Bioactive phenylethanoids and coumarines from basalmocitrus cameroonensis

Article abstract of DOI:10.1515/znb-2009-0417

Two new phenylethanoids, basalefhanoid A (1) and B (2), and one new ceramide, basalamide A (3), together with eleven known compounds (4-14) were isolated from the MeOH extract of the stem barks of Basalmocitrus cameroonensis. The structures of all compoun

Full text of DOI:10.1515/znb-2009-0417

Bioactive Phenylethanoids and Coumarines from
Basalmocitrus cameroonensis
Jean Duplex Wansia,b, Judith Liliane Djouaka Bavouac, Emmanuel Ngeufa Happia,
Krishna Prasad Devkotab, Bruno Ndjakou Lentab, Muhammed Ahmed Mesaikd,
Muhammad Iqbal Choudharyd, Zacharias Tanee Fomumc, and Norbert Sewaldb
a Department of Chemistry, University of Douala, Faculty of Science, P. O. Box 24157 Douala,
Cameroon
b Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University,
33501 Bielefeld, Germany
c Department of Organic Chemistry, University of Yaounde´ I, Faculty of Science,
P. O. Box 812 Yaounde´, Cameroon
d International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270,
Pakistan
Reprint requests to Dr. Jean Duplex Wansi. Phone: +237-77-81-77-31. E-mail: jdwansi@yahoo.fr
Z. Naturforsch. 2009, 64b, 452 – 458; received November 28, 2008
Two new phenylethanoids, basalethanoid A (1) and B (2), and one new ceramide, basalamide A
(3), together with eleven known compounds (4 – 14) were isolated from the MeOH extract of the stem
barks of Basalmocitrus cameroonensis. The structures of all compounds were determined by compre-
hensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data, chemical reactions, and
comparison with previously known analogs. Compounds 1, 2, 5, and 7 – 10 demonstrated a strong
inhibition on reactive oxygen species (ROS) production in the oxidative burst activity of whole blood
on activation with serum opsonized zymosan in the range of IC50 = 0.06 – 12.30 µg mL−1
.
Key words: Basalmocitrus cameroonensis, Rutaceae, Phenylethanoid, Ceramide, Oxidative Burst
Inhibition
Many clinical disorders are associated with the chemical investigation has been reported on this genus.
immune system. Suppression of the immune system This paper reports the isolation and structure elucida-
is required in the management and treatment of in- tion of two new phenylethanoids (1 and 2) and one
flammation and allergic diseases, while stimulation is new ceramide (3) (Fig. 1), and eleven known com-
highly desirable for the treatment of HIV, immunode- pounds (4 – 14) from B. cameroonensis, together with
ficiency and infectious diseases [1]. Immune suppres- data on the immunomodulatory activity of the isolated
sion and cytotoxic activity affecting the function of compounds.
the immune system have been reported for many syn-
thetic and natural agents [2]. Various disease condi-
tions such as infections, organ transplantation, cancer,
Results and Discussion
rheumatoid arthritis, and systemic lupus erythromato-
sus are currently treated with novel immunomodulat- powdered and extracted with MeOH. The crude ex-
ing agents [3]. tract was separated by repeated column chromatog-
The air-dried stem bark of B. cameroonensis was
In continuation of our research for bioactive raphy and preparative TLC (PTLC) to afford the
molecules from Cameroonian rainforest medicinal two new phenylethanoids 1 and 2, one new ce-
plants, Basalmocitrus cameroonensis (Rutaceae), a ramide (3), and eleven known compounds iden-
shrub or small tree with young thorny seedlings, was tified as (2S,3S,4R,5R,14E)-1,3,4,5-tetrahydroxy-2-
studied. This plant is used as food and as remedy [(2ꢀR)-2ꢀ-hydroxy octadecanoylamino]tetracos-14-ene
for several diseases in Cameroon. The seeds of B. (4), (R)-byakangelicin (5), trans-fagaramide (6),
cameroonensis are prepared to obtain oil and the ad- 4,5- dimethoxysalicylaldehyde (7), marmesin (8),
hesive [4]. To the best of our knowledge, no phyto- scopoletin (9), marmin (10), stigmasterol-3-O-β-D-
c
0932–0776 / 09 / 0400–0452 $ 06.00 ꢀ 2009 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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J. D. Wansi et al. · Bioactive Phenylethanoids and Coumarines from Basalmocitrus cameroonensis
453
Fig. 1. Structures of com-
pounds isolated from B.
cameroonensis.
glucopyranoside, stigmasterol, betulinic acid, and lu- (br s, OH-4ꢀ) exchangeable with D2O. This inference
peol [5 – 10].
was supported by the 13C NMR and DEPT data, which
Basalethanoid A (1) and B (2) were obtained as showed characteristic signals of a p-disubstituted ben-
white amorphous powders, showing a positive reac- zene ring at δ = 115.4 (C-3ꢀ, C-5ꢀ), 128.2 (C-1ꢀ), 129.7
tion with FeCl3 indicating their phenolic nature. Their (C-2ꢀ, C-6ꢀ) and 155.7 (C-4ꢀ), and the CH2CH2O unit
UV spectrum exhibited two absorption maxima at 279 at δ = 65.0 (C-1) and 34.2 (C-2) [13]. Furthermore,
1
and 235 nm characteristic of phenylethanoids [11, 12]. in the H NMR spectrum, a terminal methyl at δ =
The presence of hydroxyl and ester functions was in- 0.89 (t, J = 6.9 Hz) and methylenes at δ = 2.29 (t, J =
dicated by two IR bands at 2915 and 1730 cm−1, re- 7.5 Hz, -CH2-CO-), 1.58 (m, CH2-CH2-CH2-CO-), and
spectively. From the R-ESI-MS, the molecular compo- 1.25 (br s, nH) were also observed. These data sug-
sition was found to be C40H72O3Na by [M+Na]+ at gested the presence of a long chain linked to a 4-
m/z = 623.5380 (calcd. 623.5385) and C38H68O3Na hydroxyphenylethanol moiety. The presence of a long
by [M+Na]+ at m/z = 595.5070 (calcd. 595.5072), re- chain was further confirmed by the 13C NMR spec-
spectively.
trum, which showed characteristic signals at δ = 173.7
The H NMR spectra of 1 and 2 showed the typi- (C-1ꢀꢀ), 34.1 (C-2ꢀꢀ), 31.8 – 29.3 (CH2)n, 24.8 (C-3ꢀꢀ),
1
cal AAꢀXXꢀ system of a p-disubstituted benzene ring and 14.0 (CH3) [14].
at δ = 7.04 (d, J = 8.1 Hz, H-2ꢀ, H-6ꢀ) and 6.79 (d,
To determine the linkage between the long chain and
J = 8.1 Hz, H-3ꢀ, H-5ꢀ), the presence of a CH2CH2O the 4-hydroxyphenylethanol moiety, an HMBC exper-
unit at δ = 4.22 (t, J = 6.9 Hz, H-1), and 2.84 (t, J = iment was used. In the HMBC spectrum, correlation of
6.9 Hz, H-2), and a free hydroxyl group at δ = 8.53 H-1 (δ = 4.22) with C-1ꢀꢀ (δ = 173.7), C-1ꢀ (δ = 128.2)
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J. D. Wansi et al. · Bioactive Phenylethanoids and Coumarines from Basalmocitrus cameroonensis
and C-2 (δ = 34.2) suggested that the long chain is
linked to the 4-hydroxyphenylethanol moiety by an es-
ter function.
The methanolysis of 1 and 2 yielded methyl dotria-
contanoate (1a) (identified by ESI-MS which indicated
a pseudomolecular ion at m/z = 517 [M+Na]+, corre-
sponding to a molecular formula C33H66O2), methyl
triacontanoate (2a) (identified by ESI-MS which indi-
cated a pseudomolecular ion at m/z = 489 [M+Na]+,
corresponding to a molecular formula C31H62O2)
and 4-hydroxyphenylethanol. The latter was identi-
fied by 1H NMR and EI-MS. From these spectro-
scopic data, basalethanoid A (1) and B (2) were char-
acterized as 2(4-hydroxyphenethyl)dotriacontanoate
and 2(4-hydroxyphenethyl)hentriacontanoate, respec-
tively. Phenylethanoids have previously been described
in the genus Citrus and Fagara (Rutaceae) [11, 12].
Basalamide A (3) was obtained as a brown amor-
phous powder. The IR spectrum showed an absorp-
tion band at 3480 cm−1 due to the OH functions, a
strong absorption band at 1665 cm−1 indicating the
presence of a secondary amide group [15], and at 2950,
2900 and 1505 cm−1 (aliphatic) suggesting it to be
a fatty acid amide. The molecular composition was
found to be C43H85NO6Na by ESI-MS (m/z = 737,
[M+Na+2H]+) and HR-ESI-MS ([M+Na]+ at m/z =
734.6270, calcd. 734.6275). The EI-MS of 3 exhibited
a peak at m/z = 693 due to the loss of a water molecule
from the molecular ion.
Fig. 2. Selected HMBC correlations for compound 3.
correlations with the olefinic signals at δ = 5.37 (m,
H-16 and H-17). Since the chemical shifts of allylic
methylene carbons are different when alkene double
bonds are cis-oriented (δ < 27 ppm) and trans-oriented
(δ > 30 ppm) [19], the double bond in 3 was assigned
an E-configuration.
In order to determine the lengths of the sphin-
gosine and fatty acid chains, the position of the
double bond, and the absolute configuration of 3,
the acid methanolysis method of Gaver and Swee-
ley was used [18]. A sphingosine (3a) and a fatty
acid methyl ester (3b) were obtained by methanol-
ysis of compound 3. The 1H NMR data of 3a
and 3b indicated that the double bond was located
in the sphingosine unit in 3. The length of the fatty
acid chain 3b was determined by EI-MS, which
showed significant fragment ion peaks at m/z = 297
[CH3(CH2)16CHOHCO]+, 312 [CH3(CH2)16CHOH-
CONH]+, and 356 [CH3(CH2)16CHOHCONHCH2-
CH2OH]+. The length of sphingosine 3a was de-
termined by the characteristic ions at m/z = 355
The 1H NMR spectrum of 3 displayed a downfield [CH3(CH2)6CH=CH(CH2)10(CHOH)3]+, 325 [CH3-
doublet at δ = 7.37 (d, J = 9.4 Hz, NH), a very strong (CH2)6CH=CH(CH2)10(CHOH)2]+ and by the base
aliphatic methylene band at δ = 1.26– 1.46, as well as peak m/z = 278 [CH3(CH2)6CH=CH(CH2)10CH]+ in
the signals of six protons at δ = 0.87 (t, J = 6.9 Hz, the EI-MS [20]. The peak at m/z = 278 [CH3(CH2)6-
H-19ꢀ and H-24) and five free hydroxyl groups at δ = CH=CH(CH2)10CH]+ and the typical fragment ion
3.98, 4.24, 4.42, 4.47 and 4.50, all exchangeable with at m/z = 472 [CH3(CH2)6CH=CH(CH2)10(CHOH)3-
D2O. The 13C NMR and DEPT spectral data of 3 were CH(CH2OH)NHCOH=COH]+ formed by elimination
supportive of the above analysis, showing a carbonyl of heptadecene from the molecular ion at m/z = 711
group at δ = 172.2 (C-1ꢀ), one double bond at δ = through McLafferty rearrangement confirmed that the
130.1 (C-16 or C-17) and 130.7 (C-16 or C-17), oxy- double bond is on the long chain base. In order to
genated carbons at δ = 75.7 (C-3), 75.1 (C-2ꢀ), 72.4 determine the position of the double bond, MS-MS
(C-5), 71.5 (C-4), 60.9 (C-1), and 51.9 (C-2), aliphatic fragmentations of 3a were studied, and the results
methylenes at δ = 22.5– 32.8, and two methyls at δ = indicated the double bond to be between C-16 and
14.3 (C-19ꢀ and C-24) [16, 17]. The correlations of δ = C-17 (Fig. 3). Cross peaks in the 1H-1H COSY spec-
7.37 (NH) with δ = 172.2 (C-1ꢀ), 75.7 (C-3) and 51.9 trum were observed between an amide proton (δ =
(C-2) observed in the HMBC spectrum (Fig. 2), which 7.37) and H-2 (δ = 4.08), which, in turn, was cou-
confirmed the presence of an amide function in com- pled to three protons at δ = 3.57 (H-1a), δ = 3.55
pound 3, suggested it to be a ceramide [18]. The chem- (H-1b), and δ = 3.38 (H-3). Furthermore, H-3 showed
ical shifts of the allylic methylene carbons in 3 were correlations with H-2 and with H-4, and finally H-4
assigned at δ = 32.5 (C-15 or C-18) and δ = 32.4 showed correlation with H-5 (δ = 3.38). No cross
(C-15 or C-18) based on the clearly observed HMBC peaks were observed of the signal at δ = 3.97 (H-2ꢀ)
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J. D. Wansi et al. · Bioactive Phenylethanoids and Coumarines from Basalmocitrus cameroonensis
455
Fig. 3. Selected mass fragmen-
tation patterns for compound 3.
Table 1. Effect of compounds 1, 2, 5 and 7 – 10 on oxidative
to any downfield proton signals, but in the HMBC
spectrum it showed strong correlation with C-1ꢀ (δ =
172.2). This suggested that the fifth hydroxyl group
is present at C-2ꢀ of the fatty acid chain. The posi-
tions of the four hydroxyl groups in the long chain
base were further confirmed by the mass fragmenta-
tion pattern (Fig. 3) as well as by the HMBC correla-
tions. Thus the long chain base and fatty acid of 3 must
be (E) 2-aminotetracos-16-ene-1,3,4,5-tetraol and 2-
hydroxynonadecanoic acid, respectively. On the basis
of this evidence, the structure of basalamide A (3) was
determined to be 2-hydroxy-N-((E)-1,3,4,5-tetrahydr-
oxytetracos-16-en-2-yl)nonadecanamide.
burst of whole blood.
Compounds
IC50 (µg mL−1
11.70 2.20
7.00 1.40
0.08 0.01
0.07 0.01
)
Compounds
IC50 (µg mL−1
12.30 3.50
0.09 0.02
0.06 0.01
11.20 1.90
)
1
2
5
7
8
9
10
Ibuprofen
in vitro for oxidative burst studies of whole blood.
Compounds 7 – 14 showed significant effects on the
oxidative burst of the whole blood (IC50 range 0.06–
0.09 µg mL−1), while compounds 1, 2, and 5 showed
inhibition activity (IC50 range 7.00 – 12.30 µg mL−1
)
)
equal to the control (Ibuprofen IC50 =11.20 µg mL−1
The stereochemistry at the chiral centers C-1 to
C-4 has already been established in nubenamide [19].
By comparison of the optical rotation value of 3a
{[α]2D8 = +8.4 (c = 0.20, MeOH)} with the literature
value of nubenamide, the absolute configuration
of basalamide A (3) was found to be 2S, 3S, 4R,
and 5R. In the same manner, the optical rotation value
of 3b [α]2D8 = +22.8 (c = 0.60, CHCl3) suggested
the absolute configuration of C-2ꢀ of the fatty acid to
be R by comparison with the literature values of titho-
niamide B [21]. Thus, the structure of compound 3
was assigned as (2S,3S,4R,5R,16E)-1,3,4,5-tetrahydr-
oxy-2-[(2ꢀR)-2ꢀ-hydroxynonadecanoylamino]tetracos-
16-ene.
(Table 1). The test compounds exhibited a clear sup-
pressive effect on phagocyte oxidative burst response
upon activation with serum opsonized zymosan in a
dose-dependent manner.
Experimental Section
General
Optical rotations [α]D(MeOH or CHCl3, c in g mL−1
)
were determined by using a JASCO digital polarime-
ter (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 an APEX III
(Bruker Daltonik) 7 Tesla (ESI-FT-ICR-MS). EI-MS spec-
tra were recorded on a Finnigan MAT 95 spectrome-
ter (70 eV) with perfluorkerosene as reference substance
for HR-EI-MS. The 1H and 13C NMR spectra were
recorded at 500 and 125 MHz, respectively, on a Bruker
AMX 500 NMR spectrometer. Methyl, methylene and me-
thine carbons were distinguished by DEPT experiments.
Ceramides, which are mostly found in cell walls of
plants and animals, have already been reported in the
literature [16, 18 – 21].
Compounds 1, 2, 5, and 7 – 10 were screened over
a wide range of concentrations (3.1– 50 µg mL−1) for
their immunomodulatory potential. These compounds
were shown to possess inhibitory activity upon activa-
Homonuclear 1H connectivities were determined by us-
tion with serum opsonized zymosan, which was tested ing the COSY experiment. One-bond H-13C connectivities
1
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J. D. Wansi et al. · Bioactive Phenylethanoids and Coumarines from Basalmocitrus cameroonensis
were determined with HMQC gradient pulse factor selec- oxyphenethyl)hentriacontanoate (2) (19.1 mg). Fraction C
tion. Two- and three-bond 1H-13C connectivities were de- was chromatographed over a silica gel 60 column with a
termined by HMBC experiments. Chemical shifts are re- hexane-CH2Cl2-MeOH gradient. A total of 50 fractions of
ported in δ (ppm) using TMS as internal standard and ca. 100 mL each were collected and combined on the basis
coupling constants (J) are measured in Hz. Column chro- of TLC. Fractions 5 – 30 were further chromatographed over
matography was carried out on silica gel (70 – 230 mesh, silica gel 60 with a mixture of hexane-CH2Cl2 (1 : 4) to yield
Merck) and flash silica gel (230 – 400 mesh, Merck). TLC 4,5-dimethoxysalicylaldehyde (7) (15 mg), marmin (10)
was performed on Merck precoated silica gel 60 (F254
Merck), and spots were visualized by using ceric sul- and (R)-byakangelicin (5) (18.0 mg). Fraction D was chro-
fate spray reagent. All reagents used were of analytical matographed over a silica gel 60 column with a CH2Cl2-
,
(125.1 mg), marmesin (8) (69.0 mg), betulinic acid (15.2 mg)
grade.
MeOH (2 : 1) gradient. A total of 35 fractions of 100 mL
each were collected and combined on the basis of TLC. Frac-
tions 1 – 12 yielded the β-D-glucopyranoside of stigmaster-
ol (15.0 mg) and (2S,3S,4R,5R,16E)-1,3,4,5-tetrahydroxy-
2-[(2ꢀR)-2ꢀ-hydroxynonadecanoylamino]tetracos-16-ene (3)
(18.9 mg). Fractions 18 – 20 yielded (2S,3S,4R,5R,14E)-1,3,-
4,5-tetrahydroxy-2-[(2ꢀR)-2ꢀ-hydroxy octadecanoylamino]-
tetracos-14-ene (4) (25.0 mg).
Collection and identification
The stem barks of B. cameroonensis was collected and
identified by Mr. Nana Victor of the National Herbar-
ium, Cameroon, in April 2006 at Batouri locality, Eastern
Cameroon. The herbarium specimen documenting the collec-
tion has been deposited in the National Herbarium, Yaounde´,
Cameroon (Ref. No. 6159 SRF/CAM).
Chemical derivatives
Methanolysis of compounds 1 and 2
Extraction and isolation
Compounds 1 and 2 (5.0 mg each) were added to a mix-
ture of HCl (3.5 mL, 1 N) and dry MeOH (6.0 mg) and re-
fluxed for 16 h with magnetic stirring. Then H2O (10.0 mL)
was added to the refluxed mixture, which was extracted with
n-hexane (3 × 10 mL). The fatty acid methyl esters (1a,
2.0 mg and 2a 1.4 mg) were obtained after the purification of
the n-hexane extract over a silica gel column with n-hexane-
CH2Cl2 (9 : 1) as solvent.
Air-dried, powdered stem barks of B. cameroonensis
(5.5 kg) were extracted with MeOH at r. t. for 72 h. After re-
moving the solvents by evaporation under reduced pressure,
the obtained crude extract (165.0 g) was chromatographed
over silica gel 60 (230 – 400 mesh), using hexane, ethyl ac-
etate and MeOH in increasing polarity order. A total of 123
sub-fractions (ca. 250 mL each) were collected and com-
bined on the basis of TLC analysis leading to four main frac-
tions A – D.
Fraction A (22.0 g) consisted of the combined sub-
fractions 1 – 15 eluted with a mixture of hexane-ethyl ac-
etate (9 : 1). Fraction B (13.0 g) was constituted of the sub-
fractions 16 – 45 eluted with a mixture of hexane-ethyl ac-
etate (7 : 3), and main fraction C (55.0 g) of sub-fractions 46 –
85 eluted with hexane-ethyl acetate (1 : 1). Fraction D
(25.0 g) was constituted of sub-fractions 86 – 123 eluted with
ethyl acetate and with a mixture of ethyl acetate-MeOH
(9 : 1).
Methanolysis of compound 3
Compound 3 (7.5 mg), was added to a mixture of HCl
(3.5 mL, 1N) and dry MeOH (6.0 mg), and refluxed for 16 h
with magnetic stirring. Then H2O (10.0 mL) was added to
the refluxed mixture, which was extracted with n-hexane
(3 × 10 mL). The fatty acid methyl ester (3b, 2.0 mg) was
obtained after the purification of the n-hexane extract over
a silica gel column with n-hexane-CH2Cl2 (9 : 1) as solvent.
The MeOH/H2O phase was evaporated under reduced pres-
sure. The residue obtained was also purified over a silica gel
column and eluted with CH2Cl2-MeOH (15 : 1) to yield a
sphingosine (3a, 1.5 mg).
Fraction A was chromatographed over a silica gel 60 col-
umn with a hexane-CH2Cl2 gradient. A total of 33 fractions
of ca. 100 mL each were collected and combined on the
basis of TLC. Fractions 1 – 20 yielded lupeol (155.5 mg),
fractions 21 – 33 yielded a mixture of sterols (stigmasterol +
sitosterol) (30.0 mg) and fatty acids. Fraction B was chro-
matographed over a silica gel 60 column with a hexane-CH2-
2(4-Hydroxyphenethyl)dotriacontanoate (1)
White amorphous powder. – UV (MeOH): λmax (log ε) =
Cl2 gradient. A total of 20 fractions of ca. 100 mL each 279 (3.8), 235 (3.9) nm. – IR (CHCl3): νmax = 2915, 2850,
were collected and combined on the basis of TLC. Frac- 1730, 1614, 1392 cm−1. – 1H NMR (500 MHz, CDCl3): δ =
tions 1 – 15 were further chromatographed over silica gel 60 0.89 (t, J = 6.9 Hz, 3H, 31ꢀꢀ-H), 1.25 (br s, 56H, 4ꢀꢀ-30ꢀꢀ-H),
with the mixture of hexane-CH2Cl2 (3 : 1) to yield trans- 1.58 (m, 2H, 3ꢀꢀ-H), 2.29 (t, J = 7.5 Hz, 2H, 2ꢀꢀ-H), 2.84 (t,
fagaramide (6) (10.5 mg), scopoletin (9) (40.0 mg), 2(4-hydr- J = 6.9 Hz, 2H, 2-H), 4.22 (t, J = 6.9 Hz, 2H, 1-H), 6.79
oxyphenethyl)dotriacontanoate (1) (13.4 mg) and 2(4-hydr- (d, J = 8.1 Hz, 2H, 3ꢀ-H and 5ꢀ-H), 7.04 (d, J = 8.1 Hz, 2H,
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2ꢀ-H and 6ꢀ-H), 8.53 (br s, 1H, OH). – 13C NMR (125 MHz, 3-OH), 4.50 (br s, 1H, 1-OH), 5.37 (br s, 2H, 16-H and
CDCl3): δ = 14.0 (C-19ꢀ), 24.8 (C-3ꢀꢀ), 29.3 (methylenes), 17-H), 7.37 (d, J = 9.4 Hz, 1H, -NH). – 13C NMR (125 MHz,
34.1 (C-2ꢀꢀ), 34.2 (C-2), 65.0 (C-1), 115.4 (C-3ꢀ and C-5ꢀ), [D6]DMSO): δ = 14.3 (C-19ꢀ and C-24), 22.5 (C-18ꢀ and
128.2 (C-1ꢀ), 129.7 (C-2ꢀ and C-6ꢀ), 155.7 (C-4ꢀ), 173.7 C-23), 25.9 (C-4ꢀ), 31.2 – 29.0 (methylenes), 31.7 (C-6), 32.4
(C-1ꢀꢀ). – MS ((+)-ESI): m/z = 623 [M+Na]+. – HRMS ((+)- (C-18), 32.5 (C-15), 32.8 (C-3ꢀ), 51.9 (C-2), 60.9 (C-1), 71.5
(C-4), 72.4 (C-5), 75.1 (C-2ꢀ), 75.7 (C-3), 130.1 (C-16),
130.7 (C-17), 172.2 (C-1ꢀ). – MS ((+)-ESI): m/z = 737
[M+Na+2H]+. – HRMS ((+)-ESI): m/z = 734.6270 (calcd.
734.6275 for C43H85NO6Na, [M+Na]+). – MS (EI, 70 eV)
data are illustrated in Fig. 3.
ESI): m/z = 623.5380 (calcd. 623.5385 for C40H72O3Na,
[M+Na]+).
Methyl dotriacontanoate (1a)
White powder. – MS (EI, 70 eV): m/z = 494. – 1H NMR
(500 MHz, CDCl3): δ = 0.73 (t, J = 7.5 Hz, 3H, -CH3), 1.40 –
1.09 (br s, -(CH2)n-), 2.06 (t, J = 7.8 Hz, 2H, 2-H), 3.80 (s,
3H, -OCH3).
Sphingosine (3a)
Brown oil. – [α]2D8 = + 8.4 (c = 0.2, MeOH). – IR
(CH3OH): νmax = 3530, 3050, 2900, 1505 cm−1. – 1H NMR
(500 MHz, MeOD): δ = 0.85 (t, J = 7.0 Hz, 3H, 24-H), 1.26
(methylenes), 1.94 – 2.00 (m, 4H, 15-H and 18-H), 3.40 (br
s, 1H, 3-H), 3.50 (br s, 1H, 1b-H), 3.57 (br s, 1H, 1a-H), 3.70
(m, 1H, 4-H), 3.97 (br s, 1H, 5-H), 4.26 – 4.65 (br s, 4H, 4 ×
OH), 5.37 (br s, 2H, 16-H and 17-H).
2(4-Hydroxyphenethyl)hentriacontanoate (2)
White amorphous powder. – UV (MeOH): λmax (log ε) =
280 (3.5), 245 (3.7) nm. – IR (CHCl3): νmax = 2915, 2848,
2359, 1732, 1467 cm−1. – 1H NMR (500 MHz, CDCl3): δ =
0.89 (t, J = 6.9 Hz, 3H, 29ꢀꢀ-H), 1.24 (br s, 52H, 4ꢀꢀ-28ꢀꢀ-H),
1.59 (m, 2H, 3ꢀꢀ-H), 2.30 (t, J = 7.5 Hz, 2H, 2ꢀꢀ-H), 2.84 (t,
J = 6.9 Hz, 2H, 2-H), 4.22 (t, J = 6.9 Hz, 2H, 1-H), 6.79 (d,
J = 8.1 Hz, 2H, 3ꢀ-H and 5ꢀ-H), 7.04 (d, J = 8.1 Hz, 2H, 2ꢀ-
H and 6ꢀ-H), 8.53 (br s, 1H, -OH). – 13C NMR (125 MHz,
CDCl3): δ = 14.1 (C-19ꢀ), 24.8 (C-3ꢀꢀ), 29.3 (methylenes),
34.1 (C-2ꢀꢀ), 34.2 (C-2), 65.0 (C-1), 115.4 (C-3ꢀ and C-5ꢀ),
128.2 (C-1ꢀ), 129.7 (C-2ꢀ and C-6ꢀ), 155.7 (C-4ꢀ), 173.7
(C-1ꢀꢀ). – MS ((+)-ESI): m/z = 595 [M+Na]+. – HRMS ((+)-
ESI): m/z = 595.5070 (calcd. 595.5072 for C38H68O3Na,
[M+Na]+).
Fatty acid methyl ester (3b)
Yellow oil. – [α]D28 = + 22.8 (c = 0.6, CHCl3). – 1H NMR
(500 MHz, CHCl3): δ = 4.15 (dd, J = 4.0, 7.8 Hz, 1H,
2-H), 3.80 (3H, -OMe), 1.67 – 1.80 (m, 2H, 3-H), 1.27 – 1.24
(methylene), 0.86 (t, J = 7.0 Hz, 3H, 19-H).
Chemiluminescence assay for determination of immunomod-
ulation activity
A luminol-enhanced chemiluminescence assay was per-
formed, as described in [22]. In brief, whole blood (diluted
1 : 200) and neutrophils (1 × 107) suspended in Hank’s bal-
Methyl triacontanoate (2a)
White powder. – MS (EI, 70 eV): m/z = 466. – 1H NMR
(500 MHz, CDCl3): δ = 0.66 (t, J = 7.4 Hz, 3H, -CH3), 1.38 –
1.10 (br s, -(CH2)n-), 2.08 (t, J = 7.7 Hz, 2H, 2-H), 3.83 (s,
3H, -OCH3).
ance salt solution with calcium and magnesium (HBSS++
)
were incubated with 50 µL of each test compounds at con-
centrations of 3.1 – 50 µg mL−1 for 30 min. Then, 50 µL
(20 mg mL−1) zymosan (Sigma Chemical Co. St. Louis,
MO) followed by 50 µL (7×105 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.
1,3,4,5-Tetrahydroxy-2-[(2ꢀR)-2ꢀ-hydroxynonadecanoyl-
amino]tetracos-16-ene (3)
Brown amorphous powder. – [α]2D8 = + 9.5 (c = 0.15,
MeOH). – IR (CH3OH): νmax = 3480, 2950, 2900, 1665,
1
1505 cm−1. – H NMR (500 MHz, [D6]DMSO): δ = 0.87
Acknowledgements
(t, J = 6.9 Hz, 6H, 19ꢀ-H and 24-H), 1.26 (br s, 62H, 8-
14-H, 20-23-H, 4ꢀ-18ꢀ-H), 1.56 (m, 2H, 3ꢀ-H), 1.94 (m, 4H,
15-H and 18-H), 3.38 (br s, 2H, 3-H and 5-H), 3.55 (dd,
J = 6.3, 10.6 Hz, 1H, 1b-H), 3.57 (dd, J = 6.3, 10.6 Hz,
1H, 1a-H), 3.67 (m, 1H, 4-H), 3.97 (dd, J = 4.4, 8.8 Hz,
1H, 2ꢀ-H), 3.98 (br s, 1H, 2ꢀ-OH), 4.08 (m, 1H, 2-H), 4.24
(br s, 1H, 4-OH), 4.42 (br s, 1H, 5-OH), 4.47 (br s, 1H,
We wish to acknowledge the Alexander von Humboldt
(AvH) Foundation, Germany for post doctoral fellowships
to J. D. Wansi and K. P. Devkota at Bielefeld University, the
European Commision for the Marie Curie IIF fellowship to
B. N. Lenta, and the International Foundation for Sciences
(IFS), Sweden, for financial support (F/3978-2).
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