Oxidative burst inhibitory and cytotoxic amides and lignans from the stem bark of Fagara heitzii (Rutaceae)

Article abstract of DOI:10.1016/j.phytochem.2009.08.007

Two amides, heitziamide A and heitziamide B and two phenylethanoids, heitziethanoid A and heitziethanoid B together with thirteen known compounds were isolated from F. heitzii (Letouzey). The structures of all compounds were established by spectroscopic a

Full text of DOI:10.1016/j.phytochem.2009.08.007

Phytochemistry 70 (2009) 1442–1447
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Oxidative burst inhibitory and cytotoxic amides and lignans from the stem bark
of Fagara heitzii (Rutaceae)
Luc Meva’a Mbaze a, , Jean Alexandre Lado b,c, Jean Duplex Wansi a,d, Tze Chieh Shiao c,
*
David Dako Chiozem b, Muhammad Ahmed Mesaik e, Muhammad Iqbal Choudhary e,f
Marie-Aleth Lacaille-Dubois g, Jean Wandji b, René Roy c, Norbert Sewald d
,
a Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon
b Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
c Department of Chemistry, Université du Québec à Montréal, P.O. Box 8888, Succ. Centre-ville, Montréal (Qc), Canada H3C 3P8
d Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, 33501 Bielefeld, Germany
e Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi, Karachi-75720, Pakistan
f H.E.J Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
g Laboratoire de Pharmacognosie, UMIB, UPRES EA 3660, Université de Bourgogne, Faculté de Pharmacie, 7-Bd.Jeanne D’arc, BP 87900, 21079 Dijon Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 May 2009
Received in revised form 9 August 2009
Available online 9 September 2009
Two amides, heitziamide A and heitziamide B and two phenylethanoids, heitziethanoid A and heitzieth-
anoid B together with thirteen known compounds were isolated from F. heitzii (Letouzey). The structures
of all compounds were established by spectroscopic analysis.
Nine compounds were evaluated for oxidative burst inhibitory activity in a chemoluminescence assay
and for cytotoxicity against PC-3 prostate cancer cells.
Keywords:
Fagara heitzii
Amides
Phenylethanoids
Lignans
All compounds exhibited a clear suppressive effect on phagocytosis response upon activation with
serum opsonized zymosan at the range of IC50 = 2.0–6.5
(IC50 > 100 M).
l
M, but no cytotoxic effect was observed
l
Ó 2009 Elsevier Ltd. All rights reserved.
Oxidative burst inhibition
Cytotoxicity
1. Introduction
terpenes and steroids (Bongui et al., 2005; Ngouela et al., 1994;
Saboor, 1984). In this paper, we report the isolation, the structure
elucidation of four new compounds (1–4), and their biological
activities.
Immune suppression and cytotoxic activity affecting the func-
tion of the immune system have been reported for many synthetic
and natural therapeutic agents (Asuman and Barlas, 1984; Sell,
1987; Siddiqui et al., 2001). Various conditions such as infections,
organ transplantation, cancer, rheumatoid arthritis, and systemic
lupus erythromatosus are currently treated with novel immuno-
modulating agents (Bartlett et al., 1991).
As part of a program to discover potential antitumoral, anti-
inflammatory, antioxidant, and antimalarial agents from Cameroo-
nian rainforest medicinal plants, we investigated Fagara heitzii
(syn. Zanthoxylum heitzii) (Rutaceae). This species is a shrub of
the humid rain forests of Cameroon, where it is traditionally used
as medicinal plant against cancer, syphilis, malaria, cardiac palpita-
tions, urogenital affections as well as to prepare poisonous arrows
(Sandberg et al., 2005; Adjanohoon et al., 1988; Zirihi et al., 2005).
Previous phytochemical investigations of F. heitzii have yielded
methylnitidine chloride, nitidine, fagaramide, flindersine, lignans,
2. Results and discussion
The stem bark of F. heitzii was extracted with MeOH. The extract
was separated using repeated column chromatography and pre-
parative TLC (PTLC) to afford four new compounds (1–4) and thir-
teen known compounds (Fig. 1). By comparison with the reported
data (Mbaze et al., 2007; Chaaib et al., 2003; Ayafor et al., 1984),
the known compounds were identified as trans-fagaramide (5),
arnottianamide (6), iso-
c-fagarine (7), iso-skimmianine (8), arc-
tigenin methyl ether (9), savinin (10), (+)-eudesmin (11), (+)-ses-
amin (12), lupeol, lupeone, b-sitosterol, stigmasterol and
stigmasterol-3-O-b-D-glucopyranoside.
Heitziamide A (1) was obtained as a white amorphous powder.
Its molecular formula C24H33NO3 was deduced from EI-MS and HR-
EI-MS ([M]+, m/z found 383.2452, calc. 383.2460). The UV spec-
trum showed two maxima at 260 and 302 nm, suggesting the pres-
ence of an amide group. The presence of an amide function was
* Corresponding author. Tel.: +237 99 23 21 90.
E-mail address: lmbazze@yahoo.fr (L.M. Mbaze).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.08.007

L.M. Mbaze et al. / Phytochemistry 70 (2009) 1442–1447
1443
11
5''
1''
1'
3''
O
O 1''
2''
O
12
10
9
4''
2''
6
7
1'
3'
1
2
5
5
1
2'
7
O
O
1
(CH2)n-CH3
9
3
4
O
O
N
H
6''
3''
8
1'
4'
8
HO
2
13
4'
2
6 O
N
3'
4
3
2'
H
3 n = 32
4 n = 36
R
4'
2 R = OH
1
5 R = H
O
R1
R2
O
O
N
R1
R2
O
O
R1
O
R
O
O
N
CHO
R2
H3CO
OH CH3
OCH3CH3
OCH3
11 R1 = R2 = OCH3
12 R1, R2 = OCH2O
R1
7 R = H
6
R2
8 R = OCH3
9
R1 = R2 = OCH3
10 R1, R2 = OCH2O
Fig. 1. Structures of some isolated compounds.
Table 1
1H (500 MHz) and 13C (125 MHz) NMR assignments for 1 and 2 in CDCl3.
oxy at d 100.8, six aromatic carbons at d 107.6, 108.3, 120.8,
138.3, 146.0 and 147.6. Furthermore, the 1H NMR spectrum
showed signals characteristic of the cyclohexene ring at d 2.16–
2.24 (m, 4 H, H-9 and H-12); 2.43 (td, J = 11.3, 5.0 Hz, 1H, H-8);
2.92 (ddd, J = 13.2, 11.3, 5.6 Hz, 1H, H-7) and 5.49 (brs, 1H, H-11)
(Joshi et al., 1975). The cyclohexene ring was confirmed by the
1H–1H COSY spectrum, in which several cross-peaks were ob-
served; correlation between proton H-11 (d 5.49) and the methy-
lene H-12 (d 2.16–2.25), between H-12 and H-7 (d 2.92), and
finally between H-7 and H-8 (d 2.43), which in turn was also cor-
related to the methylene H-9 (d 2.16-2.25). The 1H NMR spectrum
also exhibited signals of an isohexenyl group at d 1.63 (s, 3H, H-500);
1.71 (s, 3H, H-600); 2.04 (m, 2H, H-200); 2.23 (m, 2H, H-100) and 5.13 (t,
J = 6.9 Hz 1H, H-300), and of an isobutylamide at d 0.66 (d, J = 6.3 Hz,
3H, H-40); 0.68 (d, J = 6.3 Hz, 3H, H-30); 1.43 (m, 1H, H-20); 2.69 (dd,
J = 6.5, 5.0 Hz, 1H, H-10a); 2.96 (m, 1H, H-10b) and 5.21 (t, J = 5.0 Hz,
NH) (Portet et al., 2007). The presence of isohexenyl and isobutyla-
mide moieties was further confirmed by the 13C NMR, DEPT and
HMQC spectra, which showed two methyls, three methylenes,
two methines, one amide (d 174.4), and one quaternary carbon (d
131.7).
To establish the connections between the above-described
structural units, an HMBC analysis was done (Fig. 2). In the HMBC
spectrum, correlations for the proton H-7 (d 2.92) to carbons C-1 (d
138.3), C-6 (d 120.8), C-2 (d 108.3), C-13 (d 174.4), C-11 (d 119.7),
and C-9 (d 33.6) and for the proton H-8 (d 2.43) to carbons C-1(d
138.3), C-13 (d 174.4), C-10 (d 136.3), and C-12 (d 32.7) established
the attachment of the methylenedioxyphenyl, the isohexenyl and
the isobutylamide moieties to the cyclohexene ring at C-7, C-10,
and C-8, respectively. The relative configurations of the two stere-
ogenic centers of 1 were elucidated on the basis of the 1H–1H cou-
pling constants and NOESY correlations as shown in Fig. 3. The
coupling constant of H-7/H-8 (J = 11.3 Hz) and the lack of the
NOESY correlation between the two protons indicated the anti
relationship of H-7/H-8 and their axial orientations (Wei et al.,
2005). Furthermore, since the optical rotation was zero and the cir-
cular dichroism (CD) spectrum exhibited no Cotton effect; com-
pound 1 was concluded to be a racemate. From the above
spectroscopic studies, therefore 1 is ( ) 6-(3,4-methylenedioxy-
phenyl)-N-isobutyl-3-(isohexenyl)cyclohex-3-enecarboxamide
named as heitziamide A.
Attribution
1
2
13C
1H
–
13C
1H
–
1
2
3
4
5
6
7
138.3
129.3
108.3 6.74 (d, J = 1.3)
106.3 7.12 (s)
147.6
146.0
–
–
140.6
135.9
148.2
–
–
–
107.6 6.72 (d, J = 8.2)
120.8 6.70 (dd, J = 8.2; 1.3)
101.4 6.80 (s)
42.8
2.92 (ddd, J = 13.2; 11.3; 5.6) 141.2 7.55 (d,
J = 15.4)
8
49.4
2.43 (td, J = 11.3; 5.0)
118.8 6.23 (d,
J = 15.4)
9
33.6
2.16 (m)
167.1
10
11
12
13
10
136.3
119.7 5.49 (brs)
32.7
174.4
46.6
–
2.25 (m)
–
2.96 (m)
46.9
3.24 (t, J = 6.3)
2.69 (dd, J = (6.5; 5.0)
1.43 (m)
0.68 (d, J = 6.3)
0.66 (d, J = 6.3)
2.23 (m)
20
28.3
19.8
19.8
26.4
37.4
28.7
20.1
20.1
1.87 (m)
0.96 (d, J = 6.6)
0.96 (d, J = 6.6)
3’
40
100
200
2.04 (m)
300
124.1 5.13 (t, J = 6.9)
400
131.7
17.7
25.7
–
500
1.63 (s)
1.71 (s)
600
–OCH2O–
100.8 5.90 (d, J = 1.3)
5.91 (d, J = 1.3)
6.01 (brs)
NH
OH
5.21 (t, J = 5.0)
5.61 (brs)
9.80 (brs)
Assignments were based on HMQC, HMBC and NOESY experiments.
further indicated by two IR bands at 3289 and 1629 cmꢀ1. The 1H
NMR spectrum (Table 1) of compound 1 exhibited signals for
methylenedioxy protons at d 5.90 (d, J = 1.3 Hz) and 5.91 (d, J =
1.3 Hz), and an ABX pattern of three aromatic protons at d 6.70
(dd, J = 8.2; 1.3 Hz, 1H, H-6); 6.72 (d, J = 8.2 Hz, 1H, H-5) and 6.74
(d, J = 1.3 Hz, 1H, H-2). These findings are in agreement with the
presence of a methylenedioxyphenyl group in compound 1. This
assumption was supported by the 13C NMR (Table 1), DEPT and
EI-MS spectra which show a characteristic signal of methylenedi-

1444
L.M. Mbaze et al. / Phytochemistry 70 (2009) 1442–1447
H
O
O
H
O
N
H
Fig. 2. Important HMBC correlations for 1.
teristic signal of the methylenedioxy group at d 101.3, together
with six aromatic carbons at d 101.4, 106.3, 129.3, 135.9, 140.6,
and 148.2.
H9
Furthermore, the 1H NMR spectrum showed signals characteris-
tic for the isobutylamide at d 0.96 (d, J = 6.6 Hz, 6H, H-30and H-40);
1.87 (m, 1H, H-20); 3.24 (t, J = 6.3 Hz, 2H, H-10) and 5.61 (brs, NH)
(Kubo et al., 1984), and an AB system of two olefinic protons at d
6.23 (d, J = 15.4 Hz, 1H, H-8) and 7.55 (d, J = 15.4 Hz, 1H, H-7).
The large coupling constant (J = 15.4 Hz) between H-7 and H-8
indicated a trans configuration at the double bond. All these
above-described structural units were confirmed by the 13C NMR,
DEPT, COSY, HMQC, and HMBC spectra, which showed characteris-
tic signals of trans-fagaramide (5) (Okorie, 1976; Kubo et al., 1984).
The absence of an ABX system of three aromatic protons in com-
pound 2, suggested that one of the aromatic carbons was hydrox-
ylated. This assumption was further supported by EIMS (m/z 263),
which was 16 mass units higher than that of trans-fagaramide (5)
(m/z 247) confirming the presence of additional oxygen in com-
pound 2. The location of the hydroxyl group at C-3 was based on
the correlations for H-7 (d 7.55) to C-2 (d 106.3), C-6 (d 101.4)
and C-9 (d 167.1) as observed in HMBC.
H7
O
CONHiBu
O
H8
H12
Fig. 3. Selected NOESY correlations for 1.
This compound is a novel cyclohexene-type amide. We propose
a possible biosynthesis for its formation based on closer examina-
tion of the structure. This compound could result from the inter-
molecular [4 + 2] cycloaddition reaction (Diels–Alder reaction)
between the monoterpene 6-methyl-2-vinylheptane-1,5-diene
(13) (which results from two isoprenyl units linked by head/tail)
and the trans-fagaramide (5) (Fig. 4). The trans-stereochemistry
of the product postulated in the biosynthetic hypothesis was iden-
tical to that of the natural compound 1.
Heitziamide B (2) was obtained as a yellow amorphous powder.
Its molecular composition was C14H17NO4 as shown by EI-MS and
HR-EI-MS ([M] +, m/z 263.11410, calc. 263.11581). The UV spec-
trum exhibited several absorption maxima at 325, 290, 235, and
at 215 nm. The presence of hydroxyl and amide functions was indi-
cated by two IR bands at 3350 and 1680 cmꢀ1, respectively. The 1H
NMR spectrum (Table 1) of compound 2, exhibited signals for
methylenedioxy protons at d 6.01 (brs, 2H), two aromatics protons
appeared as singlet at d 7.12 (brs) and 6.80 (brs), and one hydroxyl
group at d 9.80 (brs) exchangeable with D2O. These findings are in
agreement with the presence of a hydroxylmethylenedioxyphenyl
group in compound 2. This assumption was further supported by
the 13C NMR (Table 1) and DEPT spectra, which show the charac-
From the above spectroscopic studies, the structure of com-
pound 2 was assigned as trans-3-hydroxyfagaramide named heit-
ziamide B.
Heitziethanoid A (3) and B (4) were obtained as white amor-
phous powders, showing positive reactions with FeCl3 indicating
their phenolic nature. Their UV spectrum exhibited two absorption
maxima at 275 and 240 nm characteristic of phenylethanoids
(Chaaib et al., 2003; Chihiro et al., 1988). The presence of hydroxyl
and ester functions was indicated by two IR bands at 2935 and
1750 cmꢀ1, respectively. From the HR-ESI-MS, the pseudomolecu-
lar ion was found to be C44H80O3Na by ([M+Na]+ at m/
z = 679.6010, calc. 679.6005) and C48H88O3Na by ([M+Na]+ at m/
z = 735.6649, calc. 735.6631), respectively.
The 1H NMR spectra of 3 and 4 showed the typical AA‘XX’ sys-
tem of a p-disubstituted benzene ring at d 7.06 (d, J = 8.3 Hz, H-20,
H-60) and 6.80 (d, J = 8.3 Hz, H-30, H-50), the presence of an ethyl
alcohol unit at d 4.23 (t, J = 6.9 Hz, H-1), and 2.84 (t, J = 6.9 Hz, H-
O
O
O
O
O
N
H
13
1
O
N
H
Fig. 4. Biosynthesis hypothesis of compound 1.

L.M. Mbaze et al. / Phytochemistry 70 (2009) 1442–1447
1445
M)
2), and a free hydroxyl group at d 9.58 (brs, OH-40) exchangeable
Table 3
with D2O. This inference was supported by the 13C NMR and DEPT
data, which showed characteristic signals of a p-disubstituted ben-
zene ring at d 114.4 (C-30, C-50), 129.1 (C-10), 130.2 (C-20, C-60) and
156.7 (C-40), and ethyl alcohol unit at d 65.2 (C-1) and 34.3 (C-2)
(Acevedo et al., 2000). Furthermore, in the 1H NMR spectrum, a ter-
minal methyl at d 0.90 (t, J = 6.8 Hz) and methylenes at d 2.29 (t,
J = 7.5 Hz, H-200), 1.58 (m, H-300) 1.25 (brs, nH) were also observed.
These data suggested the presence of a long alkyl chains linked
to the 4-hydroxyphenylethanol moiety. The presence of a long al-
kyl chains was further confirmed by 13C NMR spectrum, which
showed characteristic signals at d 174.0 (C-100), 34.1 (C-200), 31.8-
29.3 (CH2)n 24.8 (C-300) and 14.0 (CH3) (Wandji et al., 2002).
To determine the linkage between the long chain and the 4-
hydroxyphenylethanol moiety, HMBC experiment was performed.
In the HMBC spectrum, correlation of H-1 (d 4.23) with C-100 (d
174.0), C-10 (d 129.1) and C-2 (d 34.1), suggested that the long
chain is linked to the 4-hydroxyphenylethanol moiety by an ester
function.
Effect of compounds 1, 2, 5, and 7–10 on oxidative burst of whole blood.
Compounds
IC50 (l
1
2
5
7
8
9
10
11
2.6 0.4
2.0 0.1
3.4 0.4
3.1 0.3
2.3 0.2
6.5 0.5
5.6 0.8
6.0 0.5
2.2 0.3
12.1 3.0
12
Ibuprofena
a
Standard used in the assay.
Since these compounds are weakly toxic and exhibited a clear
suppressive effect on phagocyte oxidative burst, they can be there-
fore considered as potential antioxidants drugs.
The methanolysis of 3 and 4 yielded methyl hexatriacontanoate
(identified by the ESIMS indicating a pseudomolecular ion at m/z
573 [M+Na]+, corresponding to a molecular formula C37H74O2),
methyl tetracontanoate (identified by the ESIMS indicated a
pseudomolecular ion at m/z 629 [M+Na]+, corresponding to a
molecular formula C41H82O2) and 4-hydroxyphenylethanol. 4-
hydroxyphenylethanol was identified by the 1H NMR and EI-MS.
From the above spectroscopic studies, heitziethanoid A (3) and B
(4) were characterized as 2-(4-hydroxyphenethyl)hexatriacont-
anoate and 2-(4-hydroxyphenethyl)tetracontanoate, respectively.
Phenylethanoids have previously been described in the genus
Citrus and Fagara (Rutaceae) (Chaaib et al., 2003; Chihiro et al.,
1988).
3. Experimental section
3.1. General
Optical rotations [a]
(MeOH, c in g mlꢀ1) were determined by
D
using a JASCO digital polarimeter (model DIP-3600). Infrared
spectra were recorded on a JASCO FT/IR-410 spectrophotometer.
UV spectra were determined on a Spectronic Unicam spectropho-
tometer. HR-ESI-MS were recorded on a APEX III (Bruker Dalto-
nik) 7 Tesla (ESI FT-ICR-MS). EI-MS spectra were recorded on a
Finnigan MAT 95 spectrometer (70 eV) with perfluorokerosene
as reference substance for HR-EI-MS. The 1H and 13C NMR spectra
were recorded at 500 MHz and 125 MHz, respectively, on Bruker
AMX 500 NMR spectrometers. Methyl, methylene and methine
carbons were distinguished by DEPT experiments. Homonuclear
1H connectivities were determined by using COSY experiments.
One bond 1H–13C connectivities were determined with HMQC
gradient pulse factor selection. Two- and three- bond 1H–13C con-
nectivities were determined in HMBC experiments. Chemical
shifts are reported in d (ppm) using TMS as internal standard
and coupling constants (J) were measured in Hz. Column chroma-
tography was carried out on silica gel (70–230 mesh, Merck) and
flash silica gel (230–400 mesh, Merck). TLC was performed on
Merck precoated silica gel 60 (F254, Merck), and spots were visu-
alized by using ceric sulfate spray reagent. All reagents used were
of analytical grades.
Compounds 1, 2, 5, and 7–12 were screened over a wide range
of concentrations (3.1–50 l
g mlꢀ1) for their immunomodulatory
potential. These compounds were shown to possess inhibitory
activity upon activation with serum opsonized zymosan, which
was tested in vitro for oxidative burst studies of whole blood. All
these compounds showed significant effects on the oxidative burst
of the whole blood (IC50 range 2.0–6.5
lM) compared to the con-
trol (Ibuprofen IC50 = 12.1 M) (Table 3). The tested compounds
l
exhibited a clear suppressive effect on phagocyte oxidative burst
response upon activation with serum opsonized zymosan in a
dose-dependent manner.
In addition, all these compounds were evaluated for their cyto-
toxicity against prostate cancer cell (PC-3) cell line using MTT
method and doxorubicin as positive control. However, no cytotoxic
effect was observed on prostate cancer cell (IC50 > 100 lM).
3.2. Plant material
The stem bark of F. heitzii Letouzey was collected at Mbalmayo
locality, Centre, Cameroon in June 2006 and identified by Mr. Nana
Victor of National Herbarium, Yaounde, Cameroon, where the vou-
cher specimen (Ref. No. 1482/SRFK) has been deposited.
Table 2
1H (500 MHz) and 13C (125 MHz) NMR assignments for 3 and 4 in CDCl3 + MeOD.
Attribution
3–4
13C
1H
4. Extraction and isolation
1
2
1’
65.2
34.3
4.23 (t, J = 6.7)
2.84 (t, J = 6.7)
–
7.06 (d, J = 8.4)
6.80 (d, J = 8.4)
–
The powdered stem bark of F. heitzii (4.5 kg) was extracted with
MeOH at room temperature during 72 h. After removing the sol-
vents by evaporation under reduced pressure, the obtained crude
extract (110.0 g) was chromatographed over silica gel 60 (70–
230; 230–400 mesh), using hexane, ethyl acetate, and MeOH in
increasing polarity order. A total of 162 sub-fractions (ca. 250 ml
each) were collected and combined on the basis of TLC analysis
leading to five main fractions A, B, C, D, and E.
129.6
130.2
114.4
156.7
174.0
34.1
24.8
29.4
14.0
20/60
30/50
40
100
–
200
2.29 (m)
1.58 (m)
1.25 (brs)
0.90 (t, J = 6.8)
9.58 (brs)
300
–(CH2)n–
CH3-
aOH
Fraction A (28.0 g, combined from sub-fractions 1–33) was
chromatographed on silica gel with a hexane–ethyl acetate gradi-
a
Observed in DMSO.

1446
L.M. Mbaze et al. / Phytochemistry 70 (2009) 1442–1447
ent to yield lupeol (30.6 mg), b-sitosterol (19.6 mg) and stigmas-
terol (16.1 mg).
13C NMR see Table 2; HR-ESI-MS m/z 735.6649 (calc. for
C48H88O3Na, 735.6631); ESI–MS m/z = 735 [M+Na]+.
Fraction B (32.0 g, combined from sub-fractions 34–98) was
chromatographed on silica gel with a hexane–ethyl acetate gradi-
ent. A total of 35 fractions (ca. 75 ml each) was collected and com-
bined on the basis of TLC. Fractions (7–35) were further
chromatographed on silica gel with a mixture hexane–ethyl ace-
tate (3:1) to yield lupeone (17.0 mg), savinin (10) (71.3 mg), (+)-
eudesmin (11) (11.0 mg), (+)-sesamin (12) (19.6 mg) and arctige-
nin methyl ether (9) (8.0 mg).
4.4.1. Methyl tetracontanoate
Colourless oil; ESI–MS m/z = 629 [M+Na]+; 1H NMR (500 MHz,
CDCl3): d 0.74 (t, J = 7.5 Hz, 3H, –CH3), 1.42–1.12 (brs, –(CH2)n–),
2.10 (t, J = 8.0 Hz, 2H, H-2), 3.82 (s, 3H, –OCH3).
5. Chemical derivatives
Fraction C (130.0 g, combined from sub-fractions 99–113) was
chromatographed on silica gel with a hexane–ethyl acetate gradi-
ent. A total of 32 fractions (ca. 100 ml each) was collected and com-
bined on the basis of TLC. Fractions (6–32) were further
chromatographed on silica gel with a mixture of hexane–ethyl ace-
tate (2:1) to yield arnottianamide (6) (16.6 mg), trans-fagaramide
Methanolysis of compounds (3–4): Compounds 3–4 (5.0 mg
each) were added to a mixture of HCl (3.5 ml, 1 N) and dry MeOH
(6.0 mg), and refluxed for 16 h with magnetic stirring. Then 10.0 ml
H2O was added to the refluxed mixture, which was extracted with
n-hexane (3 ꢂ 10 ml). The fatty acid methyl ester (2.5 mg and
2.8 mg, respectively) was isolated by chromatography of the n-
hexane extract over a Silica gel with n-hexane-CH2Cl2 (9:1) as
solvent.
(5) (94.4 mg), iso-c-fagarine (7) (10.3 mg) and iso-c-skimmianine
(8) (9.1 mg).
Fraction D (21.8 g, combined from sub-fractions 114–140) was
chromatographed on silica gel with a hexane – ethyl acetate gradi-
ent. A total of 41 fractions (ca. 100 ml each) was collected and com-
bined on the basis of TLC. Fractions 3–41 were further
6. Biological activities
chromatographed on silica gel to yield heitziamine
(12.2 mg), heitziamine (2) (9.0 mg), heitziethanoid
(11.9 mg) and heitziethanoid B (4) (18.3 mg).
A
A
(1)
(3)
6.1. Chemiluminescence assay for determination of
immunomodulation activity
B
Fraction E (11.0 g, combined from sub-fractions 141–162) was
chromatographed on silica gel and eluted with a mixture of ethyl
acetate–MeOH of increasing polarity. A total of 33 fractions (ca.
100 ml each) was collected and combined on the basis of TLC. Frac-
tions 1–13 were further chromatographed on silica gel with a mix-
ture to yield stigmasterol-3-O-b-D-glucopyranoside (24.6 mg).
A luminol-enhanced chemiluminescence assay was performed,
as described in (Helfand et al., 1982). In brief, whole blood (diluted
1:200) and neutrophils (1 ꢂ 107) suspended in Hank’s balance salt
solution with calcium and magnesium (HBSS++) were incubated
with 50
ll of each test compounds at concentrations of 3.1–
50 l
l
g mlꢀ1 for 30 min. Then, 50 l (20 mg mlꢀ1) zymosan (Sigma
Chemical Co. St. Louis, MO) followed by 50
l
L (7 ꢂ 105 M) luminal
(G-9382 Sigma) and then HBSS++ were added to adjust the final
volume to 0.2 ml. HBSS++ was used as a control.
4.1. Heitziamide A (1)
Amorphous powder; ½a D25
ꢁ
¼ 0 (c = 0.010 in CHCl3); UV kMmaexOH (log
e
) nm: 302 (4.82), 260 (3.86); IR (KBr) mmax: 3310, 3289, 2920,
6.2. Cytotoxicity assay
1660, 1629, 1585, 1540, 920 cmꢀ1 1H and 13C NMR see Table 1;
;
HR-EI-MS m/z 383.24520 (calc. for C24H33NO3, 383.24336); EI-MS
m/z (rel. int.): 383 (100) [M]+, 314 (31), 283 (22), 247 (33), 191
(5), 176 (5), 148 (42), 136 (12), 135 (75), 69 (54).
Cytotoxic activities of the compounds were evaluated against
the PC3 Caucasian prostate adenocarcinoma cell line by the MTT
method according to a reported protocol (Zhao et al., 2008). In
brief, freshly trypsinized cell suspensions were seeded in 96-well
microtiter plates at densities of 1x104 cells per well with test com-
pounds added from DMSO-diluted stock. After 3 days in the cul-
ture, the attached cells were incubated with MTT and
subsequently solubilized in DMSO. The absorbance at 550 nm
was then measured by using a microplate reader. The IC50 is the
concentration of agent that reduced cell growth by 50% under
experimental conditions, with doxorubicin as the positive control
4.2. Heitziamide B (2)
MeOH
Yellow amorphous powder; UV k
(log
e
m
) nm: 325 (3.41),
max: 3350, 3178,
1H and 13C NMR see Table 1;
max
290 (3.04), 235 (2.46), 215 (2.25); IR (KBr)
2920, 1680, 1620, 1581, 925 cmꢀ1
;
HR-EI-MS m/z 263.11410 (calc. for C14H17NO3, 263.11581); EI-MS
m/z (rel. int.): 263 (12) [M]+, 247 (25), 190 (90), 175 (18), 163
(20), 151 (28), 145 (55), 117 (45).
(IC50 1.5 lM).
4.3. Heitziethanoid A (3)
Acknowledgements
MeOH
White amorphous powder; UV k
(3.7); IR (CHCl3)
(log ) nm: 275 (3.8), 240
max
We acknowledge the International Foundation for Sciences (IFS)
(Sweden) for providing financial support (F/3978-2), the Alexander
von Humboldt (AvH) Foundation (Germany) for postdoctoral fel-
lowships to J.D. Wansi at Bielefeld University and the MAECI (Can-
ada) for Ph.D. scholarships to J.A. Lado at University of Quebec at
Montreal. The biological activity determinations were supported
by a Higher Education Commission Grant (20-684-R & D/2007;
Pakistan).
m ;
max: 2935, 2860, 1750, 1616, 1390 cmꢀ1 1H
and 13C NMR see Table 2; HR-ESI-MS m/z 656.6010 (calc. for
C44H80O3Na, 679.6005); ESI–MS m/z = 679 [M + Na]+.
4.3.1. Methyl hexatriacontanoate
Colourless oil; ESI-MS m/z = 573 [M+Na]+; 1H NMR (500 MHz,
CDCl3): d 0.73 (t, J = 7.6 Hz, 3H, –CH3), 1.42–1.10 (brs, –(CH2)n–),
2.08 (t, J = 7.9 Hz, 2H, H-2), 3.80 (s, 3H, –OCH3).
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MeOH
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(log
e
) nm: 277 (3.8), 240
max
(3.6); IR (CHCl3) m ;
max: 2935, 2850, 1750, 1618, 1387 cmꢀ1 1H and

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