Mai Thanh Thi Nguyen* and Nhan Trung Nguyen Faculty of Chemistry, University of Science, Vietnam National University, HoChiMinh City, Vietnam From the MeOH extract of the aerial part of
Trang 1Xanthine Oxidase Inhibitors from Vietnamese
Blumea balsamifera L.
Mai Thanh Thi Nguyen* and Nhan Trung Nguyen
Faculty of Chemistry, University of Science, Vietnam National University, HoChiMinh City, Vietnam
From the MeOH extract of the aerial part of Blumea balsamifera L., a new dihydroflavonol, (2R,3S)-(
)-4′-O-methyldihydroquercetin (1), together with seven known compounds has been isolated Their structures were
elucidated on the basis of spectroscopic data Compounds 1–4 and 6–8 displayed significant xanthine oxidase
inhibitory activity in a concentration-dependent manner, and compounds 1, 6 and 8 showed more potent inhibitory
activity, with IC 50 values ranging from 0.23 to 1.91m M , than that of a positive control allopurinol (IC 50 2.50 m M ).
Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: Blumea balsamifera; Asteraceae; (2R,3S)-( )-4′-O-methyldihydroquercetin; xanthine oxidase inhibition.
Supporting information may be found in the online version of this article (Supplementary Material)
INTRODUCTION
Xanthine oxidase (XO) is a key enzyme that catalyses
the last step in the conversion of purines to uric acid,
and plays a vital role in producing hyperuricemia and
gout Allopurinol, the medication prescribed for gout
prevention, is a xanthine oxidase inhibitor (Oettl and
Reibnegger, 1999) However, due to the unwanted side
effects of allopurinol, such as hepatitis, nephropathy
and allergic reactions, new alternatives with increased
therapeutic activity and fewer side effects are desired
Moreover, superoxide anion radicals generated by XO
are involved in various pathological states such as
hepa-titis, inflammation, ischemia-reperfusion, carcinogenesis
and aging (Cos et al., 1998) Thus, the search for novel
XO inhibitors would be beneficial not only to treat gout
but also to combat various other diseases
Blumea balsamifera L (Asteraceae) has been used in
Vietnamese traditional medicine for the treatment of
rheumatism and inflammatory diseases (Do, 2001)
Sev-eral studies on the chemical constituents of B balsamifera
have been reported and a number offlavonoids,
monoter-penes and sesquitermonoter-penes have been isolated from this
plant (Nessa et al., 2004; Fazilatun et al., 2001) Our
preliminary screening study revealed that the methanol
extract of the aerial part of B balsamifera exhibited
significant XO inhibitory activity with an IC50 value of
6.0mg/mL (Nguyen et al., 2004) Therefore,
activity-guided fractionation of the MeOH extract was carried
out and a new dihydroflavonol (1) isolated together
with seven known compounds (2–8) In the present
study, the isolation and structure elucidation of the
new compound by spectroscopic techniques is reported,
together with the XO inhibitory activity of the isolated
compounds
MATERIAL AND METHOD
General experimental procedures Optical rotations were recorded on a Perkin-Elmer 241 digital polarim-eter The IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solutions The NMR spectra were taken on a Bruker Avance III 500 spectrometer (Bruker Biospin) with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in d values The HR-ESI-MS was performed
on a Micro O-QIITOF mass spectrometer (Bruker Daltonics) The CD spectra were measured in a Jasco J-805 spectropolarimeter Analytical and preparative TLC were carried out on precoated Merck Kieselgel 60F254or RP-18F254plates (0.25 or 0.5 mm thickness)
Plant material The aerial part of B balsamifera was collected at Lam Dong province, Vietnam, in October
2008 and was identified by Ms Hoang Viet, Faculty of Biology, University of Science, National University Ho Chi Minh City A voucher sample of the aerial part (AN-2962) has been deposited at the Department of Analytical Chemistry of the University of Science, National University Ho Chi Minh City, Vietnam
Extraction and isolation The aerial part ofB balsamifera was cut into pieces and then part (2.1 kg) was extracted with MeOH (12 L, reflux, 3 h 3) to yield a MeOH extract (180 g; IC50, 6.0mg/mL) The MeOH extract was suspended in H2O and partitioned successively with hexane, CHCl3, EtOAc and H2O to yield hexane (57 g;
IC50> 100 mg/mL), CHCl3(68 g; IC50, 5.0mg/mL), EtOAc (32 g; IC50, 1.0mg/mL) and H2O (15 g; IC50,> 100 mg/mL) fractions, respectively The CHCl3 fraction (62 g) was subjected to silica gel column (9 40 cm) chromatography eluted with MeOH/CHCl3(0–30%) to give four fractions: fr.1 (5.7 g; IC50,> 100 mg/mL), fr.2 (9.1 g; IC501.4mg/mL), fr.3 (11.2 g; IC50, 10.7mg/mL) and fr.4 (15.7 g; IC50, 71.4mg/ mL) Fraction 2 was rechromatographed on silica gel with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN: MeOH: H2O = 1: 1: 3, to give 7
* Correspondence to: Dr Mai Thanh Thi Nguyen, Faculty of Chemistry,
University of Science, Vietnam National University, HoChiMinh City,
Vietnam.
E-mail: nttmai@hcmus.edu.vn
PHYTOTHERAPY RESEARCH
Phytother Res (2012)
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3710
Copyright © 2012 John Wiley & Sons, Ltd.
Received 29 June 2011 Revised 13 October 2011 Accepted 17 October 2011
Trang 2(6.7 mg) and 8 (8.3 mg) Fraction 3 was further separated
by silica gel column chromatography, followed by
reversed-phase preparative TLC with CH3CN: MeOH:
H2O = 1: 1: 3, to give 3 (9.2 mg) The EtOAc fraction
(32 g) was subjected to silica gel column (7 40 cm)
chromatography eluted with MeOH/CHCl3(0–30%) to
give six fractions: fr.1 (5.2 g; IC50, > 100 mg/mL), fr.2
(4.4 g; IC50, 3.5mg/mL), fr.3 (4.6 g; IC50, 1.0mg/mL), fr.4
(3.5 g; IC50, 0.5mg/mL), fr.5 (13 g; IC504.7mg/mL) and fr.6
(2 g; IC50, 55.9mg/mL) Fraction 2 was subjected to silica
gel column chromatography with MeOH–CHCl3, followed
by reversed-phase preparative TLC with CH3CN: MeOH:
H2O = 1: 1: 4, to give 8 (5.3 mg) and 2 (14.6 mg) Fraction 3
was separated by silica gel column chromatography with
MeOH–CHCl3, followed by reversed-phase preparative
TLC with CH3CN: MeOH: H2O = 2: 1: 3, to yield 1
(2.5 mg) Fraction 4 was separated by silica gel column
chromatography with MeOH–CHCl3, followed by
reversed-phase preparative TLC with CH3CN: MeOH:
H2O = 1: 2: 3, to yield 4 (7.2 mg) Fraction 5 was separated
by silica gel column chromatography with MeOH–CHCl3,
followed by reversed-phase preparative TLC with MeOH:
H2O = 1: 1, to yield 5 (17.4 mg) and 6 (7.2 mg)
(2R,3S)-( )-4′-O-methyldihydroquercetin (1) [a]25
D – 12.5 (c 0.5, CH3OH) IR (CHCl3) cm 1: 3400, 1660,
1605, 1450 1H- and 13C-NMR (CD3OD, 500 MHz),
see Table 1 HR-ESI-MS m/z: 319.0830 [M + H]+(Calcd
for C16H15O7: 319.0818) CD[θ]2640, [θ]295–29146, [θ]317
0, [θ]336 +13160 (c 0.5, CH3OH) (For further
informa-tion, see supplementary data)
XO inhibitory assay The XO inhibitory activity was
assayed spectrophotometrically at 290 nm under aerobic
conditions by using 96-well plates as described previously
(Nguyen et al., 2004) The XO inhibitory activity was
expressed as the percentage inhibition of XO in the above
assay system, calculated as
A
100;
where A and B are the activities of the enzyme without
and with test material The IC50values were calculated
from the mean values of data from four determinations
RESULTS AND DISCUSSION The dried aerial part of B balsamifera was extracted with
refluxing MeOH, and the MeOH extract was suspended
in H2O and partitioned successively with hexane, CHCl3, EtOAc and H2O to yield hexane, CHCl3 and H2O fractions The CHCl3and EtOAc-soluble fractions showed
XO inhibitory activity with IC50values of 5.0 and 1.0mg/
mL, respectively Further separation and purification of these fractions led to the isolation of a new dihydroflavonol (1), together with seven known compounds (2–8) The known compounds were identified as (2R,3R)-(+)-4′-O-methyldihydroquercetin (2) (Islam and Tahara, 2000), (2R,3R)-(+)-4′,7-di-O-methyldihydroquercetin (3) (Nessa
et al., 2004), (2R,3R)-(+)-7-O-methyldihydroquercetin (4) (Harborne and Mabry, 1982); 5,7,3′,5′-tetrahydroxy flava-none (5) (Nessa et al., 2004), quercetin (6) (Nessa et al., 2004), quercetin-3,7,3′-trimethyl ether (7) (Kumari et al., 1986) and quercetin-3,3′,4′-trimethyl ether (8) (Urbatsch
et al., 1976) (Fig 1) based on the spectroscopic analysis and comparison with literature data
Compound 1 showed a quasimolecular ion at m/z 319.0830 [M + H]+, corresponding to the molecular for-mula C16H15O7in HR-ESI-MS Its IR spectrum displayed the absorbance of hydroxyl (3400 cm–1), phenyl (1605,
1450 cm–1) and carbonyl (1660 cm-1) groups The 1H NMR spectrum of compound 1 (Table 1) revealed the presence of one methoxy group at d 3.89 (3H, s) and two oxymethine groups at d 5.39 (1H, d, J = 3.5 Hz, H-2) and 4.42 (1H, d, J = 3.5 Hz, H-3) In addition, it displayed three ABX type protons at d 7.03 (1H, d, J = 2.0 Hz, H-2′), 6.84 (1H, d, J = 8.5 Hz, H-5′) and 6.94 (1H, dd, J = 2.0, 8.5 Hz, H-6′) together with two singlets of a 1,2,4,5-tetra-substituted benzene ring at d 5.98 (1H, d, J = 2.0 Hz, H-6) and 6.01 (1H, d, J = 2.0 Hz, H-8) Moreover, the 13C and DEPT NMR spectra exhibited signals for an meth-oxy, two oxymethines, five oxygenated aromatic quater-nary carbons, an aromatic quaterquater-nary carbon, five aromatic carbons and one carbonyl carbon On the basis
of the analysis of the COSY and HMQC spectra (Fig 2),
1 was suggested to be a dihydroflavonol The location of methoxy group was deduced to be at C-4′, based on the HMBC correlations between methoxy protons and C-4′
On the other hand, in 2, the H-2 and H-3 protons each resonated as a doublet with J = 11.5 Hz reflecting the trans-diaxial orientation normally found in naturally occurring 3-hydroxyflavanones (dihydroflavonols) However, both H-2 at d 5.39 and H-3 at d 4.42 of 1 had a coupling constant
of 3.5 Hz which suggests that in this compound the C-ring protons are related in a cis axial-equatorial fashion (Ingham
et al., 1986) Moreover, the negative and positive CD bands
at 295 and 336 nm in 1 were similar to those of (2R,3S)-3′,5-dihydroxy-4′,7-dimethoxydihydroflavonol suggested that the absolute configuration of this compound should be
‘2R, 3S’ (Islam and Tahara, 2000) Thus, the structure of 1 was deduced as (2R,3S)-( )-4′-O-methyldihydroquercetin The isolated compounds were tested for their XO inhibitory activity (Table 2) The assay was carried out
at five different concentrations ranging from 0.2–
100mM Compounds 1–4 and 6–8 possessed significant
XO inhibitory activity in a concentration-dependent manner, and compounds 1, 6 and 8 showed more potent inhibitory activity, with IC50values ranging from 0.23 to 1.91mM, than that of a positive control allopurinol (IC50, 2.5mM), a well known XO inhibitor used clinically for treatment of gout (Oettl and Reibnegger, 1999) (Fig 3)
Table 1. 1H- and13C-NMR data for compound 1 in CD 3 OD (J
values in parentheses)
6 ′ 6.94 (1H, dd, J = 2.0, 8.5 Hz) 118.9
M.T.T NGUYEN AND N.T NGUYEN
Trang 3The activity of these compounds greatly depended
upon the nature of the substitution in the flavonoids
The compounds having the hydroxyl groups at C-5 and
C-7 and the double bond between C2and C3in the ring
C (6 and 8) displayed stronger XO inhibitory activity
than those of the other compounds (2–5, 7), except 1
These results are in agreement with the previous report
on the structure–activity relationship of flavonoids and
their XO inhibitory activity (Cos et al., 1998) All isolated
flavonols (6–8) possessed stronger activity than that of
the positive control allopurinol and the presence of methoxyl groups at C-3, C-7, C-3′/C-4′ (7 and 8) instead
of hydroxyl group in 6 showed slightly improved activity Among the five flavanones, compounds having a hy-droxyl group at C-3 (1–4) displayed strong activities with
IC50values smaller than that of 5 without the hydroxyl group at C-3 (Table 2) In addition, the presence of an a-OH group at C-3 (1) showed more potent activity than that of the b-OH group at the same position (2)
In conclusion, the traditional use of B balsamifera for the treatment of rheumatism and inflammatory diseases
in Vietnam may be attributable to the XO inhibitory activity of theflavonoid constituents
Acknowledgements This work was supported by grant 104.01.68.09 from Vietnam ’s National Foundation for Science and Technology Development (NAFOSTED).
Conflict of Interest
The authors declare no con flict of interest.
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Table 2 Xanthine oxidase inhibitory activity of the isolated
compounds
Each value represents the mean SD of four determinations.
0 20 40 60 80 100
Concentration (µM)
0 0.2 2 20 50 100
Figure 3 Dose-dependent inhibition of XO by 1, 6, 8 and allopurinol.
O
OH
R 1 O
OR2
OR3
OR4 O
OH
R 1 O
R 3
OH
R 4 H
R2
6 R1= R2= R3= R4= H
7 R1= R2= R3= CH3, R4= H
8 R1 = H, R 2 = R 3 = R 4 = CH3
1 R1= R2= H, R3= OH, R4= OCH3, R5= H
2 R1= H, R2= OH, R3= H, R4= OCH3, R5= H
3 R1= CH3, R2= OH, R3= H, R4= OCH3, R5= H
4 R1 = CH3, R 2 = OH, R 3 = H, R 4 = OH, R 5 = H
5 R1 = R 2 = R 3 = R 4 = H, R 5 = OH
R 5
Figure 1 Structures of the isolated compounds from B balsamifera.
O
O OH HO
OH
OH
Figure 2 Connectivity (bold line) deduced by the COSY spectrum
and significant HMBC correlations (arrow) observed for 1.
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M.T.T NGUYEN AND N.T NGUYEN