ANALYSIS OF THE HUSK AND KERNEL OF THE SEEDS OF MORINGA STENOPETALA

The ethanol extract of the kernel of the endemic plant Moringa stenopetala after silica gel column chromatography led to the isolation of 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate (1) and sucrose. The oil obtained by Soxhlet extraction with petrol was trans-esterified with BF3-MeOH and analyzed by GC-MS. The results showed the presence of diverse fatty acids namely palmitic (11%), palmitoleic (1.2%), stearic (11%), oleic (63%), linoleic (1.2%), arachidic (6%) and the rare behenic acids (6%). The white glossy seed husk which comprises 25% of the whole seed yielded two compounds namely 4-(σ-L-rhamnosyloxy)benzyl alcohol (2) and allantoin (3). To the best of our knowledge this is the first report of compound 2 as a natural product. Allantoin is reported here for the first time from the genus Moringa.

Moringa seeds are obtained from the pods of the Moringa tree.The seeds are covered by white fluffy husk which encapsulates the kernel (Figure 1).Removal of the husk by de-hulling yields the soft edible kernel, which when pressed yields oil.The seed cake, which is a byproduct obtained after pressing, is used for water treatment [10].4-(α-L-rhamnopyranosyloxy) benzylglucosinolate (1) was reported before from the seeds [3].Two compounds from the husk and one additional compound from the kernel are reported here.

Plant material
Seeds of M. stenopetala were collected from Arba Minch.The plant was identified by Mr. Melaku Wondafrash; voucher specimen (YM002/2015) deposited at the National Herbarium of Addis Ababa University, Ethiopia.

Chemicals and instruments
All solvents used were analytical grade.Melting points were determined in Digital Melting Point Apparatus.Analytical TLC was run on a 0.25 mm thick layer of silica gel GF 254 (Merck) on aluminum plate.Spots were detected by observation under UV light (254 nm) followed by spraying with vanillin in H 2 SO 4 .Column chromatography was performed using silica gel (230-400 mesh) Merck.Solvent was freed using Rotavapor BÜCHI, RE 121.NMR spectra were measured on Bruker Avance 400 NMR Spectrometer.UV-Vis spectra were recorded on T60 UV-Visible Spectrophotometer.Optical rotations were measured using Autopol ® IV Automatic Polarimeter.The IR spectra of compounds were recorded using a Perkin-Elmer BX Spectrometer (400-4000 cm -1 ) as KBr pellets.
GC-MS analysis were performed using Agilent Technologies 7820A gas chromatograph system equipped with HP-5 capillary column (30 m x 0.25 m; coating thickness, 0.25 µm) and Agilent technologies 5977 E mass spectroscopy ion trap detector.Analytical conditions were as follows: Injector and transfer line temperature are 220 and 260 o C, respectively; oven temperature programmed from 60 o C to 240 o C at 3 o C/min; carrier gas, helium at 1 mL/min; injection 5 µL; split ratio, 1:30.Identification of the constituents was based on search through mass\hunter\library\NIST11.L and mass\hunter\library\W9N11.L.

FT-MS
The high resolution positive and negative ion electrospray mass spectra were obtained from a Bruker Apex III Fourier transform ion cyclotron resonance mass equipped with an Infinity cell, a 7.0 Tesla superconducting magnet, an RF-only hexapole ion guide and an external electrospray ion source.Nitrogen was used as drying gas at 150 C.The sample solutions were introduced continuously via a syringe pump with a flow rate of 120 L h -1 .All data were acquired with 512 k data points and zero filled to 2048 k by averaging 32 scans.The data were evaluated by the Bruker XMASS 7.0.8software.

Preparation of fatty acid methyl esters (FAME)
M. stenopetala oil (2 g) was placed in 25 mL round bottom flask which contained 6 mL hexane to which BF 3 -MeOH (4 mL) was added.The reaction mixture was refluxed in water bath for 30 min.To the cooled mixture, 5 mL of water was added with shaking.The upper layer was separated by using separatory funnel, dried over anhydrous sodium sulphate, filtered and concentrated to afford 500 mg (25%).A small portion of the methylated fatty acids was dissolved in hexane and analyzed using GC-MS.

Extraction and isolation of compounds from the husk of M. stenopetala
The whole seed was separated first into husk (25%) and kernel.The finely powdered husk (100 g) was extracted with EtOH (600 mL), using a mechanical shaker (8 h), filtered and concentrated to afford 2 g (2%) yellowish jelly semisolid.This was adsorbed and applied on silica gel column with solvent system CH 2 Cl 2 :MeOH of increasing polarities as eluent to afford 27 fractions (each 40 mL).Fraction 8 (eluent CH 2 Cl 2 ) was identified as stearic acid.Fraction 19 eluted with CH 2 Cl 2 :MeOH (4:1) was found to be compound 2 (20 mg) while fraction 24 eluted with 100% MeOH was identified as allantoin (20 mg).

RESULTS AND DISCUSSION
The composition of the yellowish oil obtained by Soxhlet extraction of the kernel of M. stenopetala with petrol ether (36%) was determined by gas chromatography-mass spectrometry (GC-MS), after converting the oil to fatty acid methyl esters using BF 3 -MeOH.The fatty acids were identified by comparing the spectra of the components with the NIST Database.The GC-MS results showed that M. stenopetala oil contains a large proportion of double bond-containing fatty acids.The dominant fatty acid is oleic acid (63%), followed by palmitic (11%), stearic (11%), arachidic (6%), behenic (6%), palmitoleic (1.2%), linoleic (1.2%) and myristic acids (0.2%).These results are similar to that reported for oil from M. stenopetala growing in Kenya [11].The oil of the seeds of M. oleifera is known as Ben Oil owing to the presence of 5-6% of behenic acid [12].
The marc after the above Soxhlet extraction was extracted with hot ethanol and chromatographed over silica gel to afford sucrose (340 mg) and compound 1 (40 mg) as amorphous solid:broad band at 3400 cm -1 due to hydroxyl stretching, molecular formula C 20 H 29 NO 14 S 2 established from (-)-ESI-FT-MS, which displayed a molecular ion peak at m/z at 570.0960 (calcd 570.5666 for C 20 H 28 NO 14 S 2 -).The peaks at m/z 570 and 97 are characteristic of 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate [13].The 1 H-NMR spectrum of compound 1 displayed signals at δ 7.25 (2H, d, J = 8.40 Hz) and 7.04 (2H, d, J = 8.40 Hz) due to symmetrically placed protons on unsymmetrically para substituted aromatic ring.The anomeric proton signal at δ 5.30 (1H, d, J = 1.36 Hz) is characteristic of a rhamnopyranosyl group with αconfiguration [14].The presence of a rhamnosyl group was further substantiated by the appearance of terminal methyl signal at δ 1.  Sucrose was isolated as a white solid and identified using NMR ( 1 H and 13 C), and FT-MS.Further confirmation was doneusing TLC and polarimetry against reference sucrose.Sucrose, which is reported here for the first time from M. stenopetala, accounts for the slight sweet taste of the seeds when chewed.
The ethanol extract of the husk of M. stenopetala was subjected to silica gel column chromatography which led to the isolation of two compounds 2 and 3 (Figure 2).The benzylic methylene carbon signal was observed at δ 62.9 (C-7).
The heteronuclear multiple bond correlation (HMBC) spectrum showed correlation between methylene proton at δ 4.40 (H-7) with the carbon at 128.4 (C-3) and 136.2 (C-4) establishing the site of attachment of C-7 to C-4 of the benzene ring.Another key correlation observed was between anomeric proton signal at δ 5.32 (H-1') with the oxygenated aromatic carbon signal at δ 155.2 (C-1) which established the site of attachment of rhamnose to the aromatic ring.This was in agreement with the UV-Vis spectral analysis which showed the absence of phenolic hydroxyl as no bathochromic shift was observed on addition of sodium hydroxide to the cuvette containing methanolic solution of compound 2. Compound 2 was identified as 4-(α-Lrhamnopyranosyloxy) benzyl alcohol, a compound that has not been reported before.
Compound 3 was identified as allantoin, white solid, mp 220-222 o C. The asymmetric and symmetric stretching of primary amide were observed in the IR spectrum at 3438 and 3343 cm -1 , respectively.This was substantiated by the appearance of C-N stretching of primary amide at 1385 cm -1 .The intense band at 1715 cm -1 is characteristic of five membered cyclic lactams.The FT-MS showed molecular ion at m/z 181.0332 (calcd 181.10537 for C 4 H 6 O 3 N 4 Na) compatible with the molecular composition of C 4 H 6 O 3 N 4.

Figure 1 .
Figure 1.Picture of Moringa seed before and after removal of husk.