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Original Paper
Abstract: A new antifungal and radical scavenging
2-hydroxy-flavanone, named mosloflavanone, was
isolated from the di-
chloromethane extract of Mosla soochouensis together with the known
mosloflavone and moslosooflavone. Structures were established by spectroscopic
and chemical methods, as well as X-ray crystallography.
Key words: Mosla soochouensis, Lamiaceae,
2-hydroxyflavanone, radical scavenging activity, antifungal activity.
Introduction
Mosla soochouensis Matsuda (Lamiaceae), a small aromatic
herb endemic to eastern
medicine (Wu Xiang Cao) to treat common cold, tonsilitis and heatstroke
(1). In previous phytochemical studies on M. soo-
chouensis, the analysis of the essential oil constituents (2)
and the isolation of mosloflavone (3) and moslosooflavone (4)
were reported. In our continuing search for antifungal
and antioxidant constituents of plant origin, we investigated the
whole plant dichloromethane extract of M. soochouensis, which
showed antifungal activity against Cladosporium cucumerinum (5) and the
existence of free radical scavengers using 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical (6) in autographic TLC assays.
Materials and Methods
General methods
The whole herb of flowering Mosla soochouensis was
collected in September 1996 at a hill top at Xishan, near
2:1, 4l; 1:1, 5l; 1: 2, 5l; 0:1, 4l) to give 16
fractions. Fraction 10 (2 g; eluent 1: 1) was further separated by gel
filtration on
Sephadex LH-20 (2.5 . 60 cm)
with CHCl3 :MeOH (1 :2) to obtain a flavonoid fraction, which was then
repeatedly chromatographed on Lobar Diol (size B, 2.5 . 31 cm) with CHCl3 (600
ml) and n-hexane-EtOAc, 4:1 (700 ml) to afford, in the
elution order, moslosooflavone (3, 12 mg), mosloflavone (2, 47mg)
and 1 (340 mg). Rechromatography of fraction 9 afforded
more 2 (79 mg) and 3 (24 mg). Other known compounds were
isolated and characterized by spectral methods and by
comparison with literature: p-menth-8-ene-1,2-diol (6 mg) (7),
3-(3-methoxy-4,5-methylene-dioxyphenyl)-2-propen-1-ol (3.5 mg) (8) and
sitosterol- b-D-glucoside (20 mg) (9). 2,5-Dihydroxy-6,7-dimethoxy-flavanone (mosloflavanone) (1):
plates (n-hexane/EtOAc), m.p. 89.5±93.5 8C; [ a ]D:
+2.58 (MeOH, c 0.10); EI-MS: m/z (%) = 316 [M+] (100), 298 (9), 283 (10), 269
(3), 255 (3), 196 (34), 181 (25), 170 (34), 153 (10); IR: n KBr max = 3440,
1670, 1635 (sh), 1595 cm±1; UV: l MeOH max (log e ) = 203 (4.55), 235 (sh,
4.13), 289 (4.23), 346 (3.68), + NaOAc 291, 368; + NaOAc + H3BO3 280, 371; +
NaOMe 384; + AlCl3 314, 398; + AlCl3 + HCl 311, 394 nm. 1H-NMR (CD3COCD3, 500
MHz): d= 2.94/2.96 (d, J = 17 Hz, Heq-3), 3.23/3.25 (dd, J = 3 and 17 Hz,
Hax-3), 3.72/3.73 (s, CH3O-6), 3.91/3.92 (s, CH3O-7), 6.18/6.24 (s, H-8),
6.50/6.56 (d, J = 3 Hz, OH-2), 7.40±7.52(m, H-3¢,4¢,5¢), 7.72±7.78 (m,
H-2¢,6¢), 11.86/11.94 (s, OH-5);13C-NMR (CD3COCD3, 125 MHz)d (ppm) 49.48/49.51
(C-3),56.56/56.59 (CH3O-7), 60.53/61.02 (CH3O-6), 93.45/93.67 (C-8),
102.82/102.90 (C-2), 103.16/103.47 (C-10), 126.23/126.24
A New 2-Hydroxyflavanone from Mosla
soochouensis
Qi Wang1, Christian Terreaux1, Andrew Marston1, Ren
Xiang Tan2, Helen Stoeckli-Evans3, and Kurt Hostettmann1,*
1 Institut de Pharmacognosie et
Phytochimie, UniversitØ de Lausanne,
2 Department of Biological Sciences and Technology,
3 Institut de Chimie, UniversitØ de Neuchâtel,
Revision accepted:
Planta Medica 65 (1999) 729±731
_ Georg Thieme Verlag
·
ISSN: 0032-0943
Received:
l= 0.71073 .) withw/2Qscans in the 2Q range 5±518. The structure was
solved by direct methods using the programmeSHELXS-97 (10). The refinement and
all further calculations were carried out using SHELXL-97
(11). The hydroxy H-atoms
were located from difference maps and refined isotropically. The remainder of the H-atoms were
included in calculated positions and treated as riding atoms using
SHELXL-97 default parameters. The non-H atoms were
refined anisotropically, using weighted full-matrix
leastsquares on F2. The bond lengths and angles are normal within
experimental error. Further details of the crystal structure
investigation can be obtained from the Cambridge Crystallographic
Data Centre,
Dehydration of mosloflavanone (1) Compound 1 (25 mg)
was boiled for 15 min with 5ml 30% H2SO4. After cooling and addition of 5ml of
a saturated Na2SO4 solution, the reaction mixture was extracted with MeCOEt. A
quantitative reaction occurred as 1 was no longer detected by TLC. Upon
separation by Sephadex LH-20 with acetone- MeOH 2:1 and Lobar Diol with
n-hexane-EtOAc 5:1, 2 (17.2 mg) and 3 (4.5 mg) were recovered.
Activity tests
TLC autographic assays of antifungal test against
Cladosporium cucumerinum (5) and radical scavenging test using DPPH
radical (6) were applied for extract screening and
activityguided isolation. For pure compounds, a dilution test was
used for Cladosporium cucumerinum inhibition activity and
a spectrophotometric assay for DPPH radical reduction activity
(6), (12).
Results and Discussion
Compound 1 was obtained after several purification
steps of the whole plant dichloromethane extract of M. soochouensis
as a bright yellow powder and as pale yellow prisms
after recrystallization from n-hexane/EtOAc. The UV spectrum in
MeOH showed absorption maxima at 289 nm and 346 nm,
indicative of a flavanone, considering the weak intensity of the
latter maximum. The EI-MS gave a base peak as the parent ion
at m/z = 316 [M]+. However, both the 1H- and 13C-NMR
spectra showed many more signals (with significant minor peaks)
than one might expect from a low molecular weight
compound of this type. The spectral complexity, as well as the ratio between
signal intensities of major and minor peaks was strongly solvent-dependent.
Despite this complexity, the major resonances in the 1H-NMR spectrum recorded
in acetone- d6 gave much information about the structure of the dominant forms
of 1. Two signals at d = 11.86 ppm and 11.94 ppm, each integrated for one
proton were apparently originating from chelated hydroxy protons at position
C-5. Both of them, together with a pair of doublets at d = 6.56 (d, 2.5 Hz) and
6.50 (d, 3.0 Hz) were D2O-exchangeable. The shifting of the 13C-NMR signal of
C-2 in a usual flavanone from around d = 80 to a pair at d = 102.82/102.90
suggested that the second hydroxyl group was indeed located at C-2, which is
further confirmed by the observation of a weak coupling between these
hydroxy protons and Hax-3 in COSY experiments. The
multiplets centered at d = 7.75 integrated for 4 protons and at d =
7.43 for 6 protons together
with the 13C-NMR signals at d = 126.23/126.24, 129.06/129.12 (each double
density) and a single double-density peak at d = 129.52 showed a
non-substituted B-ring. Proton signals at d = 3.91/3.90 and 3.71/3.72 and 13C-NMR
signals at d = 60.53/61.02 and 56.59/56.60 indicated that, together with the
5-hydroxy unit, two methoxy groups were located on the A ring, one of them in
an ortho-disubstituted pattern. Thus, 1 should have a structure of either 2,5-dihydroxy- 6,7-dimethoxyflavanone or
2,5-dihydroxy-7,8-dimethoxyflavanone. The HMBC experiment confirmed a
correlation between H-8 and C-9 and thus supported the former as the correct
structure for the new compound 1, which was
then named mosloflavanone. The structure was confirmed by
X-ray crystallographic analysis, which indicated the presence of a racemic
mixture (Fig.1). This was in agreement with the low [ a ]D value recorded (+
2.58), implying the enantiomer ratio not to be exactly 1:1 and suggesting the
(+)-form to occur naturally in the plant and then racemizing during extraction
and isolation. 2-Hydroxyflavanones are known for their cyclo-oxo tautomerism (13),
(14). This equilibrium via a chalcone intermediate (1b) is responsible for the
presence of a racemic mixture and the cyclotautomer of 1, in this form of a
racemic 2-hydroxyflavanone, was dominant in the solution. Meanwhile, the
chalcone intermediate further underwent a keto-enol tautomerization process to
an oxo-form of diaroylmethane type (1a) (Scheme 1). Upon boiling under acidic
conditions (30% H2SO4), compound 1 yielded almost quantitatively, a mixture of
mosloflavone (2) and moslosooflavone (3), the former as the major product. Fig.1 ORTEP plot and numbering of compound 1. Planta Med. 65
(1999) Qi Wang et al. 730 These two flavones were also
obtained during the isolation procedure and have already been reported from M.
soo- chouensis (3), (4).
The minimum quantity of compound 1 needed to inhibit
the growth of Cladosporium cucumerinum in the bioautographic
TLC assay was 2 mg, while that of propiconazole as
the reference was 0.1mg. However a concentration of 1 up to 100
mg/ml could not totally inhibit the growth of the
microorganism in an agar dilution test (12). Compound 1 was also tested
against DPPH radical in a spectrophotometric assay using quercetin,
BHT (2,6-di-tert-butyl-4-methylphenol) and cinnamic
acid (negative control) as reference compounds. The radical
scavenging activity of 1 was shown to be weaker than that of quercetin but
higher when compared to BHT (Fig. 2).
Acknowledgements
The Swiss National Science Foundation is gratefully
acknowledged for supporting this work.
References
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Prof. Dr. K. Hostettmann
Institut de Pharmacognosie et
Phytochimie
BEP, UniversitØ de Lausanne
CH-1015
E-mail: Kurt.Hostettmann@ipp.unil.ch
Fax: +41 21 6924565
Scheme 1
Fig. 2 Radical scavenging activity
of compound 1.
A New 2-Hydroxyflavanone from Mosla soochouensis
Planta Med. 65 (1999) 731