Polar compounds isolated from the leaves of albertisia delagoensis (menispermaceae)

Article abstract of DOI:10.3390/molecules16119153

Aqueous infusions of the leaves of the shrub Albertisia delagoensis (Menispermaceae) are used in South Africa in traditional Zulu medicine to alleviate a variety of symptoms, including fever, and intestinal problems. We report the analysis of such an aqueous extract using the HPLC-NMR technique. A number of polar compounds were identified, including proto-quercitol, nicotinic acid, allantoic acid, 3,4-dihydroxybenzoic acid, phthalic acid and the aporphine alkaloid derivative roemrefidine. Allantoic acid and roemrefidine have been fully characterised by ¹H- and ¹³C-NMR and mass spectrometry. Earlier reports of antiplasmodial activity of roemrefidine and of A. delagoensis extracts are correlated with this study and with the antipyretic properties of neutral aqueous extracts.

Full text of DOI:10.3390/molecules16119153

Molecules 2011, 16, 9153-9160; doi:10.3390/molecules16119153
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molecules
www.mdpi.com/journal/molecules
ISSN 1420-3049
Article
Polar Compounds Isolated from the Leaves of
Albertisia delagoensis (Menispermaceae)
Geoffrey E. Hawkes 1, Helene de Wet 2,* and Jia Li 3
1
School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road,
London E1 4NS, UK
2
Department of Botany, University of Zululand, P/Bag X1001, Kwa-Dlangezwa 3880, South Africa
3
Department of Surgery and Cancer, Imperial College London, South Kensington Campus,
London SW7 2AZ, UK
* Author to whom correspondence should be addressed; E-Mail: hdewet@pan.uzulu.ac.za;
Tel.: +27-35-902-6108; Fax: +27-35-902-6391.
Received: 8 October 2011; in revised form: 21 October 2011 / Accepted: 27 October 2011 /
Published: 2 November 2011
Abstract: Aqueous infusions of the leaves of the shrub Albertisia delagoensis
(Menispermaceae) are used in South Africa in traditional Zulu medicine to alleviate a
variety of symptoms, including fever, and intestinal problems. We report the analysis of
such an aqueous extract using the HPLC-NMR technique. A number of polar compounds
were identified, including proto-quercitol, nicotinic acid, allantoic acid, 3,4-dihydroxy-
benzoic acid, phthalic acid and the aporphine alkaloid derivative roemrefidine.
1
13
Allantoic acid and roemrefidine have been fully characterised by H- and C-NMR and
mass spectrometry. Earlier reports of antiplasmodial activity of roemrefidine and of
A. delagoensis extracts are correlated with this study and with the antipyretic properties of
neutral aqueous extracts.
Keywords: Albertisia delagoensis; traditional medicine; roemrefidine; allantoic acid;
HPLC-NMR

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1. Introduction
Albertisia delagoensis N.E. Br. Forman [=Epinetrum delagoensis] is a suffrutescent of the
Menispermaceae family found in Mozambique and north-eastern KwaZulu-Natal, South Africa.
A number of Albertisia species are used medicinally throughout tropical and sub-tropical Africa [1],
however A. delagoensis is the only species found in southern Africa, and the leaves are reportedly used
as an antipyretic [2], antidiarrhoeal and antiemetic [3,4] and the roots are used for menstrual pain,
sexual performance in men, chest pain and back pain, among other uses [3-5]. An early phytochemical
report on A. delagoensis [6] described the isolation of three alkaloids from the acidified ethanolic root
bark extracts, and reported by mass spectrometry m/z (M+) values and molecular formulae for the three
compounds. On the basis of the mass spectrometry fragmentation patterns it was suggested that all
three belonged to the bis-benzylisoquinoline class of alkaloids, but chemical structures were not
suggested. The alkaloids were tested [6,7] for cytotoxicity on continuous cell lines (VERO cells) and
found to be highly active. More recently the isolation of three alkaloids, cocsoline, cocsuline and
O-methylcocsoline from dilute sulphuric acid extracts of the rhizomes of A. delagoensis, and an
additional two alkaloids, cycleanine and dicentrine, from similar extracts of the leaves was reported [3].
Subsequently De Wet et al. [4] reported that methanol extracts of the dried, milled rhizomes and leaves
exhibited a high level of antiparasitic activity on the chloroquine-resistant Gambian FCR-3 strain of
Plasmodium falciparum, but the same extracts showed low level cytotoxicity against Graham cells
(transformed human kidney epithelium cells). Crude alkaloidal extracts of A. Delagoensis leaves
showed a high level of cytotoxicity against selected breast, melanoma and renal cancer cell lines [8].
However the above studies, identifying antiplasmodial activity and cytotoxicity used compounds
extracted into acidified aqueous or alcoholic solution, whereas in traditional medicine in southern
Africa it is the neutral aqueous extracts of roots or leaves that are drunk. In the present study we
sought to analyse leaf extracts obtained in this traditional manner, and did not seek to target only
alkaloids as had been the case in previous studies.
The crude extract mixture was inspected by the hyphenated HPLC-DAD-NMR method, in an
on-flow experiment where the 1H-NMR spectrometer acted as a detector identifying components of the
mixture which were present in sufficient quantity to merit more detailed structural analysis by
2-D NMR methods. Those selected components were then separated and accumulated in multiple
HPLC-DAD runs with the selected eluent collected in loops (the loop storage method). The
HPLC-DAD-NMR and HPLC-DAD-loop storage are among an array of powerful hyphenated
analytical methods employing NMR for molecular structure determination, and have been recently and
extensively reviewed [9-11].
2. Results and Discussion
The chromatograms (HPLC conditions as for the loop storage method) of the extract showed eight
peaks which eluted within the first 25 min, were well resolved and symmetric. In addition there was a
broad peak (with tailing) with retention time 28.5 min.
The on-flow HPLC-NMR experiment on the extract showed five components of the mixture gave
1
sufficiently strong H-NMR spectra that it was considered useful to isolate these components in

Molecules 2011, 16
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sufficient quantity to enable 2-D NMR experiments for structural elucidation. These five peaks in the
chromatogram corresponded to four of the eight well-resolved peaks (retention times 2.0, 7.0, 13.2 and
16.8 min) and the broad (tailing) peak (retention time 28.5 min) identified in the shorter (40 min) run
(HPLC conditions for loop storage). The collection and accumulation of these five fractions in
capillary loops is described in Section 3.4 below.
1
The H 1-D NMR spectra of the 2.0 min fraction indicated the presence of a major and two minor
components. The 1-D 13C spectrum showed six signals, five to higher frequency (71.8, 72.2, 74.5, 75.7
and 78.0 δ), and one at 36.8 δ. The COSY, TOCSY and HSQC spectra are all consistent with this
major component being a pentahydroxycyclohexane, and the 1H- and 13C-NMR data are in agreement
with those reported [12] for proto-quercitol (2, see Figure 1). The two minor components were
identified as nicotinic acid [13] and allantoic acid (1, see Figure 1).
Figure 1. Chemical structures of allantoic acid (1), proto-quercitol (2), and roemrefidine (3).
OH
HO
O
O
O
HO
OH
H2N
N
H
N
H
NH2
(1)
HO
(2)
OH
3
4
O
3a
2
5
CH3
N
11c
1
O
11b
6a
7
CH3
11a
11
7a
(3)
10
8
9
The 1H- and 13C-NMR spectra of the 7.0 and 13.2 min fractions showed these to be 3,4-dihydroxy-
benzoic acid and phthalic acid, respectively, and these were confirmed by comparison with the spectra
of authentic samples (see also [14]).
1
The H, and 13C-NMR spectra of the 16.8 min fraction showed this to be a mixture of two
1
components, both aromatic in nature. The major component gave H-NMR signals at δ 6.96 (d,
J = 8.3 Hz), δ 7.5 (d,d J = 1.9, 8.3 Hz) and δ 7.73 (d, J = 1.9 Hz). A combination of HSQC and HMBC
spectra gave 13C-H signals at δ 121.1, 126.6 and 117.4 and non-protonated 13C signals at δ 134.5,
1
148.9, 153.7 and 174.8. Similarly the minor component gave H signals at δ 6.65 (d, J = 8.3 Hz),
13
δ 7.02 (d, J = 1.8 Hz) and δ 7.22 (d,d J = 1.8, 8.3 Hz), with corresponding C shifts measured from
the HSQC spectrum at δ 117.7, 117.3 and 125.8. There was insufficient signal/noise in the HMBC

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spectrum to give information on the non-protonated 13C shifts. However these data were not sufficient
to assign structures.
The 1H- and 13C-NMR spectra of the 28.5 min fraction indicated a structure more complex than any
of the above. An accurate m/z value of 294.1503 was measured; the molecular formula C19H20NO2 has
calculated mass 294.1489, i.e., ∆ = 4.8 ppm. Consideration of all available 1-D and 2-D NMR data led
to the candidate structure 3 (see Figure 1). This was identified as roemrefidine, which has been
reported previously [15], and is the N-methyl derivative of the alkaloid roemerine.
The 1H and 13C chemical shifts determined here for roemrefidine are given in Table 1. The 3-bond
(vicinal) 1H-1H coupling constants between the protons at positions 4 and 5, and positions 6 and 7 fall
into two ranges; 3.9 to 5.8 Hz, and 13.0 to 14.0 Hz. The smaller coupling range is typical for C-H bond
dihedral angles ca. 60° or ca. 120°, whereas the larger coupling constants are typical for dihedral
angles nearer 180°.
Table 1. 1H- and 13C-NMR data for roemrefidine (3) a.
Position δ 1H
δ 13C b
143.3 c
147.6 c
106.8
122.6
23.1
1
2
3
6.82 (s)
3a
4 (Ψe)
4 (Ψa)
5 (Ψa)
5 (Ψe)
6 (Ψa)
7 (Ψa)
7 (Ψe)
7a
3.05 (d,d J = 4.5, 18.0 Hz)
3.32 (d,d,d J = 5.8, 13.0, 18.0 Hz)
3.67 (d,t J = 4.5, 13.0 Hz)
3.76 (d,d J = 5.8, 13.0 Hz)
4.64 (d,d J = 3.9, 14.0 Hz)
3.09 (t J = 14.0 Hz)
61.2
68.2
28.1
3.44 (d,d J = 3.9, 14.0 Hz)
130.5
128.0
128.4
128.0
126.2
130.4
115.3
118.5
101.3
8
7.43 (d J = 7.4 Hz)
7.39 (t J = 7.4 Hz)
7.43 (t J = 7.4 Hz)
8.12 (d J = 7.4 Hz)
9
10
11
11a
11b
11c
OCH2O
6.07 (d J = 0.8 Hz)
6.20 (d J = 0.8 Hz)
CH3 (Ψa) 3.05 (s)
(Ψe) 3.38 (s)
42.4
53.0
a Solution in CD3CN-D2O (1:1); b Measured from HSQC and HMBC spectra, and values are 0.3 δ;
c These two assignments may be interchanged.
Accordingly an energy minimized structure for roemrefidine was generated (see section 3.8) and
this is shown in Figure 2. Relative to the average plane of the molecule it is clear that the protons at
positions 4,5,6 and 7 can be assigned as pseudo-axial (Ψa) or pseudo-equatorial (Ψe), and on this basis

Molecules 2011, 16
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the smaller vicinal coupling constants are assigned to Ψe/Ψe interactions or to Ψa/Ψe interactions, and
the larger vicinal coupling constants to Ψa/Ψa interactions.
Figure 2. Spartan energy minimized structure of roemrefidine.
The methyl signals were assigned as CH3 (Ψa) and CH3 (Ψe) since the NOESY spectrum showed a
strong correlation between the CH3 signal at 3.38 δ and 4.64 δ (H6 Ψa), but only a very weak
correlation between 3.05 δ and 4.64 δ. This indicates the methyl group giving the signal at 3.38 δ is
closer in space to the H6 (Ψa) proton, which is the case for the Ψe methyl group (see Figure 2).
3. Experimental
3.1. Plant Material
Samples of the leaves of Albertisia delagoensis were collected in Tembe Elephant Park,
Sihangwane [2732 AB], South Africa, in June 2005 and a voucher specimen [De Wet and SJ Siebert
100 (ZULU)] was deposited in the herbarium of the University of Zululand for verification purposes.
3.2. Extraction
The leaves were air-dried and powdered in a mill. Powdered leaf sample (10 g) was added to
distilled water (100 mL), heated under reflux for one hour, cooled, filtered and lyophilised, yielding a
brown powder (876 mg).
3.3. HPLC-DAD-on-flow-NMR
The hyphenated HPLC-DAD-NMR system consisted of an Agilent 1100 HPLC equipped with a
column oven, a Bruker u.v. diode array detector (DAD) in the range 200 to 700 nm, a Bruker 36 place
capillary loop storage device (BPSU36), and a Bruker DRX 600 NMR spectrometer with 3 mm
cryogenic flow probe. The system was controlled with the Bruker HyStar 2.1 software.
HPLC conditions: A Thermo Hypersil (DBS) C18 column (5 µm, 150 × 4.6 mm) with an oven
temperature 305 K. The gradient elution used D2O and CD3CN both containing 0.05% DCOOD,
started with 98:2 (v/v) D2O-CD3CN 98:2, and linearly changed to 46:54 over 267 min, then was held at
46:54 for 33 min. Flow rate: 0.15 mL/min; Injection volume 100 µL; Sample concentration: a sample
of the leaf extract (63 mg) was dissolved in D2O-CD3CN-DCOOD 98:2:0.05 (1 mL). The solution was
filtered (0.45 µm filter with centrifugation at 10,000 g for 10 min), and the clear supernatant collected.

Molecules 2011, 16
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DAD conditions: full range 200 to 700 nm. Alternative conditions used the same solvent composition
but with the linear variation over 800 min, then held for 100 min. Flow rate: 0.05 mL/min.
3.4. HPLC-DAD-loop Storage
The HPLC-DAD, column and temperature, and DAD conditions were the same as used in the
on-flow NMR experiments (see above).
HPLC conditions: Gradient elution used H2O and CH3CN both containing 0.1% HCOOH and the
same solvent compositions were used as for the on-flow experiments. The linear variation of
composition was over 40 min, then held for 5 min. Flow rate: 1 mL/min; Injection volume 20 µL;
Sample concentration: as prepared for SPE (see above), but using normal (non-deuterated) solvents
with 0.1% HCOOH.
The fractions for the specified peaks in the chromatogram were stored in separate loops. The
experiment was repeated 6 times, and the corresponding fractions were combined. Each combined
fraction was lyophilised, redissolved in D2O-CD3CN 1:1 (600 µL) containing 0.05% TSP (see below)
and transferred to a 5 mm o.d. NMR tube for NMR measurements.
3.5. NMR Spectroscopy
1
Off-line NMR measurements used a Bruker DRX 600 spectrometer with a standard 5 mm H/13C
13
1
inverse mode probe (1H-NMR at 600 MHz, C-NMR at 151 MHz). The residual H solvent signals
from deuterated water and acetonitrile were suppressed using the WET method [16] with 13C
decoupling. All spectra were measured at 292 K. The 2-D NMR experiments used were homonuclear
1
1H COSY, NOESY and TOCSY, and heteronuclear H-13C HSQC and HMBC, and these techniques
have been summarised by Braun et al. [17].
3.6. Molecular Modelling
The software used for molecular modelling was Spartan ’10 from Wavefunction Inc. (Irvine, CA,
USA).
3.7. Mass Spectrometery
The accurate m/z value was measured by the King’s College, University of London, Mass
Spectrometry service using a Bruker Apex III system with electrospray ionisation from methanol.
3.8. Allantoic Acid
An authentic sample of allantoic acid (1) was prepared by alkaline hydrolysis of allantoin, as
1
described by Behrend and Schultz [18]. H-NMR (DMSO-d6) δ: 5.20 (1H, t, J = 7.9 Hz, CH), 5.77
13
(4H, br s, NH2), 6.78 (2H, d, J = 7.9 Hz, NH). C-NMR (DMSO-d6) δ: 57.8 (CH), 157.9 (CONH2),
171.3 (CO2H).

Molecules 2011, 16
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4. Conclusions
HPLC-NMR analysis of the neutral aqueous extract of the leaves of Albertisia delagoensis
identified the compounds present including proto-quercitol, nicotinic acid, allantoic acid,
3,4-dihydroxybenzoic acid, phthalic acid, and the aporphine alkaloid derivative roemrefidine. The 1H-
and 13C-NMR spectroscopic data for allantoic acid and roemrefidine are reported for the first time. In
addition molecular modelling of the roemrefidine structure allowed stereospecific assignment of the 1H
resonances by comparison with vicinal coupling constants measured from the 1H spectrum.
Munoz et al. [19] showed that roemrefidine, isolated from the stem bark of Sparattanthelium
amazonum Martius (Hernandiaceae), is active against the malaria parasite Plasmodium falciparum in
vitro and against P. berghei in mice, but showed no cytotoxic activity against the cell lines KB, HEp-2,
and HeLa. It is aqueous extracts of the leaves of A. delagoensis that are reported [2] to be used in
traditional medicine to treat fever. Since roemrefidine is a salt (quaternary nitrogen) it is more likely to
be extracted into neutral aqueous solution than the less polar MOalkaloids [3-8], and therefore
roemrefidine must be considered a candidate for the febrifugal activity of the traditional preparations.
In addition we have recently demonstrated [20] the antimicrobial activity of roemrefidine towards
Bacillus cereus, Escherichia coli and Staphylococcus aureus, which may be relevant to the use of
A. delagoensis for the treatment of intestinal disorders.
Acknowledgments
The authors acknowledge technical assistance from Bhavin Dalal, Heather Matta and S.M. Goldup
at Queen Mary, and from William Nel at UZULU. We acknowledge financial support from Royal
Society/ National Research Foundation South Africa – UK Science Networks programme.
Conflict of Interest
The authors declare no conflict of interest.
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distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).