Ceramide and cerebroside from the stem bark of Ficus mucuso (Moraceae)

Article abstract of DOI:10.1248/cpb.58.1661

Two new sphingolipids mucusamide (1) and mucusoside (2) have been isolated from methanol soluble part of the stem bark of Ficus mucuso WELW., together with fifteen known secondary metabolites including cellobiosylsterol (3), β-sitosterol (4), stigmasterol (5), β-sitosterol 3-O-β-D- glucopyranoside (6), lupeol acetate (7), ursolic acid (8), procatechuic acid (9), 2-methyl-5,7-dihydroxychromone 8-C-β-D-glucoside (10), apigenin (11), (-)-epicatechin (12), (+)-catechin (13), N-benzoyl-L-phenylalanilol (14), α-acetylamino-phenylpropyl α-benzoylaminophenylpropionate (15), asperphenamate (16) and bejaminamide (17). Structures of compounds 1 and 2 were elucidated by spectroscopic analysis and chemical methods.

Full text of DOI:10.1248/cpb.58.1661

December 2010
Note
Chem. Pharm. Bull. 58(12) 1661—1665 (2010)
1661
Ceramide and Cerebroside from the Stem Bark of Ficus mucuso
Moraceae)
(
a,e
b,e
c
Jean Jules Kezetas BANKEU, Sufyan Awad Alkarim MUSTAFA, Anar Sahib GOJAYEV,
d
a
,a
b
Bruno Djakou LENTA, Didérot TCHAMO NOUNGOUÉ, Silvère Augustin NGOUELA,* Khalid ASAAD,
Mohammed Iqbal CHOUDHARY, Sean PRIGGE, Akif Alekper GULIYEV,
Augustin Ephrem NKENGFACK, Etienne TSAMO,* and Muhammad SHAIQ ALI
e
f
c
a
,a
e
a
Department of Organic Chemistry, Faculty of Science, TWAS Research Unit of University of Yaounde I; P. O. Box 812,
b
Yaounde, Cameroon: Medical Biochemistry Research Unit, Medicinal and Aromatic Plants Research Institute, National
Center for Research; P. O. Box 2404. Khartoum, Sudan: Biochemistry and Biotechnology Department, Biology Faculty,
Baku State University; Z. Khalilov Street 23, AZ 1148, Baku, Azerbaijan: Higher Teacher’s Training College, University
of Yaounde I; P. O. Box 47, Yaounde, Cameroon: International Center for Chemical and Biological Sciences, University
c
d
e
f
of Karachi; Karachi 75270, Pakistan: and Department of Microbiology and Molecular Immunology, The Bloomberg
School of Public Health, Johns Hopkins University; 615 N. Wolfe Street E4628, Baltimore, MD 21205, U.S.A.
Received July 30, 2010; accepted September 20, 2010; published online September 29, 2010
Two new sphingolipids mucusamide (1) and mucusoside (2) have been isolated from methanol soluble part
of the stem bark of Ficus mucuso WELW., together with fifteen known secondary metabolites including cellobio-
sylsterol (3), b-sitosterol (4), stigmasterol (5), b-sitosterol 3-O-b-D-glucopyranoside (6), lupeol acetate (7), ursolic
acid (8), procatechuic acid (9), 2-methyl-5,7-dihydroxychromone 8-C-b-D-glucoside (10), apigenin (11), (؊)-epi-
catechin (12), (؉)-catechin (13), N-benzoyl-L-phenylalanilol (14), a-acetylamino-phenylpropyl a-benzoylamino-
phenylpropionate (15), asperphenamate (16) and bejaminamide (17). Structures of compounds 1 and 2 were
elucidated by spectroscopic analysis and chemical methods.
Key words Ficus mucuso; Moraceae; sphingolipid; mucusamide; mucusoside
1
7)
18)
18)
Ficus is a genus of the family Moraceae that comprises apigenin (11), (Ϫ)-epicatechin (12), (ϩ)-catechin (13),
1)
19)
about 150 species distributed worldwide. Most of the mem- N-benzoyl-L-phenylalanilol (14),
a-acetylamino-phenyl-
10)
bers of the family are very high trees, shrubs and rarely herbs propyl a-benzoylamino-phenylpropionate (15), asperphe-
1)
19)
20)
often with milky juice. A number of Ficus species are used namate (16) and benjaminamide (17) (Fig. 1).
as food and medicines in Traditional Chinese Medicine Mucusamide (1) was isolated as a colourless powder
TCM). Their uses, however, originated and are most widely mp 125—126 °C. Its molecular formula was assigned as
(
2)
found in the Middle East. Ficus species are widely used in C H NO with the aid of its High Resolution (HR)-FAB-MS
traditional medicine in Cameroon too. A mixture of pow- which showed the pseudo-molecular ion peak [MϩH]
dered leaves of F. mucuso and palm oil are used in the treat- at m/z 682.5931 (Calcd, 682.5986 for C H NO ), contain-
4
1
79
6
ϩ
4
1
80
6
ment of epilepsy in Fongo Tongo, a village of the Western ing three degrees of unsaturation. The IR spectrum showed
3)
Region of Cameroon. A decoction of stem bark is used absorption bands for hydroxyl and amide functions (3200—
Ϫ1
Ϫ1
in the East Region of Cameroon by Baka pygmies to treat 3500 cm ) and an amide carbonyl group (1660 cm ).
4)
1
jaundice. Plants of the genus Ficus are known as sources The H-NMR spectrum of 1 (Table 1) showed signals for
of biologically active compounds such as 3-keto-urs-12-ene, an amide proton at d 8.57 (d, Jϭ9 Hz), two terminal methyls
a-amyrin, a-amyrin acetate, b-amiryn, b-amiryn acetate, at d 0.84 (6H, t, Jϭ7.2 Hz) and an oxygenated methylene at
boswellonate, b-sitosterol, b-sitosterone, stigmasterol, lupeol d 4.49 (1H, dd, Jϭ4.8, 11.1 Hz, H-1a) and 4.41 (1H, dd,
and lupenone. In the previous study on the figs of F. mu- Jϭ4.8, 11.1 Hz, H-1b), four carbinylic protons at d 4.27 (1H,
cuso, we reported the isolation of isoderrone and alpinu- m), 4.29 (1H, overlapped dd), 4.33 (1H, overlapped dd)
5,6)
7)
misoflavone as its major constituents. In continuation of our and 4.61 (1H, br dd), and a downfield signal at d 5.18 (1H,
21)
1
investigation for the search of bioactive constituents of F. m) assigned to H-2 of sphingosine. Also, the H-NMR
mucuso, we examined the methanolic extract of the stem bark.
In this paper we report the isolation and the characteriza-
tion of two new sphingolipids mucusamide (1) and mucuso-
side (2).
Results and Discussion
Repeated column chromatography of the MeOH extract of
the stem bark of F. mucuso, followed by purification on silica
and sephadex column, afforded seventeen compounds, in-
8
,9)
10)
cluding cellobiosylsterol (3), b-sitosterol (4), stigmas-
11)
12)
terol (5), b-sitosterol 3-O-b-D-glucopyranoside (6), lu-
peol acetate (7), ursolic acid (8), procatechuic acid (9),
-methyl-5,7-dihydroxychromone 8-C-b-D-glucoside (10),
13)
14)
15)
16)
2
Fig. 1. Structures of Compounds 1—3
∗
To whom correspondence should be addressed. e-mail: tsamo@cm.refer.org; sngouela@yahoo.fr © 2010 Pharmaceutical Society of Japan

1
662
Vol. 58, No. 12
1
13
Table 1. H- and C-NMR Data of 1 and 2 in Pyridine-d (d in ppm, J in Hz)
5
1
2
Position
dC
dH (multi., J Hz)
dC
dH (multi., J in Hz)
NH
—
8.57 (d, 9)
4.49(dd, 11.1, 4.8)
4.41(dd, 11.1, 4.8)
5.18 (m)
4.33 (overlapped dd)
4.29 (overlapped dd)
4.27 (m)
—
—
8.55 (d, 9)
4.69 (dd, 9.9, 6.6 )
4.49 (dd, 9.9, 6.6 )
5.27 (m)
4.44 (overlapped dd)
4.18 (overlapped dd)
2.27 (m)
1.90 (m)
1.90 (m)
—
1
a
62.0 (CH2)
70.3 (CH2)
1
2
3
4
5
b
53.0 (CH)
76.9 (CH)
73.0 (CH)
72.9 (CH)
—
51.7 (CH)
75.9 (CH)
72.5 (CH)
33.9 (CH2)
a
5b
6
a
34.0 (CH2)
2.19 (m)
2.02 (m)
—
26.6
—
29.6—33.9
—
—
—
—
27.5
130.2
130.4
27.9
6b
–
CH (7—10)
—
130.2
130.8
32.1
1.29 (m)
—
2
7
8
9
5.51 (m)
5.48 (m)
2.00 (m)
2.00 (m)
5.48 (m)
5.51 (m)
2.22 (m)
—
—
—
—
10
11
12
13
14
32.1
130.4
130.7
33.3
2.04 (m)
5.45 (m)
5.48 (m)
2.20 (m)
—
—
1
1
4—16
5—17
22.9—33.0
—
1.22—1.29 (m)
—
—
22.9—33.0
1.22—1.29 (m)
1
1
7
8
1
2
3
3
14.3
—
175.2
72.5
35.7
0.84 (t, 7.2)
—
14.3
175.7
72.4
0.84 (t, 7.2)
Ј
Ј
Јa
Јb
Ј
—
4.61 (br dd)
2.20 (m)
1.99 (m)
1.29 (m)
1.22—1.29 (m)
0.84 (t, 7.2)
7.61 (br s)
6.71 (br s)
6.28 (br s)
6.71 (br s)
—
4.55 (br dd)
2.17 (m)
35.5
1.98 (m)
4
26.8
22.9—33.0
26.7
22.9—33.0
14.2
—
1.90 (m)
5
Ј—23Ј
4Ј
1.22—1.29 (m)
0.84 (t, 7.2)
7.65 (br s)
2
14.3
—
—
—
—
—
—
—
—
—
—
—
—
OH-2Ј
OH-3
OH-4
OH-5
Glucose
—
6.84 (br s)
—
6.08 (br s)
—
—
—
—
1
2
3
4
5
Љ
Љ
Љ
Љ
Љ
—
—
—
—
—
—
—
105.4
75.1
78.4
71.5
78.5
62.6
4.92 (d, 7.8)
3.98 (dd, 7.8, 7.5)
4.15 (overlapped dd)
4.18 (overlapped dd)
3.85 (m)
6
6
Љa
Љb
4.45 (dd, 10.8, 4.8)
4.30 (dd, 11.4, 4.8)
spectrum showed four olefinic protons at d 5.48 (2H, m) and
.51 (2H, m), in addition to signals of two alkyl long chains
5
22)
13
appearing as multiplets at d 1.22—1.29. The C-NMR
spectrum of 1 (Table 1) showed characteristic carbon signals
for an amide CϭO group at d 175.2, a methine linked
to the amide N-atom at d 53.0 and four carbinylic methines
1
3
at d 76.9, 73.0, 72.9 and 72.5. The C-NMR spectrum also
displayed four olefinic methine carbons at d 130.8, 130.7,
1
30.4 and 130.2, suggesting the presence of two double Fig. 2. Some COSY and HMBC Connectivities in 1
bonds in 1. A downfield signal due to an oxymethylene (C-1)
appeared at d 62.0. The spectral data and the molecular respectively. The C–H proton at 4.29 also correlated with C-
2
2)
formula suggested that 1 have a sphingolipid type skeleton.
3 (d 76.9) and C-5 (d 72.9), suggesting the location of four
1
1
Analysis of the H– H correlation spectroscopy (COSY), het- OH groups at C-1, C-3, C-4 and C-5. This was further con-
eronuclear multiple quantum coherence (HMQC) and het- firmed by the COSY spectrum (Fig. 2), which showed corre-
eronuclear multiple bond connectivity (HMBC) spectra (Fig. lations from the N–CH group (d 5.18) to CH OH (H-1a, H-
2
2) led to the assignments of proton and carbon signals for 1. 1b) and an oxygenated C–H group at d 4.33 (H-3). The for-
Methylene protons at d 4.41 (H-1b) and 4.49 (H-1a) showed mer CH group (d 4.33, H-3) also correlated to another oxy-
connectivities with the methine at d 53.0 (C-2) which in turn genated C–H group at d 4.29 (H-4), which in turn correlated
was connected to N–H and C–H protons at d 8.57 and 4.33, with the oxygenated C–H group at d 4.27 (H-5). The fifth

December 2010
1663
Fig. 3. Important Fragmentations of the DMDS Derivative of 1
Fig. 5. Methanolysis of 2
ϩ
Fig. 4. Some COSY and HMBC Connectivities in 2
(
HR-FAB-MS), showing [MϩH] ion peak at m/z 844.6849
(
Calcd 844.6877 for C H NO ) with three degrees of un-
4
8
94
10
OH group was located at C-2Ј, which was proved by the saturation. The IR spectrum showed absorption bands for hy-
Ϫ1
Ϫ1
HMBC correlations from the C-H group at d 4.61 (br dd, H- droxyl functions (3437 cm ), an amide group (1670 cm )
Ϫ1
1
13
2
Ј) and the N–H group at d 8.57 to the CϭO of the amide and an olefinic moiety (1643 cm ). The H- and C-NMR
group (d 175.2). One of the olefinic protons at d 5.51 (m, H- spectra were typical for a sphingosine type skeleton like in 1
7
2
) showed correlations for two methylene protons each at d with a sugar unit, and possessing 2-hydroxy fatty acid and a
.02 (H-6b) and 2.19 (H-6a) and the other olefinic proton at b-glucopyranosyl moiety (Table 1).
d 5.48 (H-8). These two methylene protons were inter-corre-
Assignments of all protons and carbons in 2 could be
1
1
lated and correlated to a proton at d 4.27 (H-5), thus made by H– H COSY, HMBC and HSQC experiments (Fig.
indicating the presence of –CH(OH)–CH –CHϭCH– moiety. 4). The long range correlations allowed in clearly deducing
2
The fatty acid and sphingosine chain lengths of 1 were the presence of a sphingosine-type containing a sugar unit.
determined through methanolysis. When 1 was methanolysed The fatty acid linked to C-2 (d 51.7) of the sphingosine moi-
with methanolic hydrochloric acid, a fatty acid methyl ety was confirmed by the correlation between the NH proton
ester (FAME) was obtained together with a long-chain base and C-2. HMBC correlations of the carbonyl at d 175.7 with
(
LCB). Gas chromatography-mass spectrometry (GC-MS) the proton at d 4.55 (H-2Ј) which in turn correlated with C-
analysis of the FAME characterized methyl 2(R)-2-hydroxy- 3Ј (d 35.5), confirmed the presence of an a-hydroxy fatty
2
0
tetracosanoate {electron ionization (EI): m/z 398, [a]
Ϫ
acid side chain.
D
23)
3
.25° (in CHCl )}, and confirming the existence of two
When cerebroside 2 was treated with methanolic hy-
3
double bonds on the LCB moiety. The positions of double drochloric acid, a FAME was obtained together with LCB
bonds in the LCB part were determinated by the characteris- and methylglucopyranoside (Fig. 5). Gas chromatography-
tic mass of dimethyldisulfide (DMDS) derivative {[EI: m/z mass spectrometry (GC-MS) analysis of the FAME showed
ϩ
ϩ
20
D
1
31, [MeSCHC H ] and m/z 223, [C H NO S] } (Fig. 3). methyl 2(R)-2-hydroxytetracosanoate {EI: m/z 398, [a]
5
11
8
17
4
2
3)
The above characteristic fragments ion peaks obtained after Ϫ3.25° (in CHCl )}, and confirming the existence of a dou-
3
the cleavage of the disulfide bond, indicated that the two ble bond in the LCB moiety. The EI-MS of the dimethyldi-
double bonds in 1 were located at C-7 and C-11 (Fig. 4). sulfide (DMDS) derivative of 2 showed a characteristic frag-
ϩ
The geometry of the two double bonds was determined to be ment ion peak at m/z 131 [MeSCHC H ] due to the cleav-
5
11
trans as evidenced by the chemical shift of C-6 (34.0), age between the carbons bearing the methylthio groups.
C-9 (32.1), C-10 (32.1) and C-13 (33.3). Typically, the sig- Therefore, the double bond in LCB moiety of 2 was located
nals of a carbon next to a trans double bond appear at dϷ32, at C-12. Moreover, the cis geometry (Z) of the double bond
24)
while those of a cis double bond appear at dϷ27. The was determined from d values 27.5 (C-11) and 27.9 (C-14)
relative and absolute stereochemistry at the chiral centers C-2 of the allylic carbons.
25)
to C-5 has already been established in similar molecules.
The absolute configuration of the glucopyranosyl moiety
Based on the biogenetic considerations, the absolute stereo- was determined as b-D-form by comparing the sign of the
chemistry of C-2, C-3, C-4 and C-5 in 1 was then proposed optical rotation and spectral data of methyl-b-D-glucopyra-
1
3
as 2S, 3S, 4R, 5R. This was confirmed by C-NMR data of noside obtained from 2 with those of an authentic sam-
2
6,27)
C-2 (53.0), C-3 (76.9), C-4 (73.0) and C-5 (72.9) which were ple.
The relative stereochemistry of 2 at C-2, C-3, C-4
in agreement with those of nubenamide reported in the litera- was proposed as 2S, 3S, 4R, identical to that of D-sphingosine
2
5)
13
ture. Based on these evidences, mucusamide (1) could be on the basis of C-NMR spectral data, since the chemical
assigned the structure (2S,3S,4R,5R,7E,11E)-2-{[(2R)-2- shifts of C-2 (51.7), C-3 (75.9) and C-4 (72.5) were in agree-
28)
hydroxytetracosanoyl]amino}heptadeca-7,11-diene-1,3,4,5- ment with those reported in the literature. Therefore, the
tetrol.
Mucusoside (2) was isolated as a colourless powder, mp (2S,3S,4R,12Z)-2-{[(2R)-2-hydroxytetracosanoyl]amino}oc-
structure of 2 was elucidated as 1-O-(b-D-glucopyranosyl)-
2
09—210 °C. The molecular formula C H NO was estab- tadec-12-ene-1,3,4-triol.
4
8
93
10
lished by the fast-atom-bombardment mass spectrometry
Compound 3, sitosterol 3-O-[b-D-glucopyranosyl-1(1→3)-

1
664
Vol. 58, No. 12
1
13
Table 2. H- and C-NMR Data of 3 in CD ODϩCDCl
the solvent signals (in the case of mixture of CD ODϩCDCl , some drops
3
3
3
3
of CDCl were added to complete the dissolution of the sample and the ref-
3
3
erence was CD OD signal). EI-MS spectra were obtained on Varian MAT
3
Position
311A mass spectrometer. FAB-MS spectra were measured on a JOEL-HX
110 mass spectrometer. Column chromatography was performed on silica
gel. Fractions were monitored by TLC using Merck pre-coated silica gel
sheets (60 F254), and spots were visualized by heating silica gel plates
sprayed with ceric sulfate reagent solution.
Plant Material The stem bark of F. mucuso was collected in March,
2005 from Ngoa-Ekelle, Yaounde, Central Region of Cameroon and identi-
fied by Mr. Nana Victor, botanist at the National Herbarium, where the
voucher specimen is deposited (HN no. 41204).
Extraction and Isolation The air-dried and powdered stem bark
(5.5 kg) of F. mucuso was extracted with MeOH (15 l) (1 week, repeated
three times) at room temperature. The extract was then concentrated to dry-
ness under vacuum at 40 °C to give 290 g of dark brownish crude extract.
The extract was subjected to medium pressure liquid column chromatogra-
phy over silica gel eluting with n-hexane, n-hexane/EtOAc, EtOAc and
EtOAc/MeOH, in increasing order of polarity. One hundred and fifty sub-
fractions, each containing 500 ml, were collected and combined according to
their TLC profiles on pre-coated silica gel 60 F254 plates developed with
dC
dH (multi., J Hz)
1
2
3
4
38.6 (CH2)
30.7 (CH2)
79.8 (CH)
39.7 (CH2)
1.97 (m)
1.28 (m)
3.60 (m)
2.24 (m)
2.42 (m)
—
5.36 (m)
1.47 (m)
1.98 (m)
1.51 (m)
0.95 (m)
—
a
4
5
6
b
141.9 (C)
122.9 (CH)
33.1 (CH2)
7
a
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
b
33.3 (CH)
51.7 (CH)
37.9 (C)
22.2 (CH2)
41.1 (CH2)
43.5 (C)
58.2 (CH)
25.3 (CH2)
29.4 (CH2)
57.4 (CH)
12.3 (CH3)
19.9 (CH3)
37.5 (CH)
19.3 (CH3)
35.1(CH2)
1
1
1
1
1
1
1
1
1
1
2
2
1.50 (m)
2.01 (m)
—
1.02 (m)
1.61 (m)
1.97 (m)
1.12 (m)
0.87 (s)
0.99 (s)
1.37 (m)
0.78 (d, 8)
1.03 (m)
1.38 (m)
1.19 (m)
0.94 (m)
1.68 (m)
0.77 (d, 8)
0.88 (d, 6)
1.30 (m)
0.72 (d, 8)
—
n-hexane/EtOAc and CHCl /MeOH mixture to give 6 fractions (F1—6).
3
Fraction F1 was subjected to CC over silica gel, eluted with n-hexane/EtOAc
mixture starting from 100% n-hexane to 50% of the mixture. This resulted
in the isolation of lupeol acetate (7) (2.05 g), b-sitosterol (4) (115 mg), stig-
masterol (5) (8 mg) and ursolic acid (8) (10 mg). Fraction 2 was also sub-
jected to successive CC using the same quality of silica and eluted with a
mixture of EtOAc and n-hexane (1 : 9—100% EtOAc) to give: asperphena-
mate (16) (14 mg) and a-acetylamino-phenylpropyl a-benzoylamino-
phenylpropionate (15) (3 mg), N-benzoyl-L-phenylalanilol (14) (5 mg).
Fraction 3 was eluted with the same mixture with different polarity (3 : 7
to 100% EtOAc) to give apigenin (11) (4.5 mg). Fraction 4 was eluted with
CHCl /MeOH of increasing polarity (100% CHCl to 1 : 9) to yield sitos-
22a
22b
2
2
3
4
5
6
7
8
9
27.1 (CH2)
47.3 (CH)
30.3 (CH)
20.2 (CH3)
19.4 (CH3)
24.1 (CH2)
12.4 (CH3)
—
3
3
2
2
2
2
2
terol 3-O-b-D-glucopyranoside (6) (120 mg), (Ϫ)-epicatechin (12) (10 mg),
(ϩ)-catechin (13) (3 mg), benjaminamide (17) (10 mg) and mucusamide (1)
(21 mg). Fraction 5 was also successively eluted with CHCl /MeOH mix-
3
tures to yield procatechuic acid (9) (31 mg), 2-methyl-5,7-dihydroxy-
chromone 8-C-b-D-glucoside (10) (22 mg) and mucusoside (2) (17 mg), re-
Glucose-I
spectively. Fraction 6 was also eluted with CHCl /MeOH mixtures to yield
3
1
2
3
4
5
Ј
Ј
Ј
Ј
Ј
102.0 (CH)
73.8 (CH)
88.0 (CH)
70.0 (CH)
78.2 (CH)
64.7 (CH2)
4.42 (d, 8)
3.55 (m)
3.53 (m)
3.38 (m)
3.30 (m)
3.58 (m)
3.72 (m)
—
4.54 (d, 7.2)
3.33 (m)
3.24 (m)
3.25 (m)
3.26 (m)
3.64 (m)
3.86 (m)
sitosterol 3-O-[b-D-glucopyranosyl-(1→3)-b-D-glucopyranoside] (3) (5 mg).
Mucusamide, (2S,3S,4R,5R,7E,11E)-2-{[(2R)-2-Hydroxytetracosanoyl]-
amino}heptadeca-7,11-diene-1,3,4,5-tetrol (1): Colourless powder, mp 125—
3
0
Ϫ1
126 °C, [a]D ϩ29° (cϭ0.11, pyridine), IR (KBr) nmax cm : 3200—3500
(OH and NH), 1660 (NH–CϭO), m/z: 682.5931 (Calcd for C H NO :
4
1
80
6
ϩ
1
13
6
Јa
Јb
Glucose-II
682.5986, [MϩH] ), H- (300 MHz, pyridine-d ) and C- (100 MHz, pyri-
5
6
dine-d ) NMR data: see Table 1.
5
—
Mucusoside, 1-O-(b-
D-Glucopyranosyl)-(2S,3S,4R,12Z)-2-{[(2R)-2-hy-
1
2
3
4
5
Љ
Љ
Љ
Љ
Љ
105.3 (CH)
74.5 (CH)
75.5 (CH)
71.6 (CH)
77.5 (CH)
62.6 (CH2)
droxytetracosanoyl]amino}octadec-12-ene-1,3,4-triol (2): Colourless pow-
3
0
Ϫ1
der, mp 209—210 °C, [a] ϩ34° (cϭ0.08, pyridine), IR (KBr) nmax cm
3437 (OH), 1670 (NH–CϭO), 1643 (CϭC), m/z: 844.6849 (Calcd for
:
D
ϩ
1
13
C H NO : 844.6878, [MϩH] ), H- (300 MHz, pyridine-d ) and C- (125
4
8
94
10
5
MHz, pyridine-d ) NMR data: see Table 1.
5
6
Љa
Sitosterol 3-O-[b-D-Glucopyranosyl-1(1→3)-b-D-glucopyranoside] (3):
30
6
Љb
Colourless amorphous powder, [a] ϩ63° (cϭ0.03, pyridine), IR (KBr)
D
Ϫ1
n
cm : 3400 (OH), 1480 (CϭC) and 1084 (C–O–C), m/z: 738.4920
max
1
13
(
Calcd for C H O : 738.4918), H- (400 MHz, CD ODϩCDCl ) and C-
41 70 11 3 3
(
150 MHz, CD ODϩCDCl ) NMR data: see Table 2.
3
3
b-D-glucopyranoside] (cellobiosylsterol), was previously iso-
lated from the rice bran as a colourless amorphous powder.
Previous determination of its structure was based on chemi-
cal transformations and mass spectrometry. We reported here
Methanolysis of 1 and 2 Compounds 1 and 2 (1 mg each) were heated
separately with 5% HCl in MeOH (1 ml) at 70 °C for 12 h in a sealed small-
volume vial. The reaction mixture of each compound was extracted with
n-hexane and the hexane layer was concentrated to give FAME for GC-MS
analysis. The MeOH layer was concentrated in vacuo to give LCB-1 part for
1
13
for the first time its H- and C-NMR data, and signal 1 and the mixture LCB-2 part and methyl glycoside for 2.
assignments of 3 based on the COSY, HSQC and HMBC
connectivities (Table 2).
To determine the nature of LCB and the methyl glycoside, the process
was repeated from 10 mg of 1 and 2. The MeOH layer from the methanoly-
sis of 1 was suspended in H O and LCB-1 (3.4 mg, 73.5%) was extracted
2
ϩ
30
D
Experimental
with EtOAc. LCB-1: EI-MS m/z: 315 [M] , [a] ϩ28.7° (cϭ0.05, MeOH),
H-NMR (pyridine-d , 500 MHz) d: 7.81 (2H, d, Jϭ9 Hz, NH ), 5.44 (4H,
1
General Experimental Procedure Melting points were obtained on a
5
2
Gallenkamp melting point apparatus. Optical rotations were measured with a m, H-7, H-8, H-11, H-12), 5.16 (1H, m, H-2), 4.46 (1H, dd, Jϭ10.2, 3.9 Hz,
JASCO DIP-360 polarimeter. UV spectra were recorded on a Hitachi UV H-1b), 4.42 (1H, dd, Jϭ10.2, 4.3 Hz, H-1a), 4.35 (1H, m, H-3), 4.31 (1H, m,
3200 spectrophotometer. A JASCO 320-A spectrophotometer was used for H-4), 4.25 (1H, m, H-5), 2.20 (2H, m, H-13), 2.17 (1H, m, H-6a), 2.00 (1H,
scanning IR spectroscopy using KBr pellets. 1D- and 2D-NMR spectra were m, H-6b), 1.90 (4H, m, H-9, H-10), 1.25 (6H, br s), 0.86 (3H, t, Jϭ8.4 Hz,
run on Bruker spectrometer operating 75, 100, 125, 150, 300, 400, 500 and CH -17).
3
600 MHz where chemical shifts (d) were expressed in ppm with reference to
The aqueous methanolic layer from 2 was evaporated to dryness and the

December 2010
1665
residue separated by TLC as sphingosine base and methylated sugar. LCB-2
Planta Med., 68, 186—188 (2002).
ϩ
30
(
2.9 mg, 77.6%) EI-MS m/z: 315 [M] , [a] ϩ11.5° (cϭ0.09, CHCl3/
6) Djemgou P. C., Ngandeu F., Hegazy M. E., Nkanwen E. R., Neguim
G., Chosson E., Verité P., Tane P., Pharmacogn. Res., 1, 197—201
(2009).
D
1
CH OH); H-NMR (CDCl , 500 MHz) d: 7.02 (2H, d, Jϭ9 Hz, NH ), 5.32
3
3
2
(2H, m, H-12, H-13), 5.10 (1H, overlapped m, H-2), 4.18 (1H, dd, Jϭ4.3,
7
.4 Hz, H-1a), 3.78 (3H, m, H-1b, H-3, H-4), 2.31—2.04 (2H, m), 1.80 (1H,
7) Kezetas J. J. B., Lenta N. B., Ngouela S., Tsamo E., Sewald N., Neu-
mann B., Stammler H. G., Acta Crystallogr., Sect. E: Struct. Rep. On-
line, 63, 523—525 (2007).
8) Ohnishi M., Fujino Y., Agric. Biol. Chem., 42, 2423—2425 (1978).
9) Fujino Y., Ohnishi M., Biochim. Biophys. Acta, 574, 94—102 (1979).
m), 1.64 (4H, m), 1.25 (16H, br s), 0.88 (3H, t, Jϭ7.0 Hz, CH -18). The
presence of methyl-b-D-glucopyranoside (1.8 mg, 78.2%) was established by
comparison of its NMR data with those reported in the literature
3
26,27)
and
2
8
with an authentic sample, [a]D ϩ74° (cϭ0.1, MeOH), Rf 0.46 (EtOAc/
2
8
MeOH/H O, 5 : 2 : 0.5). Authentic sample [a] ϩ77° (cϭ0.1, MeOH), Rf 10) Xiao Y.-Q., Li L., You X.-L., Bian B.-L., Liang X.-M., Wang Y.-T., J.
2
D
0
.46.
Asian Nat. Prod. Res., 4, 73—79 (2002).
GC-MS Analysis of FAME from Compounds 1 and 2 The FAME 11) De-Eknamkul W., Potduang B., Phytochemistry, 62, 389—398 (2003).
from 1 and 2 were subjected to GC-MS [column temp. 100—250 °C (rate of
12) Nguyena A. T., Malonneb H., Dueza P., Vanhaelen-Fastrea R., Vanhae-
temp. increases 5 °C/min)]. The results were as follows: FAME-1 from 1
lena M., Fontaineb J., Fitoterapia, 75, 500—504 (2004).
ϩ
(
methyl 2-hydroxytetracosanoate), t ϭ40.62 (29), EI-MS m/z: 398 [M] , 13) Argay G., Kalman A., Kapor A., Ribar B., Petrovic S., Gorunovic M.,
R
ϩ
339 [MϪ59] . The FAME-2 from 2 gave the same fragments like the previ-
J. Mol. Struct., 435, 169—179 (1997).
ous one.
DMDS Derivatives of 1 and 2 Compounds 1 and 2 (1.0 mg each) were
14) Huang P.-L., Wang L. W., Lin C. N., J. Nat. Prod., 62, 891—892
(1999).
dissolved separately in carbon disulfide (0.2 ml) and iodine (1 mg) was 15) Koorbanally N. A., Koorbanally C., Harilal A., Mulholland D. A.,
added to the solution of each. The resulting mixtures were stored at 60 °C Crouch N. R., Phytochemistry, 65, 3069—3073 (2004).
for 40 h in two separate small-volume sealed vials. The reaction was subse- 16) Tanaka T., Orii Y., Nonaka G.-I., Nishioka I., Chem. Pharm. Bull., 41,
quently quenched with aqueous Na S O (5%), and mixtures were extracted 1232—1237 (1993).
2
2
3
with n-hexane (0.3 ml). The extracts were concentrated to give the 1 and 2 17) Oyama K., Kondo T., Tetrahedron, 60, 2025—2034 (2004).
ϩ
DMDS derivatives. EI-MS m/z: 131 (3.2) [MeSCHC H ] , 223 (4.0) 18) Jang D. S., Lee G. Y., Lee Y. M., Kim Y. S., Sun H., Kim D.-H., Kim J.
5
11
ϩ
ϩ
ϩ
[
[
C H NO S] , 279 (7.9) [C H S ] , 384 (8.1) [C H O N] , 522 (48.3)
C H O NS] and 540 (38.2) [C H O NS] for DMDS derivatives of 1 19) Clark A. M., Hufford C. D., Lloydia, 40, 146—151 (1977).
and fragments at m/z: 131 (9) [MeSCHC H ] , 163 (3) [C H O ] and 384 20) Simo F. C. C., Kouam F. S., Poumale P. H. M., Simo K. I., Ngadjui T.
8.07) [C H O N] for DMDS derivatives of 2 as characteristic fragments.
S., Chem. Pharm. Bull., 57, 397—400 (2009).
8
17
4
13 27
3
24 50
2
ϩ
ϩ
31
56
3
31 58
4
ϩ
ϩ
5
11
6
11
5
ϩ
(
B., Green I. R., Krohn K., Biochem. Syst. Ecol., 36, 238—243 (2007).
1) Garg H. S., Agrawal S., J. Nat. Prod., 58, 442—445 (1995).
2
4
50
2
2
Acknowledgments Authors are grateful to the Third World Academy of 22) Naheed R., Sarfraz A. N., Naveen M., Abdul M., Nighat A., Samar A.,
Sciences (TWAS) for the award of fellowship to J. J. K. Bankeu to work in Shafi U., Pir M., Choudary M. I., Chem. Biodivers., 4, 72—83 (2007).
the International Center for Chemical and Biological Sciences (ICCBS) and 23) Buckingham J., Dictionary of Natural Products on DVD, Company:
a research grant No. 07-41LDC/CHE/AF/ACUNESCO FR: 3240171776 to
our TWAS Research Unit.
HDS, Version 43.44.8.9, London, U.K., 2009.
24) Cateni F., Zilic J., Falsone G., Scialino G., Bioorg. Med. Chem. Lett.,
1
3, 4345—4350 (2003).
References
25) Ali M. S., Ibrahim S. A., Ahmed S., Lobkovsky E., Z. Naturforsch.:
Chem. Sci., 62b, 1333—1338 (2007).
26) Muralidhar N., Krishna N., Kumar M. M., Rao C. B., Rao D. V., Chem.
Pharm. Bull., 51, 1193—1195 (2003).
27) Jin W., Rinehart K. L., Jares-Erijman E. A., J. Org. Chem., 59, 144—
147 (1994).
28) Yang N.-Y., Ren D.-C., Duan J.-A., Xu X.-H., Xie N., Tian L.-J., Helv.
Chim. Acta, 92, 291—297 (2009).
1
)
Hutchinson J., Dalziel J. M., Keay R. W. J., “Flora of West Tropical
Africa,” Vol. 1, 2nd ed., London Crown Agents, 1958, p. 600.
Alziro de A., Helcio R. B., Jorge P. P. C., Daise L., Maria A. C. K., J.
Ethnopharmacol., 64, 255—258 (1999).
2)
3)
4)
5)
Noumi E., Fozi F. L., Pharm. Biol., 41, 330—339 (2003).
Betti J. L., African Study Monographs, 25, 1—27 (2004).
Peraza-S. S. R., Chai H. B., Shin Y. G., Santisuk T., Reutrakul V.,
Farnsworth N. R., Cordell G. A., Pezzuto J. M., Kinghom A. D.,