PIPTADEROL FROM PIPTADENIA AFRICANA

R. N. Mbouangouere^a*, P. Tane^a, D. Ngamga^a, P. Djemgou^a M. I. Choudhary^b, B. T. Ngadjui^c.

^aDepartment of Chemistry, Faculty of Science, University of Dschang, Box 67 Dschang, Cameroon
^bH.E.J. Resarch Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
^cDepartment of Organic Chemistry, Faculty of Science, University of Yaounde I, Box 812, Yaounde, Cameroon
Email : ti rouka@yahoo.fr , Tel: +237 5706965, Fax: + 237 3451735

Code Number: tc07045

Abstract

A new glyceryl derivative (Glyceryl-1-hexacosanoate) and a flavone derivative (methyletherapigenin) were isolated from the stem bark extract of Piptadenia africana, a western Cameroonian plant species. Common terpenes like sitosterol, β-amyrin and eicosane were also isolated. These compounds were identified using physical and spectroscopic methods including mp, IR, ¹H and ¹³C-NMR, DEPT, COSY, HMQC, HMBC, EI MS, HREI MS as well as some chemical transformations. The antibacterial activity of the extract, the fractions and the pure compounds is also discussed.

Key words: Piptadenia africana, Leguminoseae, Glyceryl-1-hexacosanoate, Methyletherapigenin, Chemotaxonomy, Antibacterial activity.

Introduction

The genus Piptadenia or piptadeniastrum belongs to the family Leguminoseae. This genus possesses several species including: P. peregrina, P. macrocarpa, P. rigida, P. vidiflora P. paraguayensi and P. africana (Letouzey, 1969). These species are distributed in tropical Africa from Senegal to Angola and across the Congo region to Uganda (Letouzey, 1969). Only the last species is found in Cameroon (Letourzey, 1985). P. africana is a tree, the leaves are very small and numerous, contiguous, about 3-8 mm long and 0.8-1.5 mm broad, fruits are broadly linear, elongated, 17-36 cm long and 2-3 cm broad. They are found in mixed deciduous and evergreen forest, it often stands as a single tree on farmlands (Letouzey, 1969). Previous investigation on the seeds of P. peregrina yielded bufotenine (Stromberg, 1954), work on P. rigida revealed the presence of tryptamine (Iacabucci et al., 1964) which was absent in P. rigida, P. vidiflora and P. paraguayensis (Linda et al., 1971), in the other hand Piptadenia africana was suggested not to contain alkaloids ( Paris et al., 1967) . As part of our contribution to phytochemical, chemotaxonomic and biological survey of the genus Piptadenia, we carried out the investigation of the stem bark of Piptadenia africana (Hook. f.) previously not investigated and we report herein the isolation and characterisation, of a new compound in addition to the full NMR assignment of methyletherapigenin. and sitosterol, β-amyrin and eicosane. At low concentration the activity of the crude extract for both gram positive and negative organisms was non existent however at highest concentration the crude extract showed a weak inhibition of gram negative microrganisms. The fractions and pure compounds did not show any activity.

Experimental

General

IR spectra were recorded on a Nicolet Magma 750 spectrophotometer with KBr disc. ¹H-NMR spectra were recorded on Avance 400 MHz Bruker NMR while ¹³C-NMR spectra recorded at 100 MHz. Chemical shifts are given in δ (ppm) taking tetramethylsilane (TMS) as reference and relative to the solvent used. NMR was taken in pyridine and CDCl₃. EI MS were recorded on a JEOL MS Route. Silica gel (60-230, 230-400 mesh) (Merck) was used for column chromatography. Melting points were recorded on BUCHI 535 melting point apparatus.

Plant Material

The stem bark of Piptadenia africana was collected at Matachouom, Noun Division, and Western province of Cameroon in February 2005. Plant identification was performed by Dr. Onana, Botanist at the National Herbarium, Yaoundé, Cameroon, where a voucher specimen (N° 5000/12115/HNC) has been deposited.

Extraction and isolation

The air dried and pulverized plant material (500 g) was macerated in a mixture of dichloromethane and methanol (1:1) for 3 days. Removal of the solvent in vacuo provided an organic extract (45 g). The extract (45 g) was dissolved in methanol and re-extracted with petroleum ether to yield fraction A (3 g). The resulting organic phase was then concentrated and dissolved in water. This aqueous phase was extracted with dichloromethane, ethyl acetate and butanol to yield fractions B (10 g), C (7 g) and D (11 g). Fraction B was then concentrated and subjected to column chromatography, using a mixture of n-hexane-acetone with increasing polarity as eluent. Fractions of 200 ml were collected and combined on the basic of their TLC profiles. The fraction eluted with n-hexane-acetone (8:2, 350 mg) was further purified by column chromatography on silica gel (60-230 mesh) to yield eicosane (4 mg) and 4', 7-dihydroxy-5-methoxyflavone (2, 6 mg). The combined fractions (800 mg) eluted with n-hexane-acetone (1:1) revealed the presence of sitosterol. Further purification of this fraction by column chromatography on silica gel with dichloromethane-acetone afforded β-amyrin (23 mg), sitosterol (30 mg) and glyceryl-1- hexacosanoate (1, 12 mg).

Hydrolyses of glyceryl-1-hexacosanoate (1)

5 mg of this compound was dissolved in 2 ml of pyridine in a 50 ml round flask, to which 10 ml of HCl 5%was added and refluxed at 60° C for 14 hours required for complete hydrolysis. The solution was kept for 24 hours to evaporate pyridine. The remaining mixture was concentrated and further purification yielded two main compounds: Glycerol was obtained as oil and hexacosanoic acid as a white solid (mp 88-89° C). Both products were identified by analysis of their mass spectrum and comparison with an authentic specimen available in our laboratory.

Glyceryl-1-hexacosanoate (12 mg, mp 91-93° C). White solid, (CH₂Cl₂/acetone), EI MS m/z (re lint.): 470, M⁺, 439 (2), 396 (7), 379 (5), 351 (3), 176 (3),134 (47), 98 (91) and 57 (100); IR (KBr) 2918, 2850, 1732 cm^-1, ¹H-NMR, ¹³C-NMR and HMBC, see Table 1

4΄, 7, Dihydroxy-5-methoxyflavone (6 mg, mp 279-283° C ). Colourless powder (n-hexane/acetone). EI MS: m/z (re lint.) 270 (100), 242 (11), 153 (31), 121 (24), 69 (23). ¹H-NMR and ¹³C-NMR, see Table 2

Sitosterol. C₂₉H₅₀O, cristallised from acetone as white powder, 50 mg, mp 134-136 °C. The physical and NMR data (¹³C and ¹H) are in agreement with those reported in the literature (Goad L. J. 1991).

β-Amyrin. C₃₀H₅₀O, cristallised from methanol as colourless needles, 30 mg, mp 197-199 °C. The physical and NMR (¹H and ¹³C) data are in agreement with those reported in the literature(veterberg et al.1925).

Eicosane. C₂₀H₄₂, crystallised in Ethanol as white solid 4 mg, mp 38 °C. The physical and NMR data (13C and 1H) data are in agreement with those reported in the literature.

A dried powder sample of the stem bark of P. africana was extracted by maceration in CH₂Cl₂/MeOH (1/1). Concentration yielded an extract, which was defatted with petroleum ether followed by extraction with dichloromethane, ethyl acetate and butanol. Fractionation of the CH₂Cl₂ extract was achieved and purified by column chromatography over silica gel to afford five compounds. Glyceryl-1-hexacosanoate 1, 5-methoxy-4', 7-dihydroxyflavone (methyletherapigenin) 2, sitosterol, β-amyrin and eicosane . Compound 1 was isolated as a white solid (12 mg, mp 91-93° C). Its cross formula was deduced to be C₂₉H₅₈O₄ on the basis of a molecular ion peak at m/z 470 from EIMS and was confirmed by HREI MS analyses. The peak at 469 (M-1) suggested the presence of hydroxyl group in the molecule. On the other hand, fragments could be observed at m/z 439 (M- CH₂OH), 410 (-C₃H₇), 351 (-CO), 154, 134, 98. An interesting fragment was observed at m/z 379 [M-glycerol] +, which suggested the fatty acid as hexacosanoic acid. The identity of the fatty acid was proved from HREI MS which gave a molecular ion peak at m/z 470.4342, consistence with our suggested molecular formula (C₂₉H₅₈O₄)_. The IR spectrum showed the presence of a carbonyl group at 1732 cm^-1, hydroxyl group at 2918 and 2850 cm^-1. The ¹H-NMR spectrum of 1 ( Table 1) revealed the presence of five hydrogen germinal to hydroxyl group at δ 3.97 (1H, dd, J = 6.3, 11.8 Hz, H-1a), 3.95 (1H, dd, J = 4.8, 11.8 Hz, H-1b), 3.70 (1H, q, J = 5.8 Hz, H-2), 3.46 (1H, dd, J = 4.2, 12.4 Hz, H-3a) and 3.39 (1H, dd, J = 6.1, 12.4 Hz, H-3b), three methylene

protons at δ 2.19 (2H, t, J = 15.0 Hz, H-2'), 1.46 (2H, q, J = 14.0 Hz, H-3') and 1.13 (2H, br s, H-25'), a methyl at δ 0.72 (3H, t, J = 13.0 Hz, H-26'), in addition the signal of a large number of cyclic methylene proton between δ 1.03 and 1.11 was observed. These chemical shifts are characteristic of oxomethylene proton signal for an -O-CH₂-CH(O)-CH₂-O- spin system attributable to glycerol (Nasim et al., 1999). The ¹³C-NMR spectrum (Table 1) confirmed these suggestions and showed an ester carbonyl signal at δ 173.7 ( C-1'), three carbon atoms connected to oxygen at δ 66.7 (C-1), 70.9 (C-2) and 64.3 (C-3), signals of aliphatic methylene carbon at δ 34.4 (C-2'), 25.3 (C-3') and 22.9 ( C-25'), a methyl carbon was found at δ 14.2 (C-26'), from δ 29.4 to 32.1 we had and a peak integrating for a large number of CH₂. In HMBC experiment (Table 1), the methylene protons at δ 3.97/3.95 ppm (H-1) showed a ³J correlation with the carbonyl. From these shifts the idea of a monoglyceride was evident. The results described and by comparison with those of similar compound (Nasim et al., 1999), as the NMR revealed a single linear alkyl chain this must be C-26 and accordingly, 1 is glyceryl -1- hexacosanoate which is a new natural product named as piptaderol.

Table 1: 1H and 13C-NMR data for compound 1 (400 and 100 MHz, δ values in C₅D₅N)

Table 2: 1H and 13C-NMR data for compound 2 (400 and 100 MHz, δ values in CDCl₃)

Table 3: In vitro Antibacterial bioassay of P. africana.

Key: Concentration of Sample 3mg/ml of DMSO Size of well 6mm (diameter)
^a Std. Drug Imipenum

Compound 2 was obtained as colorless needles mp 279-283°C and gave positive Mg- HCl test characteristic of flavonoids (Mabry et al., 1970). It was assigned the molecular formula C₁₆H₁₂O₅ as deduced from EI MS and HREI MS where a molecular ion peak was observed at m/z 282 and 282.1231 respectively. In addition fragments were observed at m/z 270, 242, 153, 149, 121, 83 and 69. The ¹H-NMR spectrum of 2 (Table 2) showed one singlet at δ 6.34 (1H, H-3), two doublets at δ 7.61 (1H, J = 2.8 Hz, H-6) and 6.76 (1H, J = 2.8 Hz, H-8), characteristic of meta-related proton (Dey and Harbone, 1989) along with two doublets of doublets at δ 6.26 (2H, J = 2.1, 8.8 Hz, H-2', H-6') and δ 6.09 (2H, J = 2.1, 8.8 Hz, H-3', H-5') revealing ortho coupled proton. A singlet at δ 3.70 integrating for the methoxyl proton was observed. It was clear that The compound has a flavone basic skeleton. This was confirmed by the ¹³C-NMR spectrum from which signals for sixteen carbon atoms were observed. Analysis of ¹³C-NMR, DEPT (90 and 135) and HMQC lead to their classification into a carbonyl at δ 182.4 (C-4), two unsaturated quaternary carbon link to hydroxyl group at δ 160.6 (C-7, C-4'), a carbon link to a methoxyl at δ 161.4 (C-5). Four aromatic quaternary carbon at δ 163.8 (C-2), 164.5 (C-9), 128.1 (C-10) and 122.3 (C-1'), in addition we could also found seven aromatic methine carbon atom at δ 115.8 (C-3, C-8), 115.6 (C-6), 99.1 (C-2', C-6'), 103.2 (C-3') and 104.4 (C-5'), a methoxyl at δ 53.2. As the chemical shifts of C-2' and C-6' were similar, like C-3' and C-5' were very closed, deduced the hydroxyl group to be at C-4'. The assignment of the hydroxyl group was also based on the chemical shift of the proton, their form on the spectrum, their coupling constant and according to the HMQC. Comparing our data to those of similar compounds 4',5-dihydroxy-7-methoxyflavone (Bosabalidis, 1998), as there was no signal between 10-14 ppm for OH-5 (Dey and Harborne, 1989), this flavone must be 5-methoxy-4',7-dihydroxyflavone. This compound was previously isolated from the flowers of Saccharum officinarum (Misra et al., 1979) and appears to be common in plant kingdom, but to best of our knowledge, the complete assignment of its NMR data is described for the first time.The other compounds were isolated and characterized from comparison of their physical and NMR data (¹H and ¹³C) to those reported in the literature.

Conclusion

Previous investigation of members of this genus yielded alkaloids, but we found no traces of this class of compounds. Further more, flavonoids have also been previously reported from other species of the genus. These may constitute a possible chemotaxonomic marker for the investigation of other species of the genus.

Acknowledgements

R. N. Mbouangouere is grateful to the COMSTECH and H.E.J. Research Institute of Chemistry for their financial support. Authors will like to thank Dr. Onana for plant identification and Ms. Samreen Hussein for the biological activity.

References