L-(−)-(N-trans-Cinnamoyl)-arginine, an Acylamino Acid from Glinus oppositifolius (L.) Aug. DC.

An amino acid derivative, L-(−)-(N-trans-cinnamoyl)-arginine, was isolated from the whole plant of Glinus oppositifolius (L.) Aug. DC. along with kaempferol 3-O-galactopyranoside, isorhamnetin 3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside, vitexin, vicenin-2, adenosine and L-phenylalanine. The structure determinations were based on analyses of chemical and spectroscopic methods.


Introduction
Glinus oppositifolius (L.) Aug. DC. (Mollungincaeae; Thai name: Phak-Khee-Khuang) is an annual prostrate weed commonly found in paddy fields after harvesting, especially in the Northeast areas of Thailand. The leaves are used as vegetable for cooking purposes, as well as an expectorant and antipyretic agent. This species has been also found in other tropical parts of Asia, Africa and North Australia. Plants in this genus are reported to contain triterpenoidal saponins [1][2][3][4][5][6]. In previous studies OPEN ACCESS of this plant, triterpenoidal saponins and pectin polysaccharides were reported to act as antiprotozoal [6] and immunomodulating agents [7,8], respectively. The plant extracts showed antifungal, larvicidal, molluscicidal, free scavenging and antioxidant activities [9,10].
The present paper deals with the isolation and characterization of a new amino acid derivative 1 from G. oppositifolius, together with six known compounds, including four flavonoid glycosides 2-5, one nucleoside 6, and one amino acid 7, from the water soluble fraction of the whole plant.

Results and Discussion
The methanolic extract of G. oppositifolius was suspended in H 2 O and partitioned with Et 2 O. The aqueous layer was separated by the combinations of Diaion HP20 column, silica gel column, RP-18 column and preparative HPLC-ODS column chromatography to provide seven compounds 1-7.   Compound 1 was isolated as an amorphous powder. Its molecular formula was determined as C 15 H 20 N 4 O 3 by high-resolution electrospray ionization time-of-flight (ESITOF) mass spectrometric analysis. Inspection of the IR spectrum indicated the presence of the typical amine N-H stretching absorption band at 3333 and 3165 cm −1 . The band at 1667 cm −1 is a characteristic for the C=O stretching of an amide and a carboxylic acid, 1613 cm −1 for the C=N of a quanidyl group, and the 1571 cm −1 one for amine group bending vibrations [11,12]. The 1 H-NMR spectrum exhibited a trans-cinnamoyl moiety inferred from a set of five protons corresponding to an aromatic ring at δ H 7.32 (2H), 7.35 (1H) and 7.43 (2H), in addition to two olefin protons at δ H 6.74 and 7.28 (each d). The trans configuration was assigned from the coupling constant with J = 15.8 Hz. The 13 C-NMR spectrum showed 13 carbon signals (Table 1), of which nine were assignable to a trans-cinnamoyl moiety. The remaining carbon signals belong to three methylenes (δ C 25.5, 30.2, 40.8), one methine (δ C 54.3) and two quaternary carbons (δ C 157.7, 176.6), compatible to those of the amino acid arginine [13]. Therefore, this compound was suggested to be a trans-cinnamoyl derivative of arginine. The complete assignments were established by analyzing the 2D-NMR spectra including COSY, HSQC and HMBC. In the HMBC spectrum (Table 1), the significant correlations were observed between δ H 4.13 (H-2) and δ C 164.7 (C-9'), indicating that one of the hydrogen atoms of the α-amino group was replaced by the cinnamoyl moiety. In order to determine the absolute configuration, L- ( Figure 2. The structure of this compound was initially proposed in 1986 as a synthetic compound for exploration of carboxypeptidase N functions [14]. The physical and spectroscopic data were given here, and it should be noted that this compound has been reported from the natural sources for the first time.   [15,16] The NMR spectroscopic data of compounds 4 and 5 were coincident with those of apigenin 8-C-β-D-glucopyranoside (vitexin) and apigenin 6,8-di-C-β-D-glucopyranoside (vicenin-2), respectively. [17,18] Compounds 6 and 7 were assigned to adenosine and L-phenylalanine, respectively, by comparison of physical and NMR spectral data with authentic samples [19].

Plant Material
Glinus oppositifolius (L.) Aug. DC. was collected in January 2008 from Khon Kaen province, Thailand. The plant was identified by Mr. Nopporn Nontapa of Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher specimen (TK-PSKKU-0063) has been deposited in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University.

Extraction and Isolation
Dried whole Glinus oppositifolius (1.4 kg) was extracted three times with CH 3 OH (8 L for each extraction) at room temperature. The solvent was concentrated in vacuo to give a greenish powder (226.4 g). This residue was suspended in H 2 O and defatted with Et 2 O three times (1 L each). The aqueous soluble fraction (113.6 g) was applied to a column of Diaion HP-20 and eluted successively with H 2 O, CH 3 OH, and (CH 3 ) 2 CO. The fraction eluted with CH 3 OH (20.0 g) was concentrated to dryness and subjected to a silica gel column using solvent systems I (4.0 l), II (4.0 l), III (6.0 l) and IV (5.0 l) providing six fractions (fraction A−F). Fraction B (2.6 g) was applied to a column of RP-18 using solvent system V to give 12 fractions. Fraction B-8 was purified by preparative HPLC-ODS with solvent system IX to afford compound 2 (10.9 mg). Fraction B-11 was purified by preparative HPLC-ODS with solvent system IX to give compound 4 (73.1 mg). Fraction C (3.2 g) was separated on a column of RP-18 using solvent system V to give nine fractions. Compound 6 (40.1 mg) was obtained from fraction C-3 by crystallization. Fraction D (5.0 g) was subjected to a column of RP-18 using solvent system V to give 12 fractions. Fractions D-3 and D-4 were combined and purified by preparative HPLC-ODS with solvent system VIII to afford compound 5 (317.3 mg). Fraction D-9 was further purified by preparative HPLC-ODS to yield compound 3 (13.0 mg). Fraction E (3.4 g) was chromatographed on a column of RP-18 using solvent system V to provide six fractions. Fraction E-1 was purified by preparative HPLC-ODS with solvent system IX to give compound 7 (31.7 mg). Finally, fraction E-4 was purified by preparative HPLC-ODS with solvent system VIII to obtain compound 1 (115.0 mg). (1 (1) L-(+)-Arginine (50.2 mg) was dissolved in H 2 O (2.0 mL) and the pH adjusted to 12.0 with 2 N NaOH. A solution of trans-cinnamoyl chloride (51.5 mg) in dioxane (2.0 mL) was added. After stirring at room temperature for 30 min, the reaction was concentrated and purified by preparative HPLC-ODS with solvent system VIII to obtain L-(−)-(N-trans-cinnamoyl)-arginine (15.0 mg) as an amorphous powder, [α] D 27 − 15.8° (6 N HCl, c 0.12). The NMR spectroscopic data was identical to those of the natural compound.

Conclusions
Isolated compounds of Thai plant origin were described as an acylamino acid 1, flavonoid glycosides 2-5, a nucleoside 6, and an amino acid 7. The results of our study were different from the previous mention on the chemical constituents of genus Glinus, indicating triterpenoidal saponins. The chemical variations might be due to the different geographical regions involved. The study identified new types of compounds in this species.