A new pterocarpan from the leaves of Abrus precatorius L

A pterocarpan, 2,3,4,8-tetramethoxy-6a,11a-dihydro-6H-benzo[4,5]furo[3,2-c]chromene-7,9-diol (compound 1) was isolated from the dichloromethane extract of the leaves of Abrus precatorius L. The structure of the compound was elucidated by detailed spectroscopic analysis such as H NMR, C NMR, distortionless enhancement by polarisation transfer (DEPT), heteronuclear multiple-quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC). The crude extracts displayed brine shrimp lethality and in vitro antimalarial activity. The methanolic crude extract demonstrated an A/B ratio of 9.7 signifying that it is more toxic against Plasmodium falciparum than brine shrimp.


General experimental procedures
The melting point of the pure compound was determined on a Gallenkamp melting point apparatus (Sanyo, United Kingdom) with open capillary tubes and was uncorrected.The NMR spectra were obtained from Varian Gemini 200 MHz and a Bruker Avance DPX 300 MHz machine.MS analysis was performed on pure solid samples using the direct insertion probe (DIP) on a Fission Platform Mass Spectrometer operated at 70 eV and mass range set at 38-400 a.m.u. and using Atmospheric Pressure Chemical Ionization (APCI) on a Finnigan LCQ Deca machine, in either the positive mode (gives M+H) or negative mode (gives M-H as quasi molecular ion).Pre-coated plastic sheets (Polygram ® sil G/UV 254 ) and aluminium sheets (Alugram ® sil G/UV 254 of 20 cm by 20 cm Machery-Nagel GmbH and Co., KG) were used for Thin Layer Chromatography (TLC).Column chromatography was carried out using slurry packing with Kiesegel silica gel (0.063-0.2/70-230 mesh ASTM Kobian Kenya Ltd., Nairobi).

Plant
The leaves of the plant A. precatorius L. were collected in October 2000 based on information given by various herbalists on its use for the treatment of various diseases specifically malaria from the Mombasa County (latitude -4.05°, longitude 39.67°), Kenya.It was identified by Mr. Simon Mathenge, a taxonomist at the Botany Department, University of Nairobi and a voucher specimen (CNM/SM/09/01) deposited at the Nairobi University herbarium.The plant material was dried under shade and ground using a motor grinding machine.

Extraction and isolation
The air dried powdered leaves of A. precatorius (600 g) were extracted by maceration successively in hexane, dichloromethane and methanol (each 1200 ml, 3×48 h).The combined extracts of each solvent were concentrated under reduced pressure below 50°C to get the respective crude extracts.Dichloromethane crude extract of A. precatorius (2.98 g) was chromatographed over silica gel column (30 g, 100-200 mesh), eluted with gradients of EtOAc in n-hexane and gave 17 fractions (300 ml each).Fractions (14 to 17) obtained on elution with 100% EtOAc yielded compound 1 on repeated washing with diethyl ether and crystallization from MeOH with an R f of value 0.70 (n-hexane-EtOAc, 50:50).

Hatching shrimp
Brine shrimp eggs, Artemia salina Leach were hatched in artificial seawater prepared by dissolving 38 g of sea salt (Sigma chemicals Co., UK) in 1 L of distilled water.After 48 h incubation at room temperature (22 to 29°C), the larvae (nauplii) were attracted to one side of the vessel with a light source and collected with pipette.Nauplii were separated from eggs by aliquoting them three times in small beakers containing seawater.

Brine shrimp bioassay
The bioactivity of the extracts was monitored by the brine shrimp lethality test (Wanyoike et al., 2004).Samples were dissolved in dimethylsulphoxide (DMSO) and diluted with artificial sea salt water so that final concentration of DMSO did not exceed 0.05%.Fifty microliters of sea salt water was placed in all the wells of the 96well microtiter plate.Fifty microliters of 4000 ppm of the plant extract was placed in row one and a two-fold dilution carried out down the column.The last row was left with sea salt water and DMSO only served as the drug free control.100 µl of suspension of nauplii containing about 10 larvae was added into each well and incubated for 24 h.The plates were then examined under a microscope (12.5×) and the number of dead nauplii in each well was counted.100 µl of methanol was then added and after 10 min, the total numbers of shrimp in each well were counted and recorded.Lethality concentration fifties (LC 50 values) for each assay were calculated by taking average of three experiments using a Finney Probit analysis program on an IBM computer (Wanyoike et al., 2004).

Cultures of P. falciparum
Laboratory adapted P. falciparum cultures of the international reference isolates VI/S (chloroquine resistant) were used.The strains have been cultured and maintained at the Faculty of Pharmacy, University of Nairobi, Nairobi.The culture medium was a variation of that described by Wanyoike (2004) and consisted of RPMI 1640 supplemented with 10% human serum, 25 mM N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid and 25 mM NaHCO 3 .Human type O+ erythrocytes (<28 days old) served as host cells and the cultures were incubated at 37°C in an atmosphere of 3% CO 2 , 5% O 2 and 92% N 2 .

Antiplasmodial bioassay
The in vitro semi-automated microdilution assay technique that measured the ability of the extracts to inhibit the incorporation of [G- 3 H]hypoxanthine into the malaria parasite was used (Wanyoike et al., 2004).For the test, 25 µl aliquots of culture medium were added to all the wells of a 96 well flat-bottom microculture plate (Costar Glass Works, Cambridge, UK).Aliquots (25 µl) of the test solutions were added, in duplicate, to the first wells, and a Titertek motorized hand diluter (Flow Laboratories, Uxbridge, UK) was used to make serial 2-fold dilutions of each sample over a 64-fold concentration range.Aliquots (200 µl) of a 1.5% v/v suspension of parasitized erythrocytes in culture medium (0.4% parasitemia; growth rate > 3fold per 48 h) were added to all test wells.Parasitized and nonparasitized erythrocytes were incorporated into all tests.The plates were incubated at 37°C in a gas mixture 3% CO 2 , 5% O 2 and 92% N 2 .After 48 h, each well was pulsed with 25 µl of culture medium containing 0.5 µCi of [G-3 H] hypoxanthine and the plates were incubated for a further 18 h.The contents of each well were then harvested onto glass fiber filters, washed thoroughly with distilled water, dried and the radioactivity in counts per minute (cpm) measured by liquid scintillation.
Computation of the concentration of drug causing 50% inhibition of [G-3 H]hypoxanthine uptake (IC 50 ) was carried out by interpolation after logarithmic transformation of both concentration and cpm values using the formula: where Y 50 is the cpm value midway between parasitisized and nonparasitisized control cultures and X 1 , Y 1 , X 2 and Y 2 are the concentrations and cpm values for the data points above and below the cpm midpoints (Wanyoike et al., 2004).

RESULTS AND DISCUSSION
The leaves of the plant A. precatorius were successively extracted with hexane, dichloromethane and methanol and the percent yield of each extract is as shown in Table 1.The brine shrimp toxicity and in vitro antiplasmodial activity of the extracts of the leaves are also shown in Table 1.

Brine shrimp lethality and in vitro antiplasmodial test
The crude extracts (hexane, dichloromethane and methanol) of the leaves of A. precatorius plant were subjected to brine shrimp lethality test and their LC 50 values were determined.The in vitro antimalarial activities of the methanolic crude extract against chloroquine resistant VI/S strain of P. falciparum were also investigated and its IC 50 value determined.The methanolic crude extract of A. precatorius displayed a higher activity against brine shrimp (415.3 µg/ml).The hexane (927.8 µg/ml) and dichloromethane (>1000.00µg/ml) extracts were about 2 times less active than the methanolic extract.The methanolic extract displayed an in vitro antiplasmodial activity of 43.0 µg/ml.

A/B ratio
When searching for new antimalarial agents, it is important to distinguish between specificity of action and non-selective toxicity.An estimation of therapeutic index in which the desired biological activity is compared with general toxicity is one in which selectivity of activity may be assessed (Abu et al., 2012).The brine shrimp lethality and activity against P. falciparum has been compared for the extracts and a ratio A/B for the two calculated (Monago and Alumanah, 2005).A value greater than one is considered indicative of more selective activity against P. falciparum, and a value less than one of more selectivity to brine shrimp.
The activities of the methanolic crude extract of A. precatorius against brine shrimp (A µg/ml) and P. falciparum (B µg/ml) in vitro was calculated to be 9.7.The A/B ratio of the methanolic extract of A. precatorius (9.7) demonstrates that it is more toxic against P. falciparum than brine shrimp.
The 1 H NMR spectral data of compound 1 (Table 2) suggested a pterocarpan structure due to the splitting pattern of the protons at 5.46 (d, J = 6.9 Hz, H-11a), 4.34 (dd,J = 4.8,10.8 Hz,3.55 (m,J=10.8,10.9 Hz,, related to the protons of the hetrocyclic ring B and the bridging protons of B and C rings, respectively (Tarus et al., 2002;Prathyusha et al., 2010).The chemical shifts appearing at  6.65 and 6.61 were assigned to H-1 and H-10, respectively.
The mass spectrum of compound 1 showed a peak at m/z 376 (88.6%) corresponding to the chemical formula C 19 H 20 O 8 .The suggested structure of compound 1 is 2, 3,4,5] furo [3,2-c]chromene-7,9-diol (Figure 1).It is a hitherto unknown natural product and has been isolated for the first time from the leaves of A. precatorius.

Conclusion
A new pterocarpan (compound 1) from the dichloromethane extract of the leaves of A. precatorius has been isolated and reported for the first time.The methanol extract of the leaves showed in vitro antiplasmodial activity confirming the claim by the communities for the use of this plant in treating malaria.

Table 1 .
Percent yield of extracts, brine shrimp toxicity and in vitro antiplasmodial activity of the extracts of the leaves of A. precatorius.
*Included as a positive control.

Table 2 .
1 H and 13 C NMR data for compound 1.