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O. Desire et al. / Journal of Ethnopharmacology 130 (2010) 320–328
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viruses (enteroviruses and rotaviruses), or parasites (Ascaris lumbri-
coïdes, Strongyloides stercoralis, Entomoeba hystolitica, Schistosoma
mansoni, Hymenolepis nana, Taenia saginata or Taenia solium)
(Ravaoarinoro et al., 1986; Cassel-Beraud et al., 1990a, 1990b;
Boisier et al., 2001; Buchy, 2003). Diarrhoea is often accompa-
nied by intestinal spasms that cause abdominal cramps and pain.
It is on account of these data that we investigated Mascarenha-
sia arborescens for antispasmodic activity, and tried to identify the
most likely candidate for the biological activity. Prior to our work,
no phytochemical or biological studies have been reported on this
species. The present paper describes the antispasmodic activity
on two isolated organs of the crude methanolic extract (CME) of
Mascarenhasia arborescens and of its four partitions (hexane (HeP),
methylene chloride (McP), ethyl acetate (EaP) and aqueous (AqP)),
and the isolation and identification of the bioactive compound. We
tested also several crude extracts, partitions and the pure com-
pound for their antiradical activities. Moreover, we quantified by
HPLC the bioactive compound in crude methanolic extract (CME)
and its four partitions.
Fig. 1. Chemical structure of davidigenin (DG).
gel 60 H) eluting with CH2Cl2/EtOAc (95/5 to 0/100), followed by
increasing concentrations of MeOH to give several fractions. Frac-
tion F-120 to F-130 contained pure DG (40 mg).
The ESI-mass spectrometry (ESI-MS) of compound 1 gave an
[M+H]+ at m/z 259 with the positive ion mode indicating a mass
258 compatible with the molecular formula C15H14O4. The struc-
ture of DG was confirmed by comparison with previously published
spectral data (Jensen et al., 1977).
2. Materials and methods
Davidigenin. White powder (62 mg), UV (DCM/MeOH) ꢀmax nm
(log ε) 196, 212, 280, 312; IR (KBr) ꢁmax 3442 (OH), 1626 (C O),
1597 (aromatic rings) cm−1; ESI-MS m/z 259 [MH]+ (C15H14O4
requires 258); 1H NMR (400 MHz, MeOD): ı 7.72 (1H, d, J = 8.7 Hz,
H-6ꢀ), 7.07 (2H, d, J = 8.4 Hz, H-2 and 6), 6.70 (2H, d, J = 8.4 Hz, H-3
and 5), 6.34 (1H, dd, J = 8.7, 1.8 Hz, H-5ꢀ), 6.26 (1H, s, J = 1.8 Hz, H-3ꢀ),
3.18 (2H, t, J = 7.7 Hz, H-␣), 2.91 (2H, t, J = 7.7 Hz, H-) and 13C NMR
(50 MHz, MeOD): ı 204 (C O), 165 (C-2ꢀ), 164.9 (C-4ꢀ), 155.3 (C-4),
132.3 (C-6ꢀ), 131.8 (C-1), 129.0 (C-2, 6), 114.8 (C-3, 5), 112.6 (C-1ꢀ),
107.7 (C-5ꢀ), 102.2 (C-3ꢀ), 29.62 (C-␣), 29.36 (C-).
2.1. Plant material
Mascarenhasia arborescens was collected by Odile Désiré in
June 2006 in Ankingameloka, Madagascar, located 33 km South
of the Ambanja district on road RN6. This species was identified
by Armand Rakotozafy (botanist, IMRA) by comparison with an
authentic specimen deposited at the Department of Botany, Botan-
ical and Zoological Park of Tsimbazaza (No. 325), Antananarivo,
Madagascar. A voucher specimen was deposited at the Faculty of
Sciences of Antsiranana, Madagascar, No. APO/Masc/02.
2.3. Synthesis of davidigenin
2.2. Extraction and isolation of the bioactive constituent
Davidigenin was prepared as described by Jensen et al. (1977),
in order to quantify it in crude methanolic extract and partitions
of Mascarenhasia arborescens by HPLC. DG was obtained by mixing
isoliquiritigenin (Alfa Aesar, France, 500 mg) with Pd/C 10% (Alfa
Aesar, France, 25 mg) in methanol and stirring the solution in a
flask under atmospheric pressure of H2 gas at room temperature
for 3 h. Yield obtained was 97%. The structure of the synthesized
derivative was found in accordance with the one of the previously
described dihydrochalcone DG (Jensen et al., 1977).
The plant material (5 kg) of Mascarenhasia arborescens was kept
at room temperature (25–30 ◦C) for air drying (3 weeks). The air-
dried plant material (500 g), a mixture of leaves and stems, was
powdered and extracted by repeated maceration with methanol
at room temperature (3× 1 l, 72 h each). The filtered solvent
was evaporated under vacuum and lyophilised to afford a crude
methanolic extract (CME) (54.4 g, 10.9% w/w). The residue (45 g)
was suspended in water and was partitioned by successive extrac-
tions three times with different solvents of increasing polarity to
yield hexane (HeP, 0.03 g), methylene chloride (McP, 0.27 g), ethyl
acetate (EaP, 1.81 g) and aqueous (AqP, 14.16 g) partitions. The CME
and its four partitions were stored in dark bottles at 4 ◦C after sol-
vent evaporation and freeze-drying.
The methylene chloride partition (McP) showing strong anti-
spasmodic activity on guinea pig ileum pre-contracted with
histamine was subjected to further purification. Using thin layer
chromatography (TLC), McP was loaded on preparative glass plate
(20 cm × 20 cm, 0.2 mm, SiO2 F254, Macherey–Nagel) and run seven
times using 200 ml CH2Cl2/MeOH (98/2) as developing solvent.
Nine fractions (F-1 to F-9) were obtained. Three fractions (F-1 to
F-3) were three different pure compounds. These nine fractions
were dried and dissolved in DMSO (0.1% final concentration) for
assessment of their antispasmodic activity on guinea pig ileum pre-
contracted with histamine. The compound F-1 was the most active.
Using LC–MS, 1D and 2D NMR spectroscopic analysis, the bioac-
tive compound F-1 (22 mg) was identified as davidigenin (DG), a
dihydrochalcone (Fig. 1).
2.4. Analysis of davidigenin in crude methanolic extract and
partitions of Mascarenhasia arborescens by RP-HPLC with DAD
2.4.1. Chemicals and general procedures
HPLC gradeacetonitrile was purchasedfromScharlau Chemie SA
(Spain). Deionized water was prepared by a Milli-Q Water Purifi-
cation System (Millipore, France). Before utilization, acetonitrile
and water were filtered through 45 m nylon 66 membrane fil-
ter (Supelco) and 0.22 m cellulosic filter (GE Water & Process
Technologies, France) respectively. Davidigenin was obtained by
synthesis from isoliquiritigenin (Alfa Aesar, France) as indicated in
Section 2.3.
2.4.2. Chromatographic conditions and instrumentation
HPLC analysis was performed on a Hewlett Packard Series 1100
HPLC system, equipped with G1311A quaternary gradient pump,
G1315A diode array detector (DAD), G1322A degasser, G1313A
autosampler, G1316A column communication, and ChemStation
software. Chromatography was carried out on a Hypersil Gold C18
In order to realize the different pharmacological tests, a second
batch of McP was subjected to a column chromatography (silica