Carotamine, a Unique Aromatic Amide from Daucus Carota L. Var Biossieri (Apiaceae)

The unique aromatic peptide 4-(p-aminobenzoylamino)-2-aminobenzoic acid, carotamine, together with 2,4-diaminobenzoic acid, isolated for the first time from a plant source, were identified from the aqueous alcoholic extract of the aerial parts of Daucus carota L. var. boissieri (Apiaceae). The structures were determined through conventional methods of analysis and confirmed by LC-ESI/MS and NMR spectral analysis.


Introduction
Daucus L. (Apiaceae) [1] includes about 60 species distributed mostly in Europe, Africa, West Asia and few ones in North America and Australia [2]. In Egypt, the genus Daucus L. is represented by 6 wild species [1] among which the two varieties Daucus carota boissieri [3] and Daucus carota sativus [4] are widely cultivated for their fleshy edible roots (Bailey, 1960). Daucus carota has been reported to contain several constituents such as flavonoids [6,7], essential oils [8,9], polyacetylenes [10,11] and phenylpropanoids [12]. Daucus carota is well known in the Egyptian folk medicine as a stimulant, carminative and diuretic [13]. The decoction of carrot is used for infantile diarrhoea and as an antihelmentic [14]. The fruit essential oil has been proven to be hypotensive, cardiac and CNS depressant [15], antibacterial [16], antibilharzial [17], and fungicidal [18]. Carrots also showed a significant protective activity in the alleviation of chloroform-induced hepatocellular injury in the mouse [19].
The present study reports on the isolation and identification of 2,4-diaminobenzoic acid (1) and the unique aromatic peptide, 4-(p-aminobenzoylamino)-2-aminobenzoic acid (2) or carotamine, which is the first aromatic peptide reported to occur in nature. Extensive EI and LC-ESI/MS techniques were applied together with 1 H-and 13 C-NMR spectral analysis to verify the full structure of both compounds.

Results and discussion
The aqueous alcoholic extract of the ground meal of the aerial Daucus carota parts, dried under vacuum, was defatted through exhaustive extraction with CHCl 3 . The residue left after CHCl 3 extraction was shown by two-dimensional chromatography to contain a mixture of polar compounds (high R f values in aqueous solvents and low R f values in organic solvent) mainly of phenolic nature (positive FeCl 3 test). The chromatograms also revealed the presence of two non-polar compounds that under UV light appeared as canary yellow (compound 1) and dark purple (compound 2) spots, respectively. A combination of column chromatography on Sephadex LH-20, using water saturated butanol as an eluent and preparative paper chromatography using 6% acetic acid as solvent afforded two pure samples of compounds 1 and 2.
Compound 1 was isolated as an amorphous white powder with LC/UV absorption maxima at 227, 274 and 312 nm. The IR spectral analysis revealed two intense absorption bands at ν max 3449.9 and 1661.7 cm -1 , consistent with amino and hydroxyl groups and a carbonyl group, respectively. The To resolve any ambiguity about the structure of 1, 1 H and 13 C-NMR spectral analysis were then undertaken. The 1 H-NMR spectrum (DMSO-d 6 , room temperature) revealed, in the aromatic region, the presence of a resonance pattern at δ 6.3 (d, J=2 Hz), 6.4 (dd, J=2 Hz & J=7.5 Hz) and 7.8 (d, J=7.5 Hz) ppm, typical of a 1,2,4trisubstituted benzene [20], and assigned to H-3, H-5 and H-6 in the proposed 2,4-diaminobenzoic acid structure of (1). The spectrum also revealed a downfield resonance appearing as a sharp singlet at δ 12.7 ppm attributable to a hydrogen bonded proton (between the carbonyl carboxyl group at position 1 and the o-amino group at position 2, thus confirming the structure of (1) as 2,4-diaminobenzoic acid. Further confirmation of the structure was obtained through 13 C-NMR analysis. The recorded spectrum showed seven distinct aromatic carbon resonances among which the most downfield resonance at δ 168.0 ppm was assigned to the carboxyl carbon resonance while the most upfield resonance at δ 100.1 ppm was assigned to the quaternary C-1 carbon. Assignment of the remaining carbon resonances was aided by calculating the expected chemical shifts deduced by applying the additive substituent rules to the reported chemical shifts of anthranilic acid [21]. Consequently, the carbons that bear the amino groups, C-2 and C-4, were found resonating at δ 148.9 and 152.6 ppm, respectively. The protonated carbons C-3, C-5 and C-6 gave three signals at δ 103.2, 108.5 and 134.1 ppm, respectively, which all agree well with the 2,4-diaminobenzoic acid structure proposed for 1. It should be mentioned that this is the second reported natural occurrence of this compound, which has been characterised once before as a metabolite of Streptomyces flocculus [22].
Compound 2 was isolated as an amorphous yellow powder which exhibited in its LC/UV spectrum two fused absorption maxima at 363.8 and 336 nm as well as two shoulders at 237 and 302 nm. IR spectral analysis of 2 afforded a spectrum which revealed three absorption bands at ν max 3445.7, 1659.9 and 1640.5 cm -1 , consistent with amino and hydroxyl groups, a carboxyl carbonyl group and an amide carbonyl group, respectively. Standard alkaline hydrolysis (5% aqueous KOH, 100°C, ½ hour) of compound 2 yielded 2,4-diaminobenzoic acid (1) and p-aminobenzoic acid (CoPC). The EI/MS of 2 showed a molecular ion at m/z 271 and a base peak at 270, thus suggesting that the molecule of 2 is formed by two amino acids joined by an amide linkage (also detected by alkaline hydrolysis). In this spectrum the base peak at m/z 270 is therefore due to the loss of a carboxylic hydrogen or allylic proton from the amide bridge. The LC-ESI-ve/MS of 2 exhibited a R t of 5. LC/MS analyses were performed by reversed-phase HPLC on a Purosphere STAR RP-18 endcapped column (55x2 mm, 3µm, Merck, Darmstadt) using a Waters HPLC system, consisting of a Waters 2690 "Alliance" separation module coupled to a Waters 996 scanning UV detector. Flow injection analysis was performed by injecting 10µ of the extract into a solvent stream of methanol/water (1:1 by volume). Solvent A was 100% acetonitrile (HPLC grade, Merck); solvent B was water. Elution was performed at room temperature and at flow rate of 0.8 mL/min. The gradient program started at 5% A with an isocratic hold for 3 min, followed by a fast linear increase to 95% A at 4 min. The solvent composition was held for 1 min to flush the column, then changed back to initial conditions over 1 min and equilibrated for 4 min before the next sample injection; a shorter equilibration time lead to a shift in retention times. The total run time was 10 min. The eluent of the HPLC was split at a 1:4 ratio using an AcuRate TM flow splitter (LC Packings, via Omnilab, Mettmenstetten, CH) so that approximately 200 µ/min entered the electrospray ion source of the mass spectrometer. The mass spectrometer used in this study was a Micromass Quattro-LC triple quadrupole mass spectrometer equipped with a "Z-Spray" electrospray ion source. The electrospray capillary voltage was set to 3.0 kV, the source block temperature to 120°C. The cone gas was operated at 60 I/h, desolvation gas at 520 I/h and the desolvation temperature to 150°C. Spectra were acquired in profile mode alternating with 35 and 70 V cone voltage and scanning over the range m/z 50 to 1500 per second. Data acquisition was performed using Micromass'software package MassLynx 3.4. 1 H-and 13 C-NMR spectra were obtained on a Bruker AMX 400 spectrometer. 1 H spectra were measured relative to TMS and 13 C spectra were measured at 100 MHz, relative to DMSO-d 6

Plant material, isolation and identification
Fresh aerial parts of Daucus carota L. var boissieri, were collected from Orman Botanical garden, Cairo, Egypt, during March 2000 and authenticated by Prof. Dr. Nabil El-Hadidi, Department of Botany, Faculty of Science, Cairo University, Egypt. A voucher specimen has been deposited in the Herbarium of the Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt. One kg of aerial parts of Daucus carota, dried in the shade in an air-draft, were comminuted to powder and exhaustively extracted with EtOH-H 2 O (3:1). The aqueous alcoholic extract was dried in vacuum, and completely defatted with CHCl 3 . The residue left, 10 g, was dissolved in methanol and subjected to column chromatography (CC) on Sephadex LH-20 using n-BuOH saturated with H 2 O for elution to yield 10 major fractions (I-X). Compound (1) (15 mg) was isolated from fraction IV by repeated PPC using 6% HOAc as a solvent. Compound (2) (20 mg) was obtained from fraction X by PPC using 6% HOAc as a solvent followed by Sephadex LH-20 CC using MeOH for elution.