A new antioxidant flavonoid from the lianas of gnetum macrostachyum

Article abstract of DOI:10.1080/14786410802280943

A new flavonoid, named 5,7,2-trihydroxy-5-methoxyflavone (1), was isolated from the acetone extract of the lianas of Gnetum macrostachyum together with 5,7,4-trihydroxy-3-methoxyflavanone (2) and seven known stilbenoids (3-7). The structure of the new com

Full text of DOI:10.1080/14786410802280943

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A new antioxidant flavonoid from the
lianas of Gnetum macrostachyum
Suwannee Saisin a , Santi Tip-pyang a & Preecha Phuwapraisirisan a
a Natural Products Research Unit, Department of Chemistry,
Faculty of Science , Chulalongkorn University , Bangkok, Thailand
Published online: 29 Oct 2009.
To cite this article: Suwannee Saisin , Santi Tip-pyang & Preecha Phuwapraisirisan (2009) A new
antioxidant flavonoid from the lianas of Gnetum macrostachyum , Natural Product Research:
Formerly Natural Product Letters, 23:16, 1472-1477, DOI: 10.1080/14786410802280943
To link to this article: http://dx.doi.org/10.1080/14786410802280943
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Natural Product Research
Vol. 23, No. 16, 10 November 2009, 1472–1477
A new antioxidant flavonoid from the lianas of Gnetum macrostachyum
*
Suwannee Saisin, Santi Tip-pyang and Preecha Phuwapraisirisan
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn
University, Bangkok, Thailand
(Received 28 November 2008; final version received 14 July 2008)
A new flavonoid, named 5,7,20-trihydroxy-50-methoxyflavone (1), was isolated
from the acetone extract of the lianas of Gnetum macrostachyum together with
5,7,40-trihydroxy-30-methoxyflavanone (2) and seven known stilbenoids (3–7).
The structure of the new compound was determined by interpretation of its
spectral data and chemical transformation. The isolated compounds showed
radical scavenging activity against DPPH with IC50 values in the range of 0.21–
19.90 mM.
Keywords: Gnetum macrostachyum; Gnetaceae; 5,7,20-trihydroxy-50-methoxyfla-
vone; antioxidation
1. Introduction
The genus Gnetum (Gnetaceae) consists of approximately 40 species, distributed in South
America (Amazon region), Southwest Africa, and the tropical and subtropical zones of
Asia (Ali et al., 2003). The family Gnetaceae has been recognised as an abundant source of
stilbene oligomers, in addition to the families Cyperaceae, Dipterocarpaceae, Leguminosae
and Vitaceae (Sotheeswaran & Pasupathy, 1993). Resveratol is one of the prominent
stilbenoids, which possess various intriguing biological activities, such as antioxidant,
antimutagenic, being an inducer of phase II drug-metabolising enzymes, antifungal, anti-
inflammatory, antiviral, antibacterial and antiplatelet aggregation (Huang, Lin, & Cheng,
2001; Huang, Tsai, Shen, & Chen, 2005; Kim, Saleem, Seo, Jin, & Lee, 2005; Pryce &
Langcake, 1977; Villano, Fernandez-Pachon, Moya, Troncoso, Garcia-Parrilla, 2007).
In our continuing search for antioxidant compounds, the CH2Cl2 extract of Gnetum
macrostachyum lianas exhibited potent radical scavenging towards DPPH. This article
deals with the isolation and structure elucidation of a new flavonoid, 1, including
antioxidant activity of the isolated compounds (Figure 1).
2. Results and discussion
5,7,20-Trihydroxy-50-methoxyflavone (1) was isolated as an amorphous yellow solid
[m.p. 269ꢀC (dec)]. The molecular formula was established to be C16H12O6, based on the
*Corresponding author. Email: preecha.p@chula.ac.th
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802280943
http://www.informaworld.com

Natural Product Research
1473
OMe
OH
OH
2
R
2′B
5′
OMe
1
2
O
HO
R
O
HO
7
A
4
5
OH
OH O
OH O
R
1
2
1 R = R = OH
3 R = H
2
1
2
1a R = R = OMe
4 R = OMe
1
2
1b R = OH, R = OMe
1
2
1c R = OMe,R = OH
Me
O
OH
OH
OMe
HO
OH
H
Me
O
H
O
HO
HO
H
H
H
H
HO
O
H
OH
OH
H
HO
OH
H
OH
HO
H
OH
OH
HO
5
OH
Me
7
6
MeO
HO
HO
O
H
HO
H
OH
OH
O
H
OH
HO
H
H
OMe
OH
H
H
OH
H
OH
HO
HO
8
9
Figure 1. Flavonoids and stilbenoids from G. macrostachyum.
[M þ Na]þ ion peak at m/z 323.0517 and 13C NMR data. The UV spectrum of 1 exhibited
maximal absorption bands at 270 and 345 nm, which are typical of a flavonoid moiety
(Mabry, Markham, & Thomas, 1970).
The IR spectrum showed absorptions of a hydroxyl group (3347 cmꢁ1), a carbonyl
group (1647 cmꢁ1), a carbon ether bond (1347 cmꢁ1), and an aromatic moiety (1600–
1400 cmꢁ1). The 1H NMR spectrum (Table 1) revealed the singlet resonance of a chelated
hydroxyl proton at ꢀH 12.90 (5-OH), in addition to a pair of exchangeable hydroxyl
protons (ꢀH 10.42 and 9.30). The signals in the aromatic region (ꢀH ca 6.0–7.8) were
assigned as two isolated spin systems, 6.20 and 6.46 (each 1H, d, J ¼ 2.0 Hz), for an AB
system, as well as 6.95 (1H, d, J ¼ 8.4 Hz), 7.51 (1H, d, J ¼ 8.4 Hz) and 7.53 (1H, brs) for
an ABX system. The 13C NMR spectrum showed 16 signals, three of which (ꢀC 182.1,
164.0 and 103.3) were indicative of a flavone moiety. The overall structure of 1 was
deduced mainly based on HMBC data (Figure 2). In ring A, the correlations of 5-OH/C-5,
C-6 and C-10 and 7-OH/C-6, C-7 and C-8 indicated that C-5 and C-7 were accommodated
by two hydroxyl groups. The methoxy group (ꢀH 3.95) was placed in ring B at C-50
(ꢀC 149.3), which was in turn coupled to H-30 and H-60. This assignment was further
confirmed by NOESY cross peaks of 50-OMe/H-40 and H-60/50-OMe, in addition to those
of H-30/H-40 and H-3/H-60. The presence of the hydroxyl group was also supported by the

1474
S. Saisin et al.
Table 1. 1H and 13C NMR spectral data (DMSO-d6) of 1.
Position
ꢀH (mult, J in Hz)
ꢀC
HMBC
1
2
3
4
5
6
7
8
164.0
103.3
182.1
162.0
99.0
6.65 (s)
C-2, 4, 10, 10
6.20 (d, 2.0)
6.46 (d, 2.0)
C-5, 7, 8, 10
C-6, 7, 9, 10
164.2
93.9
9
10
157.8
104.0
122.4
150.9
115.7
120.3
149.3
109.7
10
20
30
6.95 (d, 8.4)
7.51 (d, 8.4)
C-10, 20, 50
C-20, 60
40
50
60
7.53 (s)
12.90 (s)
10.42 (s)
9.30 (s)
3.93 (s)
C-2, 10, 20, 40, 60
C-5, 6, 10
C-6, 7, 8,
5-OH
7-OH
20-OH
50-OMe
55.7
C-50
H
3′
HO
H
H
4′
2′
1′
8
HO
H
O
HMBC
5′
OMe
2
9
7
6′
10
3
6
H
NOESY
4
H
5
OH
O
Figure 2. Key HMBC and NOESY correlations of 1.
formation of methyl ethers of 1. Treatment of 1 with trimethylsilyldiazomethane
(TMSCHN2) yielded 1a as the major product, together with two minor methyl ethers,
1b and 1c. Two additional methoxy groups newly generated in 1a indicated the presence of
7-OH and one hydroxyl group on ring B, in addition to chelated 5-OH. Therefore, the
structure of 5,7,20-trihydroxy-50-methoxyflavone was depicted for 1.
All isolated compounds were subjected to an examination of their radical scavenging
against DPPH (Table 2). Compound 1 showed slightly weak activity with an IC50 value of
19.9 mM. Conversely, resveratol (3) and other stilbenoids (4–9) displayed stronger
inhibition, with IC50 values in the range of 0.21–4.23 mM: of which, gnetulin (6) was the
most active. Stilbenes have been recognised as potent oxygen radical scavengers, possibly

Natural Product Research
Table 2. DPPH radical scavenging.
1475
DPPH radical
scavenging (IC50, mM)
Test compounds
1
2
19.9
1.48
3
4
5
6
7
8
0.40
0.30
0.78
0.21
2.90
4.23
0.53
0.11
9
Ascorbic acid
through an oxidative coupling reaction forming stable oligostilbenes. A recent investiga-
tion has shown that stilbenes undergo oxidation by nitrite ions (NOꢁ2 ), a key mediator in
the inflammatory response and in carcinogenesis (Panzella et al., 2006). A comprehensive
study on radical scavenging of stilbenes towards causative reactive species would provide
an insight into the prevention of pathological diseases.
3. Experimental
3.1. General experimental procedure
Melting points were determined on a Fisher–John apparatus and are uncorrected.
UV spectra were taken on a UV-160A spectrometer (SHIMADZU). IR spectra were
recorded on a Nicolet Impact 410 spectrometer. HRESIMS and LCMS were obtained by a
Micromass LCT mass spectrometer and a Micromass Quattro MicroTM API. NMR
spectra were recorded on a Variant Mercury 400 spectrometer and chemical shifts were
reported in parts per million reference to solvent residues (ꢀH 2.49 and ꢀC 39.7 ppm for
DMSO-d6 and ꢀH 7.25 and ꢀC 77.0 ppm for CDCl3).
3.2. Plant material
The lianas of G. macrostachyum were collected in April 2006 from Nakornphanom.
The plant material was identified by the Plants of Thailand Research Unit, Department
of Botany, Faculty of Science, Chulalongkorn University, and the voucher specimen
(BKF 108547) has been deposited in the Herbarium of the Royal Forest Department,
Bangkok, Thailand.
3.3. Extraction and isolation
The dried and powdered lianas of G. macrostachyum (2.5 kg) were extracted with CH2Cl2,
acetone and MeOH, respectively, in a Soxhlet extractor. A part of the acetone extract was
stirred with acetone, yielding acetone soluble and insoluble fractions. The acetone soluble
fraction (47.4 g) was fractionated on vacuum liquid chromatography (VLC) eluted with

1476
S. Saisin et al.
CH2Cl2, MeOH : CH2Cl2 (10 : 90 ! 50 : 50), MeOH, yielding seven fractions labelled ‘F1’
to ‘F7’. Fraction F2 was purified on silica gel CC using CH2Cl2, MeOH : CH2Cl2,
(5 : 95 ! 50 : 50) and MeOH, to afford six fractions, labelled ‘G1’ to ‘G6’. Fraction G3 was
subsequently purified on silica gel CC using MeOH : CH2Cl2, (0 : 100 ! 100 : 0) to afford
5,7,20-trihydroxy-50-methoxyflavone (1, 30 mg). A portion of fraction G4 (21 g) was
purified on silica gel CC using CH2Cl2, MeOH : CH2Cl2, (0 : 100 ! 100 : 0) and further
purified on Sephadex LH 20 using MeOH : CH2Cl2 (50 : 50) to give resveratol (3, 200 mg)
and 3-methoxyresveratol (4, 600 mg). Fraction G5 was purified on silica gel CC using
CH2Cl2, MeOH : CH2Cl2, (0 : 100 ! 100 : 0) and further purified on Sephadex LH 20 using
MeOH : CH2Cl2 (50 : 50) to give 5,7,40-trihydroxy-30-methoxyflavanone (2, 12 mg) and
shegansu B (5, 80 mg). Fraction F3 was purified on silica gel CC using CH2Cl2,
MeOH : CH2Cl2, (5 : 95 ! 50 : 50), MeOH to afford seven fractions, labelled ‘H1’ to ‘H7’.
Fraction H4 was purified on Sephadex LH 20 using MeOH : CH2Cl2 (50 : 50) to afford
gnetulin (6, 25 mg), gnetuhainin C (7, 35 mg), parvifolol B (8, 20 mg) and pallidol
(9, 11 mg).
5,7,20-Trihydroxy-50-methoxyflavone (1): Amorphous yellow solid; m.p. 269ꢀC (dec); UV
(MeOH) ꢁmax (log ") 227 (3.09), 270 (2.93), 345 (3.06); IR (KBr) 3347, 3082, 1647, 1621,
1
1517, 1347, 1269, 1204, 1165, 826 cmꢁ1; H NMR (DMSO-d6, 400 MHz) and 13C NMR
(DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z [M þ Na]þ 323.0517 (Calcd for C16
H12O6Na, 323.0526).
3.4. Preparation of methyl ethers of 1
Compound 1 (10 mg), dissolved in MeOH (2 mL), was added drop wise to 2.0 M
trimethylsilyldiazomethane (TMSCHN2) in hexane until yellow solution persisted. The
mixture was stirred at room temperature for 1 h. After the reaction mixture was
evaporated to dryness, the residue was purified on preparative TLC developed with 5 : 95
MeOH : CH2Cl2 to afford 1a (7 mg), 1b (2 mg) and 1c (2 mg).
1
1a: Yellow powder; H NMR (CDCl3) ꢀ 12.90 (1H, 5-OH), 3.86 (3H, s), 3.84 (3H, s),
3.79 (3H, s), 6.13 (1H, d, 2.0), 6.43 (1H, d, 2.0), 6.60 (1H, s), 7.03 (1H, d, 8.4), 7.55 (1H, d,
8.4), 7.50 (1H, s).
1
1b: Yellow powder; H NMR (CDCl3) ꢀ 12.90 (1H, 5-OH), 3.80 (3H, s), 3.82 (3H, s),
6.20 (1H, d, 2.0), 6.60 (1H, d, 2.0), 6.65 (1H, s), 7.05 (1H, d, 8.4), 7.60 (1H, d, 8.4),
7.50 (1H, s).
1
1c: Yellow powder; H NMR (CDCl3) ꢀ 12.90 (1H, 5-OH), 3.80 (3H, s), 3.85 (3H, s),
6.20 (1H, d, 2.0), 6.59 (1H, d, 2.0), 6.65 (1H, s), 6.90 (1H, d, 8.4), 7.50 (1H, d, 8.4),
7.55 (1H, s).
3.5. DPPH radical scavenging
Radical scavenging activity of the isolated compounds was validated using DPPH
colorimetric method (Phuwapraisirisan, Udomchotphruet, Surapinit, & Tip-pyang, 2006;
Wiboonpun, Phuwapraisirisan, & Tip-pyang, 2004). Briefly, a sample solution (0.25 mM,
0.5 mL) was added to 1 mL methanolic solution of DPPH (final concentration
of DPPH was 0.3 mM). The mixture was vigorously shaken and kept in the dark
for 30 min. The absorbance of the resulting solution was measured at 518 nm with a
UV spectrometer.

Natural Product Research
1477
Acknowledgements
S. Saisin is grateful to Huacheiwchalermprakiet University for supporting the doctoral scholarship.
We thank the Plants of Thailand Research Unit, Department of Botany, Chulalongkorn University
for plant identification.
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