New aristolochic acid and other chemical constituents of Aristolochia maurorum growing wild in Jordan

Abstract Investigation of the chemical constituents of Aristolochia maurorum growing wild in Jordan resulted in the isolation and characterisation of one new compound in addition to 19 known compounds. The new compound was identified as aristolochic acid II alanine amide (14). The other known compounds were the following: palmitic acid (1), β-sitosterol (2), E-ethyl-p-coumarate (3), Z-ethyl-p-coumarate (4), aristolochic acid IV methyl ester (5), aristolactam I (6), loliolide (7), (+)-dehydrovomifoliol (8), glycerol-1-palmitate (9), aristolochic acid I (10), E-p-coumaric acid (11), E-N-coumaroyltyramine (12), β-sitosteryl glucoside (13), aristolochic acid IV (15), aristolochic acid III (16), esculetin (17), uracil (18), shepherdine (19) and adenosine (20). The isolated compounds were characterised by different spectroscopic methods including NMR (1D and 2D), UV, IR and HRESIMS.


Introduction
The Aristolochiaceae family comprises seven genera and 400 species flourishing mainly in tropical and warm temperate zones of both hemispheres (Watson & Dallwitz 1992). The name of this family, Aristolochia, originates from the Greek word 'aristos' meaning 'most fitting or best' and 'lochia' which means 'delivery' in reference to its original use to expel the placenta after childbirth. Aristolochia is an important genus wildly used in traditional medicine as a result of its beneficial properties. Species belonging to this genus were used traditionally as antidote in snakebites, an action which was confirmed by modern scientific CONTACT hala I. al-Jaber hala.aljaber@bau.edu.jo, rhhjaber@yahoo.com supplemental data for this article can be accessed at http://dx.doi.org/10. 1080/14786419.2016.1226833. investigation (Vishwanath et al. 1987;Chandra et al. 2002). Some Aristolochia species were found to possess anticancer activity in several bio-screening studies (Voloudakis-Baltatziz et al. 1992;Mongelli et al. 2000;Akindele et al. 2015). In Jordan, four species of Aristolochia are reported to grow in the wild including Aristolochia maurorum l., Aristolochia parviflora Sm., Aristolochia bottae Jaub. & Spach and Aristolochia billardieri Jaub. & Spach (Al-Eisawi 1982).
A. maurorum is a perennial herb with many basal stems, spreading and forming hemispherical growth leaves with yellow spotted flowers (Al-Eisawi 1998). It grows wildly in the middle and northern parts of Jordan flowering during the period extending from March to May. literature survey revealed that among all Aristolochia species' constituents, aristolochic acids I and II are usually the most common (Goun et al. 2002). Aristolochia fordiana afforded a new alkaloid glycoside (Zhou et al. 2014) and Aristolochia indica afforded many different compounds with anti-inflammatory properties (Desai et al. 2014). However, the survey also revealed that the chemical constituents of A. maurorum were seldom investigated. In fact, one previous investigation conducted in Jordan led to the isolation of only three known alkaloids including aristolochic acid I, aristolochic acid II and aristolochic acid III (Al-Ali et al. 2006). A full investigation of the secondary metabolites of the plant was not done before, and thus, this paper describes a full study of the plant which resulted in the isolation and characterisation of one new compound in addition to 19 other known compounds. Structure elucidation of the isolated compounds was based on extensive spectroscopic techniques including NMR (1D & 2D), UV-vis, IR and HREIMS.

Results and discussion
The crude ethanolic residue extracted from the air-dried ground plant material was partitioned into aqueous methanol (AMA), hexane (AMH), butanol (AMB) and water (AMW) fractions according to the procedure described in the experimental section. Both aqueous methanol and butanol fractions were further subjected to chromatographic separation on silica gel, Sephadex lH-20 and prep-TlC. The aqueous methanol fraction of A. maururum afforded a total of 13 compounds ( Figure 1) all of which are reported for the first time from the plant.
On the other hand, the butanol fraction afforded compounds 2, 10, 14-20, of which compound 14 is reported for the first time in nature. However, compounds 3, 4, 8, 15, 17, 18 and 19 are reported here for the first time from Aristolochiaceae family (Figures 1 and 2).
Compound 14 was obtained as a yellow solid from fraction AMBIV-1 according to the procedure mentioned in the experimental section. The IR spectrum of this compound showed strong absorption bands that indicated the presence of two carbonyl groups at 1715 and 1635 cm −1 corresponding to carboxyl and amide groups, respectively. The spectrum revealed also absorption bands that indicated the presence of OH group (3415 cm −1 ) and NH group (3466 cm −1 ). A molecular formula of C 19 H 14 N 2 O 7 was determined based on the molecular ion peak observed in the HRESIMS spectrum at m/z 405.06926 [M + Na] + . The 1 H and 13 C NMR spectra of compound 14 showed signals similar to those reported for 2-(Phenanthro [3,4-d]-1,3-dioxole-6-nitro-5-carboxamido)propanoic acid methyl ester (Teresa et al. 1983) except for the presence of a carboxylic acid moiety instead of the ester. The 13 C and DEPT NMR spectra of compound 14 revealed signals for 19 carbons, 14 of which were aromatic. The different carbon signals were differentiated into one CH 3 (δ C 17.7), one methine (δ C 48.4), 6 aromatic CH's, one methylenedioxy (δ C 103.4) in addition to 10 quaternary carbons including one carboxylic acid (δ C 174.5) and one amide carbonyl (δ C 167.4). These data were in agreement with alanine and aristolochic acid moieties. The 1 H NMR spectrum revealed a characteristic signal for CONH at δ H 8.97 (1H, br s) in addition to other 4 aromatic protons resonating at 7.64 (1H, s, H-2), 7.80 (1H, t, J = 7.6 Hz, H-7), 7.90 (1H, t, J = 7.8 Hz, H-6), 8.24 (1H, d, J = 7.8 Hz, H-8), 8.52 (1H, s, H-9) and 9.11 (1H, d, J = 8.4 Hz, H-5). Moreover, the signal resonating at δ H 6.52 (d, J = 4.2 Hz, H-12) and integrating for two protons was assigned to the methylenedioxy protons. These signals were in agreement with those of aristolochic acid II (Al-Ali et al. 2006). 2D NMR experiments including 1 H-1 H COSY, HMQC and HMBC confirmed the structure of 14 and the position of the alanine moiety. The HMBC spectra proved that the alanine is bonded to the aristolochic acid II moiety through amide linkage. The strong HMBC correlations observed between H-13 (δ H 4.36, δ C 48.4) and each of C-11(δ C 167.4), C-15 (δ C 174.5) and C-14 (δ H 1.42, δ C 17.7) and those observed between NH (δ H 8.97) with C-13 (δ C 48.4) and C-11 (δ C 167.4) proved the linkage of the alanine moiety to the aristolochic acid skeleton through C-11-NH-C-13. In addition, the NH (δ H 8.97)-H-13 (δ H 4.36) strong correlation observed in the COSY spectrum further helped in confirming the structure of the compound ( Figure S1). Based on these data, the compound was identified as aristolochic acid II alanine amide. It is worth mentioning that this is the first report of the isolation and the NMR spectral data for this compound in acidic form. Different NMR spectra of compound 14 are shown in Figures 2S, 3S and 4S.
Comparison of the optical rotation of the new compound with that of its methyl ester derivative described in the literature (Teresa et al. 1983) indicates the amino acid moiety is l-(+)-alanine.

General experimental procedures
IR spectra of the isolated compounds were recorded on Thermo-Nicolt Nexus 870 FT-IR spectrophotometer (Thermo Scientific, Wisconsin, USA). The UV-spectra were recorded on Cary Bio 100 double beam UV-visible spectrophotometer. 1 H NMR spectra were recorded on a Bruker DPX-500 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany) and TMS as an internal standard. 13 C NMR spectra were recorded at 125 MHz using the same instrument. Electrospray ionisation mass spectra were recorded using Bruker Daltonics Apex IV, 7.0 T Ultra Shield Plus (Bremen, Germany). Melting points were measured without correction by an electrothermal (IA9300) digital melting point apparatus. Specific rotations were measured using MCP 200 polarometer (Anton Paar, Seelz, Germany). Separation of compounds was performed using column chromatography packed with normal phase silica gel and Sephadex lH-20. Silica gel used for CC was silica gel 60 (0.063-0.002 mm, Fluka, Steinheim, Germany). Purification of compounds was performed by thin-layer chromatography (TlC) using manually prepared plates (silica gel G-UV 254 , Macherey-Nagel). Final purification was performed on commercial glass plates G-UV 254 (0.5 mm, Macherey-Nagel, Easton, PA, USA). Routine TlC was done on silica gel G-UV 254 glass plates (0.25 mm, Macherey-Nagel). Sephadex lH-20 was used in purification of some fractions. Compounds were visualised under UV light and by spraying with anisaldehyde-sulphuric acid spray reagent followed by heating at 120 °C.

Plant material
A. maurorum was collected from different locations in Jordan including Irbid and Amman during its full flowering period (March-April, 2013). The identity of the plant was confirmed by Prof. Dr. Musa Abu Zarga, Chemistry Department, Faculty of Science, The University of Jordan. A voucher specimen (JU/AM-2013) was kept in the herbarium at the Chemistry Department, Faculty of Science, The University of Jordan.

Extraction and isolation
The whole dry and ground plant material (21.0 kg) was defatted by extraction with petroleum ether (80 l, 7 days) at room temperature. The residual plant material was then soaked several times in ethanol to extract the secondary metabolites (4 times, 75 l, 7 days each, room temperature). After evaporating ethanol under reduced pressure, the combined alcoholic residue (approximately 1.2 kg) was partitioned according to the procedure described in the literature (Al-Jaber et al. 2012;Al-Qudah et al. 2014Hasan et al. 2016) to afford the aqueous methanol (AMA, 200 g), the hexane (AMH, 220 g), water (AMW, 590 g) and the butanol (AMB, 180 g) extracts.
The aqueous methanol extract (AMA, 200 g) was adsorbed on silica gel 60 (200 g) and then loaded onto a column (63 × 5.8 cm) containing 600 g of the same adsorbent packed in chloroform. The column was eluted with CHCl 3 -MeOH gradient mixture of increasing polarity until pure methanol was used. A total of 90 fractions were collected (500 ml each) and then pooled according to their TlC behaviour into six major groups (AMA I-VI). Each fraction was separated to its components by a combination of column chromatography, Sephadex lH-20 and TlC.
Similarly, the butanol extract (AMB, 180 g) was adsorbed on 200 g silica gel and then subjected to a silica gel column (400 g) packed in chloroform and eluted with CHCl 3 -MeOH mixtures of increasing polarity until pure methanol was used. A total of 100 fractions (500 ml each) were collected which were then consolidated into six major groups (AMB I-VI) based on their TlC behaviour. Fractions AMB I-III were subjected to separation on Sephadex lH-20.  Salmoun et al. 2002) and 20 (140 mg, Ries et al. 1990).
Fraction AMB-IV (25 g) was adsorbed on 30 g silica gel G-V254 and then loaded onto a column containing 200 g of the same adsorbent (28.0 × 5.0 cm) packed in CHCl 3 : n-butanol: formic acid mixture (94:4:2, v/v). Isocratic elution (using the same solvent mixture) of this column afforded 50 fractions (250 ml each) which were then grouped according to their TlC behaviour into 9 groups. Treatment of fraction AMB-IV-1 with distilled methanol afforded the new compound 14 as a yellow solid.

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