Steroids and ceramide from the stem bark of Odyendyea gabonensis

Article abstract of DOI:10.5560/ZNB.2013-3132

Two new steroids, 22E, 24R-stigmast-22-ene-3,6,11-trione (1) and 22E, 24R-3-acetylstigmasta-5,22-diene-7,11-dione (2), and one new ceramide, (2S,3S,4R,5R) N-(1,3,4,5-tetrahydroxyundecan-2-yl)tetradecanamide (7), together with eleven known compounds were isolated from the CH₂Cl₂ extract of the stem bark of Odyendyea gabonensis. The structures of all compounds were determined by comprehensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data, chemical reactions, and comparison with previously known analogs. Pure compounds were tested for their activity against the bacteria Bacillus subtilis, Staphylococcus aureus and Escherichia coli, the fungi Mucor miehei and Candida albicans, and the plant pathogen oomycetes Aphanomyces cochlioides, Pythium ultimum and Rhizoctonia solani using the paper disk agar diffusion assay. For active compounds, MICs were determined by the broth microdilution assay. Cytotoxic activity against the human lung adenocarcinoma cell line A 549 was evaluated by the MTT assay. All compounds delivered low to missing antimicrobial activities in the agar diffusion assay and MICs > 1 mg mL^-1. The alkaloids 10 and 11 displayed cytotoxic activity against the human lung adenocarcinoma cell line A549 with IC ₅₀ 2.5 and 4.5 μm respectively.

Full text of DOI:10.5560/ZNB.2013-3132

Steroids and Ceramide from the Stem Bark of Odyendyea gabonensis
a
b
a
Emmanuel Ngeufa Happi , Simone V e´ ronique Fannang , Marie Fomani , Suzye Mireille
a
a
a,c
Moladje Donkwe , Nkoungou Yomzak Carine Nicaise , Jean Duplex Wansi , and
c
Norbert Sewald
a
Department of Chemistry, University of Douala, Faculty of Science, 24157 Douala, Cameroon
Department of Pharmaceutical Science, University of Douala, Faculty of Medicine and
b
Pharmaceutical Science, 2701 Douala, Cameroon
Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, 33501
c
Bielefeld, Germany
Reprint requests to E. N. Happi. Tel: +237-99-86-50-48. E-mail: ngeufa@yahoo.fr
Z. Naturforsch. 2013, 68b, 924 – 930 / DOI: 10.5560/ZNB.2013-3132
Received May 10, 2013
Two new steroids, 22E, 24R-stigmast-22-ene-3,6,11-trione (1) and 22E, 24R-3-acetylstigmasta-
5
2
,22-diene-7,11-dione (2), and one new ceramide, (2S,3S,4R,5R) N-(1,3,4,5-tetrahydroxyundecan-
-yl)tetradecanamide (7), together with eleven known compounds were isolated from the CH Cl2
2
extract of the stem bark of Odyendyea gabonensis. The structures of all compounds were determined
by comprehensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data, chemical reac-
tions, and comparison with previously known analogs. Pure compounds were tested for their activity
against the bacteria Bacillus subtilis, Staphylococcus aureus and Escherichia coli, the fungi Mucor
miehei and Candida albicans, and the plant pathogen oomycetes Aphanomyces cochlioides, Pythium
ultimum and Rhizoctonia solani using the paper disk agar diffusion assay. For active compounds,
MICs were determined by the broth microdilution assay. Cytotoxic activity against the human lung
adenocarcinoma cell line A 549 was evaluated by the MTT assay. All compounds delivered low to
−
1
missing antimicrobial activities in the agar diffusion assay and MICs > 1 mg mL . The alkaloids 10
and 11 displayed cytotoxic activity against the human lung adenocarcinoma cell line A549 with IC50
2
.5 and 4.5 µm respectively.
Key words: Odyendyea gabonensis, Simaroubaceae, Steroids, Ceramide, Antimicrobial and
Cytotoxicity Activities
Introduction
stigmasta-5,22-diene-3,7-dione, and 22E,24R-stig-
mast-22-ene-3,7-dione [4 – 7]. In this report, we
The plant species Odyendyea gabonensis (Pierre) describe the isolation and structural elucidation of two
Engl. (Simaroubaceae), synonym Quassia gabonensis new steroids (1, 2) and one ceramide (7), together with
Pierre [1, 2], is a huge tree growing in primary the antimicrobial and cytotoxic activities of isolated
forests of Central Africa. Bark, leaves and fruits of compounds.
O.gabonensis are used in traditional medicine as
purgative as well as for the treatment of malaria, Results and Discussion
pulmonary disorders and cancer [3]. Previous phy-
tochemical investigations on the bark and fruits
The CH Cl extract of the stem bark of O. gabo-
2 2
yielded scopoletin, 8-hydroxycanthin-6-one, ailan- nensis was separated by repeated column chromatog-
0
thinone, glaucarubinone, 2 -acetoxyglaucarubinone, raphy and preparative TLC (PTLC) to afford three new
2
0
-acetoxy-glaucarubin, excelsin, canthin-6-one, 5- and eleven known compounds (Fig. 1). The known
methoxycanthin-6-one, 4,5-dimethoxycanthin-6-one, compounds were identified as 22E,24R-stigmast-22-
1
-hydroxymethyl-β-carboline, 1-carboxamide-β-car- ene-3,6-dione (3), 22E,24R-3-acetylstigmasta-5,22-
boline, (–)-odyendane and (–)-odyendene, (–)-odyen- diene-7-one (4), β-sitosterone (5), stigmasterone (6),
danol, 9-hydroxy-5-methoxycanthin-6-one, 22E,24R- (–)-odyendene (8), (–)-odyendane (9), 9-hydroxy-5-
©
2013 Verlag der Zeitschrift f u¨ r Naturforschung, T u¨ bingen · http://znaturforsch.com
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E. N. Happi et al. · Steroids and Ceramide from Odyendyea gabonensis
925
H
O
H
O
H
H
H
O
H
O
H
H
H
H
O
H
AcO
O
3
O
1
2
H
H
H
H
H
H
H
H
H
O
O
O
AcO
HO
H
6
5
4
O
HN
MeO
O
O
OH
O
N
MeO
R
N
H
OH OH
O
O
O
OMe
H
H
7
R
O
1
1
0 R = OH
1 R = H
8
9
R =
CH2
MeO
R = CH3
7
a
Fig. 1. Structures of the isolated compounds.
methoxycanthin-6-one (10), 5-methoxycanthin-6-one 0.80 (d, J = 6.3 Hz, H-26), 0.97 (d, J = 6.3 Hz, H-27),
11), tirucalla-7,24-dien-3-one, oleanolic acid, and 3- and 1.03 ppm (d, J = 6.3 Hz, H-21), a methyl triplet
(
acetyloleanolic acid. The structures were confirmed by at δ = 0.84 (t, J = 6.3 Hz, H-29) and two methyl sin-
spectra comparison with authentic and published val- glets at δ = 0.79 and 1.19 ppm. A comparison with the
ues [7 – 10].
stigmast-22-ene-3,6-dione suggested that both com-
1
3
Compound 1 was obtained as a colorless pow- pounds had the same side chain [11]. The C NMR
der. The molecular composition was found to be spectrum (Table 1) confirmed the presence of a carbon-
+
C H O by HR-EI-MS ([M] at m/z = 440.3249, carbon double bond with signals at δ = 137.9 (C-22),
29
44
3
calcd. 440.3290). The presence of the carbonyl func- 129.7 ppm (C-23) and three carbonyl functions with
tions was revealed by strong absorptions at 1695 cm
−1
signals at δ = 211.4 (C-6), 209.2 (C-3), 209.2 ppm
1
1
in the IR spectrum. Its H NMR spectrum (Table 1) (C-11). Furthermore, the H NMR spectrum showed
showed two trans-olefinic protons at δ = 5.18 (dd, a doublet at δ = 2.59 ppm (d, J = 8.8 Hz, H-9) and an
J = 15.1; 8.2 Hz, H-22) and 5.07 ppm (dd, J = 15.1; AB system of two protons at δ = 2.43 (d, J = 10.7 Hz,
8
.8 Hz, H-23) as well as three methyl doublets at δ = H-12a) and 2.37 ppm (d, J = 10.7 Hz, H-12b) due to
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E. N. Happi et al. · Steroids and Ceramide from Odyendyea gabonensis
Attribution
1
2
Table 1. 1H (500 MHz) and
13C
38.1
39.3
1H
13C
1H
13
C (125 MHz) NMR assign-
a
1
2
37.1
28.2
ments for 1 and 2 in CDCl3 .
2.09 (m)
2.09 (m)
3
4
209.2
37.0
–
73.3
38.5
4.47 (brs)
2.03 (m)
2.06 (m)
–
5.83 (s)
–
2.61 (brs)
.40 (brs)
2.61(brs)
2
5
6
7
57.5
211.4
46.6
168.4
126.3
200.5
2.41 (dd, J = 13.2, 10.7)
2.37 (dd, J = 10.7, 8.8)
8
9
31.8
57.5
41.3
209.2
56.6
2.10 (m)
2.59 (d, J = 8.8)
45.8
56.0
37.9
207.1
53.6
2.54 (dd, J = 5.2, 5.2)
2.51 (d, J = 5.2)
1
1
1
0
1
2
–
–
–
–
2.43 (d, J = 10.7)
2.42 (brs)
2.37 (d, J = 10.7)
2.20 (brs)
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
43.0
55.8
25.4
28.4
56.7
11.9
18.9
40.4
12.3
137.9
129.7
51.2
29.1
18.7
21.1
24.0
12.0
–
–
–
–
–
40.5
51.2
23.0
28.8
55.9
12.0
19.0
39.6
12.2
138.1
129.5
51.2
31.8
20.9
19.8
24.1
12.3
168.4
21.1
–
–
–
–
–
0.79 (s)
1.19 (s)
2.09 (m)
0.76 (s)
0.78 (s)
2.00 (m)
1.03 (d, J = 6.3)
1.05 (d, J = 6.9)
5.17 (dd, J = 15.1, 8.2)
5.05 (dd, J = 15.1, 8.8)
1.57 (m)
5.18 (dd, J = 15.1, 8.2)
5.07 (dd, J = 15.1, 8.8)
1.56 (m)
1.57 (m)
1.57 (m)
0.80 (d, J = 6.3)
0.97 (d, J = 6.3)
1.40 (m)
0.87 (d, J = 5.6)
0.95 (d, J = 5.6)
1.39 (m)
0.83 (d, J = 6.3)
–
9
0.84 (d, J = 6.3)
0
1
2
0
2.19
a
Assignments were based on HMQC, HMBC and NOESY experiments. Multiplicities and coupling
constants in Hz in parentheses.
the presence of the protons adjacent to the third car- loss of the side chain and the cleavage mechanisms
bonyl group. The positions of the three carbonyl func- (bonds C-9/C-11 and C-8/C-14) involving transfer
tions were determined by the HMBC and EI-MS spec- of hydrogen from C-9 to the charge-retaining frag-
tra. In the HMBC spectrum, correlations between H-8 ment, confirming the position C-11 for the third car-
(
δ = 2.10) and C-6 (δ = 211.4), C-11 (δ = 209.2), bonyl [12]. Furthermore, the molecular fragment at
C-13 (δ = 43.0) and C-10 (δ = 41.3) and between H- m/z = 177 (C H O ) resulted from the hydrogen
5
1
1
13
2
(δ = 2.61) and C-6 (δ = 211.4), C-3 (δ = 209.2), transfer from C-7 to C-9 and from C-9 to C-14, fol-
C-7 (δ = 46.6) and C-10 (δ = 41.3) indicate the po- lowed by the cleavage of the bond C-9/C-11, indi-
sition of the three carbonyl groups at C-3, C-6 and cating that the second carbonyl group is located at C-
C-11. This assignment was in agreement with the EI- 6 [12]. Finally, according to biogenetic considerations
MS, which showed two prominent fragment ion peaks of steroid and ion fragments at m/z = 55 (C H O), 69
3
3
at m/z = 260 (C H O ) and 257 (C H O ) result- (C H O) and 81 (C H O) observed in the EI-MS, the
1
6
20
3
16 17
3
4
6
5
6
ing from the loss of the side chain and D-ring carbons. first carbonyl function is located at C-3 [13, 14]. From
These ion peaks indicate that the three carbonyl func- the above spectroscopic studies, the structure of com-
tions are located on the rings A, B or C. The frag- pound 1 was determined as 22E,24R-stigmast-22-ene-
ment ion peak at m/z = 124 (C H O) arises from the 3,6,11-trione.
8
12
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E. N. Happi et al. · Steroids and Ceramide from Odyendyea gabonensis
927
Compound 2 was obtained as a colorless pow- (bonds C-9/C-11 and C-8/C-14) involving transfer
der. The molecular composition was found to be of hydrogen from C-9 to the charge-retaining frag-
+
C H O by HR-EI-MS ([M] at m/z = 482.3390, ment, confirming the position C-11 for the third car-
31
46
3
calcd. 482.3396). The presence of the carbonyl and bonyl [12]. Furthermore, the molecular fragments at
enone functions was revealed by strong absorptions m/z = 194 (C H O ) and 152 (C H O ), arise from
1
1
14
3
9
12
2
−1
at 1735 and 1655 cm in the IR spectrum and con- the RDA fragmentation and the loss of an acetyl group,
firmed by a strong absorption at 245 nm in the UV suggesting that the second carbonyl is placed at C-
spectrum [15]. Its H NMR spectrum (Table 1) showed 7 [16]. The spectroscopic studies identify the structure
1
three olefinic protons at δ = 5.83 (s, H-6), 5.17 (dd, of compound 2 as 22E,24R-3-acetylstigmasta-5,22-
J = 15.1; 8.2 Hz, H-22) and 5.05 ppm (dd, J = 15.1; diene-7,11-dione.
8.8 Hz, H-23) as well as three methyl doublets at
Compound 7 was obtained as a colorless amorphous
δ = 0.87 (d, J = 5.6 Hz, H-26), 0.95 (d, J = 5.6 Hz, powder. The IR spectrum showed an absorption band
−
1
H-27) and 1.05 ppm (d, J = 6.9 Hz, H-21), a methyl at 3450 cm due to OH functions, a strong absorption
−1
triplet at δ = 0.83 ppm (t, J = 6.3 Hz, H-29) and two band at 1660 cm indicating the presence of a sec-
methyl singlets at δ = 0.76 and 0.78 ppm. A compar- ondary amide group [17], and additional absorption
−1
ison with the spectrum of stigmasterol suggested that bands at 2950, 2910 and 1507 cm (aliphatic) sug-
both compounds possess the same side chain [13]. The gesting the compound to be a fatty acid amide. The
13
C NMR spectrum (Table 1) confirmed the presence molecular composition was found to be C H NO
of an enone group with signals at δ = 168.4 (C-5) by ESI-MS (m/z = 446; [M+H] ) and HR-ESI-MS
and 126.3 ppm (C-6), a carbon-carbon double-bond at ([M+H] at m/z = 446.3840, calcd. 446.3845).
2
5
52
5
+
+
1
δ = 138.1 (C-22) and 129.5 ppm (C-23), two carbonyl
The H NMR spectrum of 7 displayed a down-
functions at δ = 200.5 (C-7) and 207.1 ppm (C-11), field doublet at δ = 7.21 ppm (d, J = 8.8 Hz, NH),
one oxymethine at δ = 73.3 ppm (C-3), and an ace- a very strong aliphatic methylene signal at δ =
tate group at δ = 168.4 (C-1 ) and 21.1 ppm (C-2 ). 1.15 – 1.33 ppm, as well as signals of two methyl pro-
Furthermore, the H NMR spectrum showed four sig- tons at δ = 0.76 (t, J = 6.9 Hz, H-11 and H-14 ) and
nals at δ = 2.54 (dd, J = 5.2; 5.2 Hz, H-8), 2.51 (d, five methines at δ = 3.80 (m, H-2), 3.74 (dd, J =
J = 5.2 Hz, H-9), 2.42 (brs, H-12a), and 2.20 ppm (brs, 6.9; 4.7 Hz, H-3), 3.48 (dd, J = 10.7; 5.2 Hz, H-1a),
H-12b) due to protons adjacent to the carbonyl groups 3.44 (dd, J = 10.7; 5.2 Hz, H-1b), 3.27 (m, H-4), and
and one additional methyl group at δ = 2.19 ppm (s) 3.25 ppm (m, H-5). The C NMR and DEPT spec-
confirming the presence of an acetate function. In ad- tra data of 7 supported the above analysis, showing
dition, it showed a signal at δ = 4.37 ppm (brs) at- a carbonyl group at δ = 173.9 ppm (C-1 ), four oxy-
0
0
1
0
1
3
0
tributed to H-3, confirming the location of the acetate genated carbons at δ = 75.1 (C-4), 71.6 (C-3), 71.6
group at C-3 position, in agreement with the reported (C-5), and 61.1 ppm (C-1), one methane carbon at
data [15]. The positions of the two carbonyl func- δ = 51.9 ppm (C-2), aliphatic methylenes in the region
tions were determined by HMBC and EI-MS spectra. δ = 22.4 – 32.4 ppm, and two methyls at δ = 14.3 ppm
0
The HMBC spectrum reveals correlations between H- (C-11 and C-14 ) [18]. The correlations of δ = 7.21
0
8
(δ = 2.54) and C-11 (δ = 207.1), C-6 (δ = 126.3), (NH) with δ = 173.9 (C-1 ), 71.6 (C-3) and 51.9 (C-2)
C-5 (δ = 168.4) and C-10 (δ = 37.9), and between H- observed in the HMBC spectrum confirmed the pres-
(δ = 2.51) and C-11 (δ = 207.1), C-7 (δ200.5), C-5 ence of an amide function and suggested compound 7
δ = 168.4), C-12 (δ = 53.6) and C-14 (δ = 51.2), to be a ceramide [19].
indicating the position of the two carbonyl groups at In order to determine the lengths of the sphingo-
9
(
C-7 and C-11. This assignment is in agreement with sine and fatty acid chains and the absolute configu-
the EI-MS, which showed two prominent fragment ion ration of 7, the acid methanolysis method of Gaver
peaks at m/z = 302 (C H O ) and 300 (C H O ) and Sweeley was applied [19]. Fatty acid methyl es-
1
8
22
4
18 20
4
resulting from the loss of the side chain and D-ring car- ter 7a was obtained by methanolysis of compound
bons. These ion peaks indicated that the two carbonyl 7. The length of the fatty acid chain in 7a was de-
functions are located on the rings A, B or C. The frag- termined by EI-MS, which showed significant ion
+
ment ion peak at m/z = 124 (C H O) arises from the peaks at m/z = 242 [CH (CH ) COOCH ] and 211
8
12
3
2 12
3
+
1
1
loss of the side chain and the cleavage mechanisms [CH (CH ) CO] . Cross peaks in the H- H COSY
3
2 12
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28
E. N. Happi et al. · Steroids and Ceramide from Odyendyea gabonensis
spectrum were observed between an amide proton (δ = out on silica gel (70 – 230 mesh, Merck). TLC was performed
7
.21) and H-2 (δ = 3.80), which, in turn, was cou- on Merck precoated silica gel 60 F254 aluminum foil, and
spots were detected using ceric sulfate spray reagent. Pheno-
pled to three protons at δ = 3.48 (H-1a), δ = 3.44
(
lic compounds were detected using FeCl reagent. The purity
of the compounds was investigated by means of H NMR and
H-1b) and δ = 3.74 (H-3). Furthermore, H-3 showed
3
1
correlations with H-2 and H-4, as well as H-4 with
H-5 (δ = 3.25) suggesting that the hydroxyl groups
are present at C-1, C-3, C-4 and C-5 of the sphingo-
sine chain. On the basis of this evidence, the struc-
ture of compound 7 was determined to be N-(1,3,4,5-
tetrahydroxyundecan-2-yl)tetradecanamide.
ESI-MS. The degree of purity of all tested compounds was
>
95%, and of the positive control (Doxorubicin) 99.9%. All
other substances, if not specified ortherwise, were purchased
from Sigma-Aldrich (Germany). All reagents used were of
analytical grade.
The stereochemistry at the stereogenic centers C- Plant material
2
to C-5 has already been established in basalamine
The stem bark of O. gabonensis was collected at the
A [20]. Comparison of the optical rotation value
Mbalmayo locality in Central Cameroon in August 2007 and
identified by Mr. Nana Victor of the National Herbarium,
Yaound e´ , Cameroon, where a voucher specimen (ref. 9861
of 7 {[α]2 = +25.5 (c = 0.20, MeOH)} with
8
D
the published value of basalamine A revealed the
absolute configuration of compound 7 to be 2S, SRF/CAM) was deposited.
3
S, 4R, and 5R. Thus, the structure of com-
Extraction and isolation
pound 7 was assigned as (2S,3S,4R,5R)-N-(1,3,4,5-
tetrahydroxyundecan-2-yl)tetradecanamide.
The air-dried and powdered stem bark (3.5 kg) of O.
Tests of the pure compounds on paper disk diffu- gabonensis was extracted with CH Cl at room temper-
2
2
sion agar against the bacteria Bacillus subtilis, Staphy- ature for 72 h. After evaporation under reduced pressure,
lococcus aureus and Escherichia coli, the fungi Mucor 46.5 g of crude extract was obtained. The CH2Cl2 extract
was purified by column chromatography over silica gel 60
miehei and Candida albicans and the plant pathogen
(230 – 400 mesh) and preparative TLC using a gradient sys-
oomycetes Aphanomyces cochlioides, Pythium ulti-
mum and Rhizoctonia solani resulted in missing or low
tem of petroleum ether, CH Cl , acetone and MeOH. 200
2
2
−1
sub-fractions (ca. 250 mL each) were collected and pooled
on the basis of TLC analysis leading to five main fractions
activities, the latter with MIC values > 1 mg mL
.
However, alkaloids 10 and 11 displayed moderate
cytotoxic activity against the human lung adenocarci-
noma cell line A 549 (IC50 2.5 and 4.5 µm).
(
A–E).
Fraction A (10.2 g, combined from sub-fractions 1 – 50)
was chromatographed over a silica gel 60C column with
a petroleum ether-CH Cl gradient. A total of 30 fractions
2
2
Experimental Section
of ca. 100 mL each was collected and combined on the basis
of TLC. Fractions 5 – 10 were further chromatographed
on silica gel 60H with a mixture of petroleum ether-
General experimental procedures
Ultraviolet spectra were recorded on a Hitachi UV 3200 CH Cl (4 : 1) for elution to yield tirucalla-7,24-dien-3-one
2
2
spectrophotometer in MeOH. Infrared spectra were recorded (10.5 mg), stigmasterone (6) (8.5 mg) and β-sitosterone (5)
on a Jasco 302-A spectrophotometer. ESI-HR mass spectra (4.5 mg). Fractions 11 – 25 were chromatographed on silica
were recorded on a Bruker FTICR 4.7 T mass spectrome- gel 60H with a mixture of petroleum ether-CH Cl (4 : 1)
2
2
ter. EI-MS were recorded on a Finnigan MAT 95 spectrome- for elution to yield 22E,24R-stigmast-22-ene-3,6-dione
ter (70 eV) with perfluorokerosene as reference substance for (3) (8.4 mg) and 22E,24R-3-acetylstigmasta-5,22-diene-
1
13
HR-EI-MS. The H and C NMR spectra were recorded at 7-one (4) (11.5 mg). Fraction B (7.0 g, combined from
00 MHz and 125 MHz, respectively, on Bruker DRX 500 sub-fractions 51 – 70) was chromatographed over a silica
NMR spectrometers. Methyl, methylene and methine car- gel 60C column with a petroleum ether-CH Cl gra-
5
2
2
bons were distinguished by DEPT experiments. Homonu- dient. A total of 20 fractions of ca. 100 mL each was
1
clear H connectivities were determined by using the COSY collected and combined on the basis of TLC. Fractions
experiment. One-bond 1H- C connectivities were deter- 7 – 15 were further chromatographed over a silica gel
13
mined with HMQC gradient pulse factor selection. Two- 60H with a mixture of petroleum ether-CH Cl (5 : 2) to
2
2
and three-bond 1H- C connectivities were determined by yield 22E,24R-stigmast-22-ene-3,6,11-trione (1) (15.2 mg)
HMBC experiments. Chemical shifts are reported in δ (ppm) and 22E,24R-3-acetylstigmasta-5,22-diene-7,11-dione (2)
using TMS as internal standard and coupling constants (J) (7.5 mg). Fraction C (6.5 g, combined from sub-fractions
were measured in Hz. Column chromatography was carried 71 – 100) was chromatographed on a silica gel 60C column
13
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E. N. Happi et al. · Steroids and Ceramide from Odyendyea gabonensis
929
with a petroleum ether-CH Cl2 gradient. 25 fractions of (2S,3S,4R,5R)-N-(1,3,4,5-tetrahydroxyundecan-2-yl)-
2
ca. 100 mL each were collected and combined on the basis tetradecanamide (7)
of TLC. Fractions 16 – 25 were further chromatographed
Colorless amorphous powder; R = 0.60, silica gel 60
over silica gel 60H with petroleum ether-CH Cl (1 : 1)
f
2
2
2
8
F254, CH Cl -MeOH (9 : 1). – [α] = +25.5 (c = 0.20,
to yield (–)-odyendene (8, 60.0 mg) and (–)-odyendane (9,
2
2
D
MeOH). – IR (CH OH): ν = 3450, 2950, 2910, 1660,
7.8 mg). Fraction D (9.5 g, combined from sub-fractions
3
−
1
1
1
(
507 cm . – H NMR (500 MHz, [D ]DMSO ): δ = 0.76
t, J = 6.9 Hz, H-11 and H-14 ), 1.15 – 1.31 (brs, H-6–H10,
H-4 –H12 ), 1.50 (m, H-2 ), 1.54 (m, H-6 and H-3 ), 3.25 (m,
1
01 – 130) was chromatographed on a silica gel 60C column
6
0
with a petroleum ether-CH Cl (1 : 3) and CH Cl -aceton
2
2
2
2
0
0
0
0
(3 : 1) gradient. As a result, 40 fractions of ca. 100 mL
H-5), 3.27 (m, H-4), 3.44 (dd, J = 5.2; 10.7 Hz, H-1b), 3.48
each were collected and combined on the basis of TLC.
Fractions 1 – 15 were further chromatographed over silica
(
3
dd, J = 5.2; 10.7 Hz, H-1a), 3.74 (dd, J = 4.7; 6.9 Hz, H-
0
13
), 3.80 (m, H-2), 7.21 (d, J = 8.8 Hz, -NH). – C NMR
gel 60H with petroleum ether-CH Cl2 (1 : 4) to yield
2
0
(
(
(
(
125 MHz, [D ]DMSO ): δ = 14.2 (C-11 and C-14 ), 22.4
9
-hydroxy-5-methoxycanthin-6-one (10, 5.5 mg) and 5-
6
0
0
C-10 and C-13 ), 25.8 (C-3 ), 31.7 – 32.4 (methylenes), 51.9
C-2), 61.1 (C-1), 71.6 (C-5), 71.6 (C-3), 75.1 (C-4), 173.9
C-1 ). – MS ((+)-ESI): m/z = 446 [M+H] . – HRMS ((+)-
methoxycanthin-6-one (11, 7.5 mg). Fractions 16 – 40 were
further chromatographed over silica gel 60H with a mixture
0
+
of CH Cl -aceton (1 : 2) to yield 3-acetyloleanolic acid
2
2
ESI): m/z = 446.3840 (calcd. 446.3845 for C H NO ,
(
10.0 mg), oleanolic acid (12.0 mg) and (2S,3S,4R,5R)-
N-(1,3,4,5-tetrahydroxyundecan-2-yl)tetradecanamide (7,
6.5 mg). Fraction E (10.5 g) was combined from sub-
fractions 131 – 200 and eluted with CH Cl -acetone (4 : 1)
25 52
5
+
[
M+H] ).
1
Fatty acid methyl ester 7a
2
2
and acetone-MeOH (5 : 1).
1
Colorless oil. – H NMR (500 MHz, CHCl ): δ = 3.75
3
(
3H, OMe), 1.67 – 1.80 (m, H-3), 1.27 – 1.24 (methylene
band), 0.87 (t, J = 7.0 Hz, H-14).
Chemical derivatives
Methanolysis of compound 7
Biological activities
Compound 7 (6.5 mg) was added to a mixture of HCl
3.5 mL, N) and dry MeOH (6.0 mL), and refluxed for 16 h
Antimicrobial assays
(
with magnetic stirring. 10.0 mL H O was added, and the
2
The studies on agar diffusion test plates with the bacte-
ria Bacillus subtilis and Escherichia coli (on peptone agar),
Staphylococcus aureus (Bacto nutrient agar) and the fungi
Mucor miehei and Candida albicans (Sabouraud agar) as
test strains were performed as previously described [21].
For the plant pathogen oomycetes Aphanomyces cochlioides,
Pythium ultimum and Rhizoctonia solani, squares of
refluxed mixture was extracted with n-hexane (3 × 10 mL).
The fatty acid methyl ester (7a, 2.0 mg) was obtained after
purification of the n-hexane extract over a silica gel column
with n-hexane-CH Cl (9 : 1) as solvent. The MeOH/H O
2
2
2
phase was evaporated under reduced pressure.
2E,24R-Stigmast-22-ene-3,6,11-trione (1)
Colorless powder (CHCl ); m. p. 180 – 183 C; R = 0.75,
2
0
.5 × 0.5 cm were cut with a microbiological hook from the
◦
3
f
growth margins of mycelial mats grown on PDA plates, inoc-
ulated onto the centers of fresh plates and cultivated for 24 h
at 28 C to initiate radial growth.
silica gel 60 F254, petroleum ether-CH Cl2 (6 : 1). – IR
2
−1
1
(
KBr): ν = 3005, 1700, 1640, 1300, 1045 cm . – H NMR
and C NMR data, see Table 1. – MS ((+)-EI): m/z(%) =
40 (8) [M] , 410 (30), 260 (15), 257 (40), 177 (25), 124
◦
1
3
The compounds were dissolved in CH Cl -MeOH (9 :
2
2
+
4
1
), and paper disks (∅ 9 mm) were each impregnated
(
4
45), 109 (31), 69 (75), 55 (68). – HRMS ((+)-EI): m/z =
with 40.0 µg, dried for 1 h under sterile conditions and ar-
ranged evenly on the pre-made agar test plates, wheras
for oomycetes, the disks were placed around the mycelial
+
40.3249 (calcd. for C H O , 440.3290, [M] ).
29 44 3
2
2E,24R-3-Acetylstigmasta-5,22-diene-7,11-dione (2)
squares at a distance of 30 mm. Bacteria and fungi plates
◦
◦
Colorless powder (CHCl ); m. p. 198 – 200 C; R = 0.75, were kept in an incubator at 37 C for 15 h, oomycetes at
3
f
◦
silica gel 60 F254, petroleum ether-CH Cl2 (6 : 1). – UV 28 C for 48 h. The diameter of inhibition zones (in mm)
2
(
3
MeOH): λmax(logε) = 245(3.45)nm. – IR (KBr): ν = was measured directly or calculated from the radius. Nystatin
−
1
1
350, 1735, 1655, 1560, 1300, 1062, 845 cm . – H NMR (Maneesh Pharmaceutic PVT. Ltd., Govandi, Mumbai, 400
1
3
and C NMR data, see Table 1. – MS ((+)-EI): m/z(%) = 043, India) was used as positive control for fungi and gen-
4
+
82 (20) [M] , 439 (15), 302 (18), 300 (36), 287 (38), 245 tamycin (Jinling Pharmaceutic (Group) corp., Zhejang Tieng
(
44), 194 (33), 151 (25), 124 (34), 55 (100). – HRMS ((+)- Feng Pharmaceutic Factory, No. 11 Chezhan Road, Huzhou
+
EI): m/z = 482.3390 (calcd. 482.3396 for C H O , [M] ). City, Zhejang, China) for bacteria.
46
31 3
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9
30
E. N. Happi et al. · Steroids and Ceramide from Odyendyea gabonensis
Microdilution broth assay
185) by the MTT method according to a reported proto-
col [23]. Freshly trypsinized cell suspensions were seeded
The MICs of compounds were measured by the microdi-
lution broth susceptibility assay [22]. The inocula of bacterial
and fungal strains were prepared from 12 h broth cultures,
and suspensions were adjusted to 0.5 McFarland standard
turbidity. The compounds were dissolved in 10% DMSO and
serial two-fold diluted in 96-well microtiter plates in dupli-
cate, using BHI broth for bacterial and sabouraud dextrose
broth for fungal tests. Standardized inocula of test strains
4
into 96-well microtiter plates at densities of 1 × 10 cells
per well, and test compounds were added from DMSO-
diluted stock. After 3 days, the attached cells were incu-
bated with MTT and subsequently solubilized in DMSO.
The absorbance at 550 nm was measured by using a mi-
croplate reader. The IC50 is the concentration of agent that
reduced cell growth under experimental conditions to 50%,
with doxorubicin as positive control (IC50 1.5 µm).
◦
were added and incubated at 37 C for 24 h on a rotary shaker
at 200 rpm. The MIC is read as the lowest concentration that Acknowledgement
inhibits test strain growth.
We wish to acknowledge the Alexander von Humboldt
AvH) Foundation for postdoctoral fellowships to J. D.
Wansi. We also thank the International Foundation for
(
Cytotoxicity assay
Cytotoxic activities of the compounds were evaluated Sciences (IFS, Sweden) for providing financial support
against the human lung carcinoma cell line A 549 (#CCL- (F/3978-2).
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