Chemical composition of the pods of Albizia polyphylla

Abstract In this study, we report for the first time the presence of alkaloids belonging to β-carboline type in the pods of the endemic Albizia polyphylla from Madagascar. Three major alkaloids were isolated and structurally identified as: 1-methyl-β-carboline, (+)-(R)-1-methyl-1,2,3,4-tetrahydro-β-carboline and (–)-(S)-1,2-dimethyl-1,2,3,4-tetrahydro-β-carboline.


Introduction
The genus Albizzia (also written as Albizia) is a member of the Mimosoideae subfamily and belongs to the Fabaceae family (formerly Leguminosae). It comprises 150 species, mostly found as trees or shrubs, distributed in tropical regions (Du Puy 2002). A number of Albizia species are used in traditional medicine especially in India and Africa (Kokila et al. 2013). In India, all parts of Albizia odoratissima are used for the treatment of leprosy, ulcers and cough (Rao et al. 2002). Albizia versicolor stem barks are employed in Kenya to treat several venereal diseases, including cough, joint pains and fever (Rukunga & Waterman 2001).
Previous bio-guided extractions reported that methanol extract of Albizia lebbeck leaves possesses diuretic effect in rodents (Velmurugan et al. 2013), antibacterial activities (Manjari et al. 2012) and mast cell stabilizing effects comparable to that of the reference drug, disodium chromoglycate (Kumar et al. 2013). The total alkaloidal fraction from leaves of Albizia inopinata was found to reduce the locomotor activity, ptosis as well as amphetamine aggregate toxicity in mice and rats (Assis et al. 2001). Spermine alkaloids with antimalarial activity were isolated from the root bark of Albizia gummifera and from the stem bark of Albizia adinocephala (Ovenden et al. 2002;Rukunga et al. 2007). The seeds of A. odoratissima showed high antioxidant activity (Yadav & Yadava 2013). Recently, phytochemical investigation of Albizia ferruginea (Mimosoideae) revealed the presence of novel flavones along with known saponosides with moderate antibacterial activity (Kamga et al. 2014).
In Madagascar, 30 Albizia species are known and 24 of which are endemic (Randrianarivo et al. 2014). Previously, we have reported that seed extracts from nine Albizia endemic species were shown to exert antimicrobial activity (Rakoto et al. 2011). Furthermore, extracts from seeds of five endemic species of Albizia displayed significant dose-dependent inhibition on vegetable seedling growth (Rakoto et al. 2012). Albizia polyphylla is an endemic species distributed throughout West and South Madagascar for which no phytochemical investigations have been reported so far. Herewith, we report the extraction of pods from A. polyphylla and the occurrence of indole alkaloids.

Results and discussion
In this study, two alkaloidal extracts were obtained by following two extractions procedures in either basic or acidic conditions (see Supplementary Material). TLC analysis of the two extracts showed the presence of three major compounds, common to both extracts. The two extracts were gathered and subjected to purification by flash chromatography, providing the three pure components, referenced as Ap1, Ap2 and Ap3 with an overall yield (w/w) of 0.035, 0.07 and 0.055%, respectively (Figure 1).
The characterisation of the isolated compounds was carried out using high-resolution mass spectrometry (HRMS), 1D and 2D NMR spectroscopy (Supplementary Material) and confirmed by comparison with literature data.
Compound Ap1 was isolated as a brown solid with a molecular formula of C 12 H 10 N 2 as determined by HReSIMS and from 13 C NMR spectra. The 1 H NMR spectrum displayed one methyl signal (δH 2.84 ppm) and six aromatic proton signals (δH between 7.26 and 8.31 ppm). 13 C NMR showed the presence of five aromatic quaternary carbons. The presence of an exchangeable singlet at 9.45 ppm is compatible with an aromatic N-H group. HMBC correlations found at the aromatic region suggested the presence of 2,3-disubstituted indole ring system. The remaining structural elements were composed of 4 carbons including a methyl group, one nitrogen and five protons (including the three protons of the methyl group). The presence of a third aromatic ring was deduced on the basis of the degree of unsaturation, COSY, HMQC and HMBC correlations. Hence, the structure of AP1 was deduced to be 1-methyl-β-carboline (Figure 1). The structure was confirmed by comparison with NMR data with literature data (Ghosal et al. 1971).
Compound Ap2 was assigned the molecular formula C 13 H 16 N 2 as determined by HReIMS.
1 H NMR displayed 4 aromatic protons, an exchangeable singlet at 7.91 ppm, attributed to an N-H group fused in an aromatic moiety (δH 7.91 ppm), and two methyl groups (δH 2.52 and 1.45 ppm) in which the upfield methyl group was observed as a doublet (J = 7 Hz) consistent with a linkage to a C-H. A part of the aromatic protons of Ap2 was similar to those of Ap1. In addition, analysis of the COSY and HMBC correlations of Ap2 revealed similar spin systems and connectivity with those found in Ap1. Using 2D, NMR allowed the identification of the full structure of Ap2 to be 1,2-dimethyl-1,2,3,4-tetrahydro-β-carboline. The structure of alkaloid Ap3 was easily established as it presents identical spin systems connectivity and very similar 1 H and 13 C NMR signals as AP1 and AP2. Finally, the absolute configuration of C-1 in alkaloids Ap2 and Ap3 was assigned by measuring the optical rotation and comparing the obtained values to literature data (Yamada & Akimoto 1969;espinoza-Moraga et al. 2010).
The easy and practical access to the pods of Albizzia polyphylla and assuming that the extraction procedure could be considerably enhanced would provide higher quantities of the β-carbolines described above that can be used as scaffolds in building semisynthetic analogues for pharmacological studies in pharmacological fields where β-carbolines were reported to be active (Cao et al. 2007;Zhang & Sun 2015).

Conclusions
In summary, this work was focused on the alkaloidal composition of endemic A. polyphylla pods that has never been investigated. A simple extraction and purification procedure allowed the access to three β-carbolines. Although the isolated alkaloids are known, the investigation could contribute to the medicinal use and misuse of fruits of this endemic plant in the fields where β-carbolines are reported to be active.

Disclosure statement
No potential conflict of interest was reported by the authors.