Antimicrobial Glycosides and Derivatives from Roots of Picralima nitida

Phytochemical screening was performed on the roots of Picralima nitida, resulting in the isolation of three new coumestan glycosides, 3-hydroxy-9-methoxy-2-[2’(E)-3’-methyl-4’-O-β-D-galactopyranosylbutenyl]-8isoprenylcoumestan (1), 3-hydroxy-9-methoxy-2-[2’(E)-3’-methyl-4’-O-β-D-glucopyranosylbutenyl]-8-[2”(E) -3”-methyl-4”-oxobutenyl]coumestan (2), and 3-hydroxy-9-methoxy-4-[2’(E)-3’-methyl-4’-O-β-Dglucopyranosylbutenyl]-8-[2”(E)-3”-methyl-4”-oxobutenyl]coumestan (3). Acid hydrolysis of 1, 2 and 3 afforded three new coumestan, 3-hydroxy-9-methoxy-2-[2’(E)-4’-hydroxy-3’-methylbutenyl]-8isoprenylcoumestan (4), 3hydroxy-9-methoxy-2-[2’(E)-4’-hydroxy-3’-methylbutenyl]-8-[2”(E)-3”-methyl-4”oxobutenyl]coumestan (5), and 3-hydroxy-9-methoxy-4-[2’(E)-4’-hydroxy-3’-methylbutenyl]-8-[2”(E)-3”methyl-4”-oxobutenyl]coumestan (6), respectively. Structures of these compounds were elucidated on the basis of spectroscopic data and chemical transformations. Compounds 1 6 showed antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Proteus vulgaris.


Introduction
Picralima nitida Stapf (Apocynaceae) is an entirely glabrous shrub of 3-10 m high.Its fruits are ovoid and yellowish at maturity (Adjanohoun et al. 1996).This plant is widely distributed throughout Africa forest regions.Throughout its distribution area the seeds, bark and roots of P. nitida have a reputation as a febrifuge and remedy for malaria ( Kouitcheu et al. 2008).They are also extensively used for pain relief and to treat chest and stomach problems, pneumonia and intestinal worms.Usually, the seeds or bark are crushed or chewed and eaten for this purpose, or a decoction from the roots, seeds or bark is drunk (Adjanohoun et al. 1996;Ezeamuzieet al. 1994).As part of our continuing study on searching for the bioactive flavonoids constituents from West Africa medicinal plants (Kazie et al. 2009), phytochemical screening was performed on the roots of P. nitida, resulting in the isolation of three new coumestan glycosides 1, 2 and 3. Acid hydrolysis of these products yielded three new coumestan derivatives 4, 5 and 6, respectively.These six compounds showed antimicrobial activities against E. coli, S. aureus, and P. vulgaris.We herein describe the isolation, structural elucidation and biological activities of these compounds.

General Procedures
Melting points were determined on X-4 digital micro-melting point apparatus and were uncorrected.Optical rotations were measured with a Perkin-Elmer 341 digital polarimeter.The NMR spectra were recorded with a Bruker AMX-500 (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR).Samples were run in DMSO-d 6 or CDCl 3 .Chemical shifts were given in (ppm) with tetramethylsilane as an internal standard (0.00 ppm).The HRFABMS spectrum was obtained with a Kratos MS 25 instrument with a DS-55 data system, and collision gas Xe (ion gun conditions 6KV and 10 mA).The EI mass spectra (at 70 eV) were recorded on a JEOLMSRoute mass spectrometer.IR spectra were run from KBr pellets on a Perkin-Elmer 577 spectrometer.HPLC was performed by using a system comprised of a CCPM pump, a CCP PX-8010 controller, an RI-8010 detector and a Shodex OR-2 detector, and a Rheodyne injection port with a 20 μl sample loop.Sephadex LH-20, Si gel GF 254 (Merck) and Si gel 60 (70-2 mesh ASTM) (Merck) were used for CC.Silica gel 60 (0.25 mm, Merck) was used for TLC.

Plant Material
Roots of P. nitida were collected in Zo-Etélé, south region of Cameroon in January 2002.The plant was identified at the National Herbarium, Yaounde, where a voucher specimen is deposited (No.2136/SRFK)

Extraction and Isolation of Compounds
Dried ground roots of P. nitida (13 Kg) was immersed in MeOH (35 L) and kept for 72 h at 25 o C.After filtration the solvent was removed by rotary evaporator under reduce pressure.The extract obtained (302 g) was partitioned with 2%H 2 SO 4 /EtOAc.The EtOAc phase (102 g) was chromatographed over silica gel (400 g).

Acid Hydrolysis and Identification of Sugar
Compounds 1, 2 and 3 (16.5 mg each) were separately refluxed with 15% HCl/MeOH (12 ml) at 80°C for 4 h.After cooling, each reaction mixture was concentrated and the residue partitioned with CHCl 3 /H 2 O.The organic layer was concentrated to dryness to yield 9.8 mg of white material (M1) from 1, 10.1mg of white material(M2) from 2 and 9.9 mg of white material(M3) from 3. After purification by preparative-TLC, using silica gel and MeOH/CH 2 Cl 2 (0.25:9.75) as development solvent, M1, M2 and M3 yielded compounds 4 (8.1 mg), 5 (7.2 mg) and 6 (7.8 mg), respectively.Each aqueous layer was evaporated and the residue was analysed by HPLC under the following conditions: column, Aminex HPX-87H (7.8 mm i.d.x 300 mm); solvent, 5 mM H 2 SO 4 ; flow rate, 0.6ml/min; detection, refractive index and optical rotation.The sugars were confirmed as D-galactose, and D-glucose by comparison of their retention times and optical rotations with those of authentic samples: retention times (min), 9.62 (D-galactose, positive optical rotation), 8.99 (D-glucose, positive optical rotation).

Antibacterial Assay
Antibacterial activity was determined by the paper disk method.A paper disk (Φ 6 mm, from Whatman number one filter paper), with the sample was incubated on an agar plate containing E. coli, S. aureus, or P. vulgaris at 25 °C.Paper discs containing Amikacin (30 µg), Vancomycin (30µg) and Penicilin (10µg), respectively, were used as positive control.The result recorded for each bioassay was the average of three tests.

Structural Elucidation of Compounds 2 and 5
Compound 2, obtained as white powder, had a molecular formula of C 32 H 34 O 12 on the basis of its HRFABMS (m/z = 610.1912[M] + ) and 13 C-NMR data (Table 2) (32 signals).The IR and UV bands indicated the coumestan nature of 2. Furthermore, the 1 H-and 13 C-NMR spectroscopic data of 2 (Table 1 and 2) revealed the presence of a sugar residue in addition to a coumestan aglycone moiety.Upon acid hydrolysis (note 2), 2 afforded D-glucose and compound 5.

Structural Elucidation of Compounds 3 and 6
Compound 3 was obtained as white powder.Its molecular formula was derived as C 32 H 34 O 12 by the HRFABMS spectrum, showing an [M] + ion at m/z = 610.1912,and broad band-decoupled 13 C-NMR spectrum (Table 1) (32 carbon signals).Its IR, UV and NMR data indicated that it was a coumestan glycoside.Acid hydrolysis (note 2) yielded D-glucose along with compound 6.

Antibacterial Assay
Compounds 1 -6 showed antimicrobial activities against E. coli, S. aureus, and P. vulgaris at the concentration of 13 g/dis (Table 4).

Conclusion
Picralima nitida is known as a rich source of alkaloids.Some of them have been reported to show biological activities (Corbett, et al. 1996;Fakeye et al. 2000;Ramirez et al. 2003;Ezeamuzie et al. 1994).However, this paper preliminarily studies the neutral constituents of P. nitida roots.The separated products are flavonoids.The use of the roots of P. nitida as anti-infective agent may be explained by the presence of antimicrobial coumestan derivatives.
Compound 4 had the molecular formula C 26 H 26 O 6 on the basis of the HREIMS, exhibiting an [M] + peak at m/z = 434.1591,and 13MR spectrum (Table1) (26 carbon signals).A distortionless enhancement by polarization transfer (DEPT) NMR experiment permitted differentiation of the 26 resonances into four methyl, three methylene, six methine, and thirteen quaternary carbons.Its UV and IR spectra were similar to those of other coumestans.The 1 H-NMR spectrum(Table2) displayed two para-coupled aromatic protons at δ ) revealed the presence of a sugar residue in addition to a coumestan aglycone moiety.Upon acid hydrolysis (note 2), 1 afforded D-galactose and compound 4.

Table 3 .
The HMQC and HMBC Data of Compounds 4 -6

Table 4 .
Results of Antibacterial Activities of Compounds 1-6