (0.5 g) and hydroalcoholic
(2.3 g) extracts after solvent evaporation. MPLC on silica gel of the CHCl3
extract (1.6 g, hexane/EtOAc gradient) gave 15 pooled fractions (A1-A15). Fraction
A2 (41.0 mg) was submitted to preparative TLC (CHCl3/acetone/HOAc
90:9:1) and gave compound 2 (12.2 mg). Fraction A3 (32.1 mg), obtained
from the elution with hexane/EtOAc 4:1, was further purified by preparative
TLC (CHCl3/acetone/HOAc 90:9:1) and gave compound 1 (6.8 mg).
Fraction A6 (88.1 mg) was recrystallized in MeOH to afford compound 3
(25.5 mg). CC on Sephadex LH-20 of the EtOAc fraction (200.0 mg) eluted with
MeOH resulted in 42 subfractions which were pooled after TLC analysis (B1-B4).
Fr. B2 (38.7 mg) was submitted to preparative TLC (CHCl3/MeOH/H2O/HOAc
79:18:2:1) and gave compound 4 (7.1 mg). Purification of fraction B3
(45.1 mg) by preparative TLC (CHCl3/MeOH/H2O/ HOAc 36:12:1:1)
afforded compound 5 (14.0 mg). The hydroalcoholic fraction (200.0 mg)
was submitted to CC on Sephadex LH-20 eluted with MeOH resulting in 10 fractions
(C1-C10). Purification of fraction C-8 (37.7 mg) by RP-HPLC (C18 Luna-Phenomenex
column, MeOH/H2O/ HOAc 55:43:2, 8 mL min-1, detection
at 280 nm) gave compound 6 (8.1 mg).
Bleaching of b-carotene
on TLC plates
Samples were eluted on TLC plates which were
then dried and sprayed with 0.02% solution of b-carotene
(Aldrich) in CH2Cl2. Plates are placed under natural light
until discoloration of background. The remaining yellow spots indicate the presence
of antioxidants.13
Reduction of 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH)
The free radical scavenging capacities of the
isolates and standard compounds rutin and BHT were evaluated from their ability
to reduce the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Each compound
(1 mg) was solubilized in methanol (10 mL) to yield a stock solution. Several
dilutions of each compound from 5 µmol L-1
to 100 µmol L-1were then prepared in methanol
and to each sample a solution of DPPH (0.004%) was added. Absorbance at 517
nm was determined after 30 min on a Milton Roy 20D spectrophotometer and the
percentage of activity was calculated as DA= (Ao-A)/Ao.11,
(7S*,8R*,1'R*,3'R
*)-3,4,5'-trimethoxy-3,7'-cyclo-8,1'-neolig-8'-ene-2,4'(3'H)-dione (1).
Light brown oil. IR(film) nmax/cm-1:
, , ; HRMS [ESI(+)-MS]: C21H25O5
[M + H]+ m/z, calc. 357.1702, found 357.1614; tandem ESI(+)-MS-MS
from selected ion of m/z 357, m/z (%): 316 (24), 341 (8), 301
(22), 219 (42), 179 (100), 151 (9). LRMS [ESI(+)-MS], m/z (%): 357 (41),
325 (18), 316 (68), 219 (42), 179 (100); 1H and 13C NMR
spectral data are given in .
Acknowledgements
This work was supported by the State of S&o
Paulo Research Foundation (FAPESP) within the BIOTA/FAPESP - The Biodiversity
Virtual Institute Program (),
CNPq (Conselho Nacional de Desenvolvimento Cient&fico e Tecnol&gico
- Brazil) and CAPES. A.B.R. thanks CAPES for providing him a scholarship.
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Received: March 9, 2004
Published on the web: March 4, 2005
FAPESP helped in meeting the publication costs of this article.