L-(-)-(N-trans-cinnamoyl)-arginine, an acylamino acid from Glinus oppositifolius (L.) Aug. DC.

Article abstract of DOI:10.3390/molecules15096186

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, v

Full text of DOI:10.3390/molecules15096186

Molecules 2010, 15, 6186-6192; doi:10.3390/molecules15096186
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molecules
ISSN 1420-3049
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Article
L-(−)-(N-trans-Cinnamoyl)-arginine, an Acylamino Acid from
Glinus oppositifolius (L.) Aug. DC.
Poolsak Sahakitpichan 1, Wannaporn Disadee 1, Somsak Ruchirawat 1,2 and
Tripetch Kanchanapoom 3,*
1
Chulabhorn Research Institute and Chulabhorn Graduate Institute, Vipavadee-Rangsit Highway,
Bangkok 10210, Thailand; E-Mails: poolsak@cri.or.th (P.S.); wannapor@cri.or.th (W.D.)
2
The Center of Excellence on Environmental Health, Toxicology and Management of Chemicals,
Vipavadee-Rangsit Highway, Bangkok 10210, Thailand; E-Mail: somsak@cri.or.th
3
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
* Author to whom correspondence should be addressed; E-Mail: trikan@kku.ac.th;
Tel.: +66-43-362092; Fax: +66-43-202379.
Received: 28 July 2010; in revised form: 27 August 2010 / Accepted: 30 August 2010 /
Published: 2 September 2010
Abstract: 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.
Keywords: Glinus oppositifolius; Molluginaceae; L-(−)-(N-trans-cinnamoyl)-arginine;
arginine derivative; flavonol glycoside; flavone C-glycoside
1. 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 [1-6]. In previous studies

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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.
2. Results and Discussion
The methanolic extract of G. oppositifolius was suspended in H2O and partitioned with Et2O. 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.
Figure 1. Structures of isolated compounds 1–7.
OH
NH
O
3'
5
3
1
6
2
2'
HO
O
4'
5'
H2N
N
OH
Glc : β-
D
-glucopyranosyl
4
H
8'
Gal : β-
D
-galactopyranosyl
HN
9'
1'
6'
7'
O-Gal
Xyl : β-D-xylopyranosyl
O
OH O
1
2
OH
OH
OH
Glc
Glc
HO
O
HO
O
HO
O
OCH3
Xyl
2
Glc
O-Gal
OH O
OH O
OH O
5
4
3
NH2
O
N
N
N
OH
N
O
NH2
HO
6
7
OH OH
Compound 1 was isolated as an amorphous powder. Its molecular formula was determined as
C15H20N4O3 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
1
1571 cm−1 one for amine group bending vibrations [11,12]. The H-NMR spectrum exhibited a
trans-cinnamoyl moiety inferred from a set of five protons corresponding to an aromatic ring at δH
7.32 (2H), 7.35 (1H) and 7.43 (2H), in addition to two olefin protons at δH 6.74 and 7.28 (each d). The
trans configuration was assigned from the coupling constant with J = 15.8 Hz. The 13C-NMR spectrum
showed 13 carbon signals (Table 1), of which nine were assignable to a trans-cinnamoyl moiety. The

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remaining carbon signals belong to three methylenes (δC 25.5, 30.2, 40.8), one methine (δC 54.3) and
two quaternary carbons (δC 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 δH 4.13 (H-2)
and δC 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-(−)-(N-trans-cinnamoyl)-
arginine ([α]2D7 − 15.8° in 6 N HCl) was synthesized by acylation of L-(+)-arginine ([α]2D7 + 24.5° in 6 N
HCl) with trans-cinnamoyl chloride (see Experimental) [12] to compare the optical rotation value.
Since the isolated compound showed the optical rotation value at [α]2D7 − 14.9° in 6 N HCl, the absolute
configuration at C-2 position was concluded to be (S). Consequently, compound 1 was identical to the
synthetic compound, L-(−)-(N-trans-cinnamoyl)-arginine. Furthermore, the electron ionization mass
spectrometric analysis showed the fragment ions of this compound at m/z 131 (100), 146 (59), 103
(45), 113 (35) as illustrated in 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.
13
Table 1. NMR spectroscopic data of compound 1 (in DMSO-d6, C-NMR: 100 MHz and
1H-NMR: 400 MHz).
Position
δC
δH
HMBC
Amino acid
1
2
3
176.6
54.3
30.2
4.13 (1H, m)
1.62 (1H, m)
1.75 (1H, m)
1.48 (2H, m)
3.05 (2H, m)
1, 3, 4, 9'
1, 2, 4, 5
1, 2, 4, 5
2, 3, 5
4
5
25.5
40.8
3, 4, 6
6
157.7
2-NH
8.21 (1H, d, J = 8.0 Hz)
1, 2, 3, 8', 9'
Cinnamoyl moiety
1'
135.5
127.8
129.2
129.6
138.4
123.3
164.7
2', 5'
3', 6'
4'
7'
8'
7.43 (2H, dd, J = 8.0, 1.7 Hz)
7.32 (2H, m)
7'
1'
7.35 (1H, m)
7.28 (1H, d, J = 15.8 Hz)
6.74 (1H, d, J = 15.8 Hz)
1', 2', 8', 9'
1', 7', 9'
9'

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Figure 2. Possible major fragment ions of compound 1.
NH
O
a
− CO
H2N
N
H
OH
b
HN
O
a
m /z =103
m /z =131
O
b
NH
O
N
or
H
H2N
N
OH
H
O
m /z =146
NH
NH
− CO2
− H
H2N
N
H2N
N
H
H
COOH
m /z =113
Compounds 2 and 3 were identified as kaempferol 3-O-β-D-galactopyranoside and isorhamnetin
3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside, respectively. [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].
3. Experimental
3.1. General
1H- and 13C-NMR spectra were recorded in DMSO-d6 using a Bruker AV-400 (400 MHz for
1H-NMR and 100 MHz for 13C-NMR) or a Varian Gemini 2000 (200 MHz for 1H-NMR and 50 MHz
for 13C-NMR) spectrometer. IR spectra were obtained from a Perkin Elmer Universal FTIR
spectrometer. Low resolution mass spectrometry (LRMS) was performed using a Finnigan Polaris
mass spectrometer. High resolution mass spectrometry (HRMS) was performed using a Bruker Micro
TOF-LC mass spectrometer. Optical rotations were measured with a Jasco P-1020 digital polarimeter.
For column chromatography, Diaion HP-20 (Mitsubishi Chemical Industries Co. Ltd.), silica gel 60
(70-230 mesh, Merck), and RP-18 (50 μm, YMC) were used. HPLC was carried out on an ODS
column (20 X 250 mm i.d., YMC) with a Jasco RI-2031 refractive index detector. The flow rate was
6 mL/min. The solvent systems were: I) EtOAc-MeOH (9:1); II) EtOAc-MeOH-H2O (40:10:1); III)
EtOAc-MeOH-H2O (70:30:3); IV) EtOAc-MeOH-H2O (6:4:1); V) 10−80% aqueous MeOH; VI) 2%

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aqueous MeCN; VII) 5% aqueous MeCN; VIII) 15% aqueous MeCN; and IX) 20% aqueous MeCN.
The spraying reagent used for TLC was 10% H2SO4 in 50% EtOH.
3.2. 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.
3.3. Extraction and Isolation
Dried whole Glinus oppositifolius (1.4 kg) was extracted three times with CH3OH (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 H2O and defatted with Et2O 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 H2O, CH3OH, and (CH3)2CO. The fraction eluted with CH3OH (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).
3.4. L-(−)-(N-trans-cinnamoyl)-Arginine (1).
Amorphous powder: [α]27 − 14.9° (6 N HCl, c 0.22); IR (UATR) νmax 3333, 3165, 3047, 1667, 1613,
D
1571, 1400, 1245, 979 cm−1; 1H- and 13C-NMR (DMSO-d6): Table 1; EIMS: m/z 131 (100), 146 (59),
103 (45), 113 (35); Negative HRMS(ESITOF), m/z: 303.1468 [M − H]– (calcd for C15H19N4O3,
303.1463).
3.5. Synthesis of L-(−)-(N-trans-cinnamoyl)-Arginine (1)
L-(+)-Arginine (50.2 mg) was dissolved in H2O (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

Molecules 2010, 15
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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]2D7 − 15.8° (6 N HCl, c 0.12). The NMR spectroscopic data was identical to those of the
natural compound.
4. 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.
Acknowledgements
This study was financially supported by Chulabhorn Research Institute and the Center of Excellence
on Environmental Health, Toxicology and Management of Chemicals, Thailand.
References
1. Hamed, A.I.; Springuel, I.; El-Emary, N.A.; Mitome, H.; Miyaoka, H.; Yamada, Y. Triterpenoidal
saponin glycosides from Glinus lotoides var. dictamnoides. Phytochemistry 1996, 43, 183-188.
2. Hamed, A.I.; El-Emary, N.A. Triterpene saponins from Glinus lotoides var. dictamnoides.
Phytochemistry 1999, 50, 477-480.
3. Endale, A.; Wray, V.; Murillo, R.; Schmidt, P.C.; Merfort, I. Hopane-type saponins from the
seeds of Glinus lotoides. J. Nat. Prod. 2005, 68, 443-446.
4. Biswas, T.; Gupta, M.; Achari, B.; Pal, B.C. Hopane-type saponins from Glinus lotoides Linn.
Phytochemistry 2005, 66, 621-626.
5. Hamed, A.I.; Piacente, S.; Autore, G.; Marzocco, S.; Pizza, C.; Oleszek, W. Antiproliferative
hopane and oleanane glycosides from the roots of Glinus lotoides. Planta Med. 2005, 71,
554-560.
6. Traore, F.; Faure, R.; Ollivier, E.; Gasquet, M.; Azas, N.; Debrauwer, L.; Keita, A.; Timon-David,
P.; Balansard, G. Structure and antiprotozoal activity of triterpenoid saponins from Glinus
oppositifolius. Planta Med. 2000, 66, 368-371.
7. Inngjerdingen, K.T.; Debes, S.C.; Inngjerdingen, M.; Hokputsa, S.; Harding, S.E.; Rolstad, B.;
Michaelsen, T.E.; Diallo, D.; Paulsen, B.S. Bioactive pectic polysaccharides from Glinus
oppositifolius (L.) Aug. DC., a Malian medicinal plant, isolation and partial characterization.
J. Ethnopharmacol. 2005, 101, 204-214.
8. Inngjerdingen, K.T.; Kiyohara, H.; Matsumoto, T.; Petersen, D.; Michealsen, T.E.; Diallo, D.;
Inngjerdingen, M.; Yamada, H.; Paulsen B.S. An immunomodulating pectic polymer from Glinus
oppositifolius. Phytochemistry 2007, 68, 1046-1058.

Molecules 2010, 15
6192
9. Diallo, D.; Marston, A.; Terreaux. C.; Touré, Y.; Paulsen, B.S.; Hostettmann, K. Screening of
Malian Plants for antifungal, latvicidal, molusscicidal, antioxidant and radical scavenging
activities. Phytother. Res. 2001, 15, 401-406.
10. AsokKumar, K.; UmaMaheswari, M.; Sivashanmugam, A.T.; SubhadraDevi, V.; Subhashini, N.
Free radical scavenging and antioxidant activities of Glinus oppositifolius (carpet weed) using
different in vitro assay systems. Pharmaceut. Biol. 2009, 47, 474-482.
11. Silverstein, R.M.; Bassler, G.C.; Morrill, T.C. Spectrometric Identification of Organic
Compounds, 5th ed.; John Wiley & Sons, Inc.: New York, NY, USA, 1991; pp. 91-164.
12. Pretsch, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds: Table of
Spectral Data, 3rd ed.; Springer: New York, NY, USA, 2000; pp. 71-160, 245-312.
13. Wauven, A.V.; stalon, V. Occurrence of succinyl derivatives in the catabolism of arginine in
Pseudomonas cepacia. J. Bacteriol. 1985, 164, 882-886.
14. Fisher, G.H.; Ryan, J.W.; Chung, A.; Plumer, T.H., Jr. Synthetic inhibitors of carboxypeptidase N.
Adv. Exp. Med. Biol. 1986, 198A, 405-410.
15. Barberá, O.; Sanz, J.F.; Sánchez-Parareda, J.; Marco, J.A. Further flavonol glycosides from
Anthyllis onobrychioides. Phytochemistry 1986, 25, 2361-2365.
16. Sikorska, M.; Matlawska, I. Kaempferol, isorhamnetin and their glycosides in the flowers of
Asclepias syriaca L. Acta Pol. Pharm. 2001, 58, 269-272.
17. Agrawal, P.K.; Bansal, M.C. Carbon-13 NMR of flavonoids. In Flavonoid Glycosides; Agrawal,
P.K., ed.; Elsevier: Amsterdam, Netherlands, 1986; pp. 283-336.
18. Kanchanapoom, T.; Kasai, R.; Yamasaki, K. Iridoid glucosides from Tunbergia laurifolia.
Phytochemistry 2002, 60, 769-771.
19. Kanchanapoom, T.; Kamel, M.S.; Kasai, R.; Picheansoonthon, C.; Hiraga, Y.; Yamasaki, K.
Benzoxazinoid glucosides from Acanthus ilicifolius. Phytochemistry 2001, 58, 637-640.
Sample Availability: Samples of the compounds are available from the authors.
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