Three New Derivatives and Others Constituents from the Roots and Twigs of Trilepisium?madagascariense DC

Article abstract of DOI:10.1002/hlca.201600073

Three new compounds, trilepisflavene (1), trilepisdepsidone (2), and daturadiol stearate (3), together with nine known compounds, 2-hydroxy-4-[(4-hydroxy-2-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoic acid (4), lichexanthone (5), naringenin (6), 3′,4′,5,7-tetrahydroxyflavanone (7), 2-hydroxybenzoic acid (8), methyl 2,4-dihydroxy-6-methylbenzoate (9), β-amyrin (10), eurothridiol palmitate (11), and β-sitosterol (12), were isolated from the AcOEt extract of the twigs and the roots of Trilepisium?madagascariense. Acetylation of eurothridiol palmitate was carried out and a new acetylated derivative (13) was obtained. The structures of the isolated and acetylated compounds were elucidated on the basis of spectroscopic analysis. Antimicrobial activity of all these compounds was evaluated using Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) method. Trilepisdepsidone, 2-hydroxy-4-[(4-hydroxy-2-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoic acid, 3′,4′,5,7-tetrahydroxyflavanone, and naringenin exhibited moderate to weak antimicrobial activity.

Full text of DOI:10.1002/hlca.201600073

Helv. Chim. Acta 2016, 99, 1 – 8
1
201600073
FULL PAPER
Three New Derivatives and Others Constituents from the Roots and Twigs of
Trilepisium madagascariense DC
a
by Yves P. Ango ), Gilbert D. W. F. Kapche* ), Victor Kuete ), Renameditswe Mapitse ), Samuel O. Yeboah ), and
b
c
d
d
a
Bonaventure T. Ngadjui )
a
)
Department of Organic Chemistry, Faculty of Science, University of Yaound ꢀe 1, P.O. Box 812, Yaound ꢀe , Cameroon
Department of Chemistry, Higher Teacher Training College, University of Yaound ꢀe I, P.O. Box 47, Yaound ꢀe , Cameroon
phone: +237 77 66 49 73; fax: +237 22 23 53 96; e-mail: dkapche2002@yahoo.com)
Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon
Department of Chemistry, Faculty of Science, University of Botswana, Block 237, Private Bag, 0022 Gaborone, Botswana
b
d
)
(
c
)
)
Three new compounds, trilepisflavene (1), trilepisdepsidone (2), and daturadiol stearate (3), together with nine known
compounds, 2-hydroxy-4-[(4-hydroxy-2-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoic acid (4), lichexanthone (5), naringenin
0 0
(
6), 3 ,4 ,5,7-tetrahydroxyflavanone (7), 2-hydroxybenzoic acid (8), methyl 2,4-dihydroxy-6-methylbenzoate (9), b-amyrin (10),
eurothridiol palmitate (11), and b-sitosterol (12), were isolated from the AcOEt extract of the twigs and the roots of
Trilepisium madagascariense. Acetylation of eurothridiol palmitate was carried out and a new acetylated derivative (13) was
obtained. The structures of the isolated and acetylated compounds were elucidated on the basis of spectroscopic analysis.
Antimicrobial activity of all these compounds was evaluated using Mueller–Hinton broth (MHB) and Mueller–Hinton agar
0 0
(
MHA) method. Trilepisdepsidone, 2-hydroxy-4-[(4-hydroxy-2-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoic acid, 3 ,4 ,5,7-
tetrahydroxyflavanone, and naringenin exhibited moderate to weak antimicrobial activity.
Keywords: Trilepisium madagascariense, Trilepisflavene, Trilepisdepsidone, Daturadiol stearate, Depsidone, Depside,
Antimicrobial activity.
Introduction
Results and Discussion
Trilepisium (family: Moraceae) previously known as Bos- The air-dried and powdered leafless twigs (500 g) and
queia is a monotypic genus represented by Trilepi-
the dried powdered roots (400 g) of T. madagascariense
were extracted with MeOH/CH Cl 1:1 at room temper-
ature for 48 h and MeOH for 8 h. The combined
extending on to the borders of savanna [1]. T. madagas- extracts (25 g from the twigs) and (15 g from the roots),
cariense is found in Cameroon, Democratic Republic of
were separately subjected to extensive column chro-
Congo, and Madagascar [2]. The leaves of T. madagas- matography to yield a new flavene, trilepisflavene (1), a
sium madagascariense DC, a deciduous tree of up to
2
2
3
0 m height, of the middle storey of the high-forest,
cariense are used as vegetable; other parts of the plant
are traditionally used as pain killers and to treat venereal
diseases, arthritis, rheumatism, diarrhea, dysentery, stom-
ach troubles, malnutrition, debility, and cutaneous and
subcutaneous parasitic infections [1] and this plant was
reported to possess antidiarrheal and bacteriostatic activi-
ties [3]. Our previous investigations on the leaves and
new depsidone, trilepisdepsidone (2), a new triterpene,
daturadiol stearate (3), together with nine known com-
pounds: 2-hydroxy-4-[(4-hydroxy-2-methoxy-6-methylben-
zoyl)oxy]-6-methylbenzoic acid (4) [5], lichexanthone (5)
0
0
[6], naringenin (6) [7], 3 ,4 ,5,7-tetrahydroxyflavanone (7)
0
[8], 2 -hydroxybenzoic acid (8) [9], methyl 2,4-dihydroxy-
6-methylbenzoate (9) [10], b-amyrin (10) [11], eurothrid-
stem barks of T. madagascariense have led to the isola- iol palmitate (11) [12], and b-sitosterol (12) [13]. The
tion and characterization of a new flavan (trilepisflavan)
and a new ester derivative of caffeic acid (trilepisuimic
acid) together with 10 other compounds [4]. In continua-
structures of the isolated compounds are presented in
Fig. 1. This paper describes the isolation and structure
elucidation of the new compounds, as well as the evalua-
tion of our systematic studies of plants from the tion of the antimicrobial activity of some of the pure
Moraceae family, the twigs and the roots of T. madagas- isolated compounds and the acetylated derivative of
cariense have been investigated.
eurothridiol palmitate.
DOI: 10.1002/hlca.201600073
© 2016 Wiley-VHCA AG, Z u€ rich

2
Helv. Chim. Acta 2016, 99, 1 – 8
Fig. 1. Chemical structures of compounds 1 – 13.
Trilepisflavene (1) was obtained as brown oil. Its
molecular formula C H O was deduced on the basis of
HR-EI-MS, from the ion peak at m/z 256.0807 (calc.
Trilepisdepsidone (2) was obtained as a brown amor-
ꢀ
phous powder. The ESI-MS (-ve) showed [M ꢀ H] ion
1
5
12
4
peak at m/z 387.1075 corresponding to the molecular for-
mula C H O , containing 11 double-bond equivalent.
2
3
56.0796). Its IR spectrum showed absorption bands at
ꢀ1
2
0
20
8
ꢀ1
1
249 cm (OH) and 1613 – 1513 cm (aryl absorptions
The H-NMR spectrum displayed two singlets at d(H)
1
13
and overtones). The H and C-NMR data of 1 (Table 1) 6.49 (s, 1 H) and 6.66 (s, 1 H) for aromatic H-atoms. It
showed signals of one CH group at (d(H)/d(C) 3.95 (br.
also showed three singlets of three H-atoms each at d(H)
3.69, 3.92, and 3.97 for three MeO and two other singlets
2
s)/33.6) together with one 1,4-disubstituted (d(H)/d(C)
6
.83 (d, 8.7)/115.3 and 7.15 (d, 8.7)/129.8) and one tetra- for three H-atoms each at d(H) 2.03 and 2.39 for two aro-
1
3
substituted (d(H)/d(C) 6.94 (s)/97.6 and 6.95 (s)/105.0) matic Me groups. The C-NMR spectrum revealed the
benzene rings suggesting, according to the coupling presence of 20 C-atoms of which two were ester C=O
pattern and the chemical shifts, that 1 is a trisubstituted groups (d(C) 167.0 and 168.1), two Me groups (d(C) 8.7
flavene [14]. Further analysis of the C spectral data with
two quaternary C-atom signals at d(C) 141.9.4 and d(C)
1
3
and 20.2), three MeO groups (d(C) 52.2, 55.8, and 56.3),
and one CH signal (d(C) 65.9, DEPT). From HSQC and
2
1
6
43.0 and HMBC between the H-atom signals at d(H) HMBC data (Table 1), the presence of two aromatic Ph
.94 and 6.95 with these C-atoms was consistent with the groups was concluded. HMBC data indicated that the first
0
fact that 1 is the 6-hydroxy derivative of 4 ,7-dihydroxy- Ph group (ring A) contained two Me groups (d(H) 2.03
flav-2-ene [14]. Therefore, 1 was identified as 4 ,6,7-trihy- and 2.39) in correlation with C-atoms at d(C) 117.0
0
droxyflav-2-ene, a new natural product to which the (C(3)), 159.4 (C(4)), 151.5 (C(2)) and C-atoms at d(C)
trivial name trilepisflavene is proposed.
119.1 (C(1)), 109.4 (C(5)), 135.7 (C(6)). This ring also
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Helv. Chim. Acta 2016, 99, 1 – 8
Table 1. H- (300 MHz), C-NMR (75 MHz), and HMBC spectral data of 1 (in acetone) and 2 (in CD
3
1
13
3
OD)
Position
1
2
d(H)
d(C)
HMBC (H ? C)
d(H)
d(C)
HMBC (H ? C)
1
2
3
4
5
6
7
8
9
–
–
–
–
–
–
–
119.1
151.5
117.0
159.4
109.4
135.7
167.6
20.2
149.3
102.6
33.6
105.0
143.0
141.9
97.6
157.1
120.8
128.7
129.8
115.3
156.0
115.3
129.8
–
–
C(2), C(10)
6.26 (d, J = 0.9)
3.95 (br. s)
C(9)
C(6), C(7)
–
6.49 (s, 1 H)
6.95 (s)
–
–
6.94 (s)
C(1), C(3), C(8)
–
–
–
C(6), C(7)
–
2.39 (s, 3 H)
C(1), C(5), C(6)
C(1), C(3), C(4)
C(4)
–
–
–
–
–
–
2.03 (s, 3 H)
3.92 (s, 3 H)
8.7
55.8
1
0
0
1
–
–
–
–
105.8
155.5
136.5
152.9
100.2
132.5
168.1
65.9
0
0
0
0
0
0
0
0
0
0
2
7.15 (d, J = 8.7)
6.83 (d, J = 8.7)
–
6.83 (d, J = 8.7)
7.15 (d, J = 8.7)
–
–
–
–
C(4 ), C(9)
3
4
5
6
7
8
9
0
–
–
0
0
0
0
0
C(2 )
C(4 ), C(5 )
6.66 (s, 1 H)
–
–
4.44 (s, 2 H)
3.97 (s, 3 H)
3.69 (s, 3 H)
C(3 ), C(4 ), C(1 )
0
–
–
–
–
–
–
–
0
56.3
52.2
C(2 )
C(7 )
0
1
contained one MeO group, in correlation with C-atom at
d(C) 159.4 (C(4)). The second Ph group (ring B) was
revealing compound 2 to be a new depsidone, to which
the name trilepisdepsidone is proposed.
Compound 3, obtained as a white amorphous powder
shown to bear only one single H-atom at d(H) 6.66 (H–
0
C(5 )) exhibiting long-range correlations with C-atoms at gave a positive test with Liebermann–Burchard reagent
0
0
0
d(C) 152.9 (C(4 )), 136.5 (C(3 )), 105.8 (C(1 )); one CH
characteristic of triterpenes. Its HR-ESI-MS showed the
+
2
0
group at d(C) 132.5 (C(6 )) and one MeO group at d(C)
155.5 (C(2 )). The presence of another C–O bond was
deduced from the correlation observed for the H-atom at
pseudomolecular ion [M + Na] peak at m/z 731.6286 cor-
responding to the molecular formula C H O . The C-
0
13
4
8
84
3
NMR spectrum (Table 2) of compound 3 showed methine
2
and quaternary C-sp signals (d(C) 121.6 and 145.2) char-
0
d(H) 6.66 (H–C(5 )) with the quaternary C-atom at d(C)
0
1
36.5 (C(3 )). This correlation together with a HMBC cor-
0
acteristic of oleanene triterpenes [22]. Detailed analyses
of this spectrum indicated that the triterpene moiety was
esterified with a fatty acid. This observation was sup-
ported by the presence in this spectrum of peaks of an
additional methyl (d(C) 14.1) and of acyl groups (ca. d(C)
179.9) whose C=O group was downshielded (Dd ca. 3.2)
relative to 3b-acetylamyrin. The C(2) of the triterpene
moiety in this compound was shielded (Dd 4), while the
oxymethine C-atoms showed downshielding effect in com-
0
relation between H–C(5 ) and C(1 ), and between H–
0
0
C(10 ) and C(7 ) led to the complete deduction of the
substitution pattern of ring B. Out of the all possible iso-
mers with permutation of the substituents in ring B, only
two types have been found in nature: the four angular
depsidones with the skeleton of deoxystictic acid [15]
were easily excluded on the basis of the HMBC pattern
and chemical shifts. Our data for ring B were in agree-
ment with excelsione isolated from Phomopsis sp. parison to C(3) of b-amyrin [22]. In addition, the C(3) sig-
CAFT69 [16]. It should be mentioned that two different nal appeared at higher frequency (d(C) 81.4) than that
0
0
chemical shift assignments for C-atoms C(2 ) and C(3 ) observed for b-amyrin (d(C) 78.9). The molecular ion
have been published [1,17][18]. The chemical shift of C
0
recorded on the MS at m/z 708 was conclusive of the
1
esterified nature of 3. The H-NMR spectrum of 3 exhib-
(
3 ) is in better agreement with our own measurements
and predicted values. Our assignment (Table 1) and the ited one characteristic triplet at d(H) 5.10 corresponding
1
2
linkage between the two units were proved by comparison
with data published by Talontsi et al. [16]. This was also
supported by comparison with previously reported NMR
to an olefinic H-atom and characteristic of the D H-
atom in the pentacyclic triterpenes [23], and two doublets
of doublets at d(H) 4.48 and d(H) 3.92 revealing the pres-
data [19 – 21]. Contrary to the downfield shift observed ence of two oxymethine. The observation of the addi-
0
0
for the O -bonded carbons C(2), C(3 ), and C(4 ), an
2
tional oxymethine C-atom peak at d(H) 68.7 and
1
13
upfield shift was observed for C(1) (d(C) 119.1), thus sug-
gesting its connection to a C=O C-atom. Consequently,
both Ph groups are connected by a seven-membered ring
containing an ether linkage and an ester bridge [21],
comparison of the H and C-NMR data of this com-
pound with the data described for methylsumaresinolate
[23] allowed us to establish the b relative configuration
for the OH group at C(6) in compound 3 [22]. The
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2016 Wiley-VHCA AG, Z u€ rich
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4
Helv. Chim. Acta 2016, 99, 1 – 8
1
13
Table 2. H- (300 MHz) and C-NMR (75 MHz) Spectral data (in
CDCl ) of 3. d in ppm, J in HZ.
doublets at d(H) 6.59 (1 H, d, J = 2.1), 6.54 (1 H, d,
J = 2.1), 6.38 (1 H, d, J = 2.1), and 6.33 (1 H, d, J = 2.1)
accounting for aromatic H-atoms. It also showed one
singlet at d(H) 3.85 for MeO group and two singlets at d
3
Position
d(H)
d(C)
13
1
2
3
4
5
6
7
8
9
41.6
28.4
81.4
37.8
55.2
68.2
41.7
39.8
47.6
36.9
23.5
121.6
145.2
39.8
27.0
22.8
36.6
38.2
46.8
31.1
32.6
32.0
28.1
16.8
15.6
16.8
26.0
26.1
33.3
23.6
172.9
34.7
31.9
22.7
29.6 – 29.7
22.7
14.1
(H) 2.35 and 2.62 for two aromatic Me groups. The C-
NMR spectrum revealed ester C=O groups at d(C) 164.8
4.48 (dd, J = 1.8, 9.0)
1
and an acid carbonyl group at d(C) 168.1. Based on H-
–
1
3
NMR, C-NMR, HMBC, and mass spectrum, compound
4 was identified as 2-hydroxy-4-[(4-hydroxy-2-methoxy-6-
methylbenzoyl)oxy]-6-methylbenzoic acid. This compound
isolated for the first time from natural source belongs to
the depside family and, was previously synthesized by
Elix et al. [5].
3.92 (dd, J = 4.5, 11.6)
2.42 – 2.47 (m), 2.29 – 2.37 (m)
–
10
11
12
13
14
15
16
17
18
19
20
21
22
–
Compounds 2 and 4 belong to the group of depside
and depsidones, which are typically found in lichens [25]
5.24 (t, J = 3.3)
–
–
[26]. Only a few depsidones have been isolated from
non-lichen sources, such as auranticins A and B, from
the mangrove fungal Preussia aurantiaca [27], emeguising
from the ascomycete Emericella unguis [20], depsidones
garcinisidone B – F from two species of Garcinia plants
[28], and Botryorhodines A – D from Botryosphaeria
rhodina, an endophyte of the medicinal plant Bidens pi-
losa [29]. Some few years ago, depsidone from non-
lichens and non-endophytic plant have been reported
–
–
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
Me(28)
Me(29)
Me(30)
0.83 (s)
0.83 (s)
1.06 (s)
1.06 (s)
1.11 (s)
0.78 (s)
0.89 (s)
0.89 (s)
–
[30].
Part of erythrodiol palmitate (11) obtained in bulk
amount was acetylated in order to study the effect of
acetylation on antimicrobial activity. A new acetylated
eurothridiol palmitate derivative (13) was obtained.
The results of the antimicrobial activity summarized
in Table 3 showed that compounds 5 and 9 were able to
prevent the growth of all the eight studied microbial
strains, including bacteria and fungi, within a MIC range
of 64 – 512 lg/ml. Other compounds showed selective or
no activity. Compound 11 was previously tested and was
shown to display moderate antimicrobial activities
against Klebsiella pneumoniae (MIC of 512 lg/ml), Sal-
monella typhi (MIC of 512 lg/ml), Escherichia coli
AG100 (MIC of 512 lg/ml), Staphylococcus aureus (MIC
0
1
0
2
2.29 – 2.37 (m)
0
3
0
4
0
0
5
1
1
– 16
1.18 (br. s)
0
7
8
0
0.88 (t, J = 7.1)
position of the OH group at C(6) was confirmed by the
HR-EI-TOF-MS with the ion peak at m/z 218.2035 of 256 lg/ml), and E. coli ATCC8739 (MIC of 128 lg/
þꢁ
(
100%, C16H ) arising from a retro Diels–Alder cleavage
ml) [4]. We are not aware of any previous report on the
of ring C (Fig. 2). The mass spectrum recorded for the antimicrobial activities of compounds 2, 3, 4, 5, and 13.
2
6
transesterification product of 3 made it possible to recog-
nize stearic acid esterified with –OH at the C(3) position
The activity recorded for active samples could be consid-
ered moderate to weak, compared to chloramphenicol or
of the triterpene through the molecular ion at m/z 298 nystatin. It can be observed that terpenoids compounds
methyl stearate). This transesterification of 3 also con- mostly showed poor activity than phenolics. When
(
firmed the structure of the triterpene moiety as 3b,6b- regarding the structure–activity relationship, acetylation
dihydroxyolean-12-ene (m/z 442) known as daturadiol seems to decrease both the antibacterial and anticandidal
and firstly isolated from Datura innoxa by Kocor et al. in activity as the results obtained for compounds 11 and 13
1
973 [24]. Consequently, compound 3 was identified as shows.
6b)-hydroxyolean-12-en-3-yl stearate, a new esterified
triterpenoid to which the name daturadiol stearate is Y. P. A. is grateful to the Organisation for the Prohibition
proposed.
of Chemical Weapons (OPCW) for three months financial
(
Compound 4 was obtained as a brown amorphous support (Travel grant and maintenance allowance). The
+
powder. The ESI-TOF-MS (+ve) showed [M + Na] ion Network of Analytical and Bioassay Services in Africa
peak at m/z 355.0788 corresponding to the molecular for-
(NABSA) is acknowledged for providing research
1
mula C H O . The H-NMR spectrum displayed four facilities.
1
7
16
7
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Helv. Chim. Acta 2016, 99, 1 – 8
5
Fig. 2. Mass fragmentation of 3.
Table 3. Antimicrobial activity of the tested compounds and reference antibiotics
a
Samples )
b
Microorganisms ), strains, and MIC [lg/ml]
P. stuartii
P. aeruginosa
K. pneumoniae
S. aureus
S. typhi
E. coli
C. albicans
ATCC29916
PA01
ATCC11296
ATCC25922
ATCC6539
AG100
ATCC8739
ATCC 9002
c
4
2
3
5
512
> 512
–
512
–
512
256
32
– )
256
> 512
–
256
–
512
256
4
256
> 512
–
128
–
512
128
4
–
512
–
128
–
–
256
4
512
512
–
512
–
–
512
4
256
512
–
256
–
512
256
4
–
64
512
–
128
–
–
512
64
–
256
–
512
512
16
1
1
9
2
3
RA
a
b
Samples (RA or reference antibiotics: chloramphenicol for bacteria and nystatin for C. albicans). ) Microorganisms (Providencia stuartii
)
(
P. stuartii); Pseudomonas aeruginosa (P. aeruginosa); Klebsiella pneumoniae (K. pneumoniae); Staphylococcus aureus (S. aureus); Salmonella
c
typhi (S. typhi); Escherichia coli (E. coli); Candida albicans (C. albicans)). ) MIC > 512 lg/ml.
Corp., Kyoto, Japan); kmax (log e) in nm. IR Spectra:
Shimadzu FTIR-8700 and Shimadzu 8900 infrared spec-
Supplementary Material
ꢀ
1
Supporting Information for this article is available on the
WWW under http://dx.doi.org/10.1002/hlca.201600073.
trometer with KBr pellets; ~v in cm . NMR Spectra:
Bruker Avance 300 (Bruker Daltonics Inc., Billerica,
1
13
MA, USA) at 300 MHz ( H) and 75 MHz ( C) and
1
Bruker Avance 600 at 600 MHz ( H) and 150 MHz
Experimental Part
1
(
3
C), with the residual solvent peaks as internal refer-
ences; d in ppm, J in Hz. GC/MS: GC chromatograph
Varian mod. Saturn II (Varian Inc., Walnut Creek, CA,
General
TLC: Precoated plates of silica gel GF254; spots were USA) coupled to an ion trap mass detector, employing
detected under UV light and spraying with anilsalde- a DB1-MS column (30 m 9 0.32 mm 9 1 lm); in m/z.
hyde/sulfuric acid in EtOH or H O/sulfuric acid. Col-
HR-ESI-MS: micrOTOF mass spectrometer (Bruker
2
umn chromatography (CC): silica gel 60 (SiO2, Daltonik GmbH, Bremen, Germany); in m/z. EI-TOF-
0.04 – 0.063 mm; Merck, Darmstadt, Germany) and
Sephadex LH-20 (Sigma, Dorset, UK). UV Spectra:
MS (pos.): Finnigan SSQ-7000 spectrometer (Finnigan
Corp., San Jose, CA, USA) by direct inlet (70 eV); in
Shimadzu UV-210 IPC spectrophotometer (Shimadzu m/z (rel. %).
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2016 Wiley-VHCA AG, Z u€ rich
www.helv.wiley.com

6
Helv. Chim. Acta 2016, 99, 1 – 8
flask. The mixture was magnetically stirred at r.t. for 6 h.
After completion of acetylation, toluene was added and the
Plant Material
The twigs and the roots of T. madagascariense DC were soln. was concentrated under vacuum. Evaporation of the
collected at Nkolnkoumou, Yaound ꢀe , Center region of
solvent gave the corresponding acetate derivative (13)
Cameroon in April 2012, and was identified by Mr. Victor which is a new acetylated eurothridiol palmitate derivative.
Nana of the National herbarium, Yaound ꢀe , Cameroon,
where a voucher specimen (number 1778/SRFK) is depos-
Transesterification of Compound 3
ited.
Compound 3 was refluxed in dry MeOH (20 ml) with
sodium methoxide (20 mg) for 1 h. The reaction product
Extraction and Isolation
was extracted with H O and CH Cl . The org. phase was
2
2
2
The air-dried and powdered leafless twigs (500 g) of
T. madagascariense were extracted with MeOH/CH Cl
separated, dried (Na SO ), and evaporated. The Me ester
2 4
of stearic acid was obtained (7.6 mg). Addition of HCl
(1%) to the remaining aq. phase, followed by extraction
2
2
1
:1 at r.t. for 48 h and MeOH for 8 h. The combined
extracts (25 g) were subjected to CC over SiO 60 (100 g) with CH Cl yielded the triterpene moieties 3b,6b-dihy-
2
2
2
and eluted with petroleum ether (PE)/CHCl and CHCl / droxyolean-12-ene. Transesterification products were
3
3
MeOH of increasing polarity. Thirty fractions of 200 ml
each were collected, concentrated, monitored by TLC and Trilepisflavene (= 4 ,6,7-Trihydroxyflavene; 2-(4-Hydroxy-
similar ones pooled to give a total of four fractions (Frs. phenyl)-4H-1-benzopyran-6,7-diol; 1). Brown oil. UV
characterized by GC analysis.
0
A – D). Fr. B (6 g, PE/CHCl 50:50 – 0:100), were resub- (acetone): 329 (1.13), 326 (1.11). IR (KBr): 3447, 3249,
3
1
13
jected to CC on SiO (40 g) eluted with PE/CHCl (1:0 – 1613, 1513. H- and C-NMR: see Table 1. HR-EI-MS:
:7) and the obtained fractions were sometimes passed
2
3
+
þ
3
256.0807 (M , C15H12O ; calc. 256.0736).
4
through Sephadex LH-20 eluted with CHCl /MeOH 70:30 Trilepisdepsidone (= Methyl 8-(Hydroxymethyl)-3,6-di-
3
and AcOEt/MeOH 90:10 to give daturadiol stearate (3; methoxy-1,4-dimethyl-11-oxo-11H-dibenzo[b,e][1,4]dioxe-
1
0 mg), trilepisdepsidone (2; 15 mg), lichexanthone (5; pine-7-carboxylate; 2). Brown amorphous powder. UV
5 mg), eurothridiol palmitate (11; 150 mg), b-sitosterol (acetone): 217.8 (1412). IR (KBr): 3264.3 (H–O), 2922.0,
2
(
12; 25 mg), and b-amyrin (10; 12 mg). Fr. C (5 g, CHCl3/ 1740.1 (C=O), 1601.9, 1492 (C=C), 1326.5, 1151.6, 1065.3.
1
13
H- and C-NMR: see Table 1. HR-ESI-MS: 387.1075
MeOH 95:5 to 85:15), was resubjected to CC on SiO2
(
ꢀ
ꢀ
40 g) eluted with CHCl /MeOH (100:0 to 80:20) and the
3
([M ꢀ H] , C H O ; calc. 387.1085).
20 19
8
obtained fractions passed through Sephadex LH-20 eluted Daturadiol Stearate (= (3b,6b)-6-Hydroxyolean-12-en-3-yl
with CHCl /MeOH 70:30 to give 2-hydroxy-4-(4-hydroxy- Octadecanoate; 3). White amorphous powder. IR (KBr):
-methoxy-6-methylbenzoyloxy)-6-methylbenzoic acid (4; 2922.9, 2853.1, 1715.7 (C=O), 1603.8, 1463 (C=C), 1045.5,
3
2
1
1
13
5 mg), 2-hydroxybenzoic acid (8; 10 mg). Fr. A (7 g, PE/
756.0. H- and C-NMR: see Table 2. HR-EI-TOF-MS
þꢁ
(pos.): 466.3784 (68, C32H50O ), 218.2035 (100, C16H ),
2
þ
207.0329 (78, C14H23O ), 203.1808 (56, C15H ), 189.1640
2
þꢁ
CHCl3 100:0 to 50:50) and Fr. D (5 g, CHCl /MeOH
3
26
þ
80:20 – 70:30) were found to contain mostly hydrocarbons
or tannins and were not investigated further.
23
þ
+
(40,
C14H ).
HR-ESI-MS: 731.6286 ([M + Na] ,
2
1
þ
The dried powdered roots (400 g) of T. madagas-
cariense were extracted with MeOH/CH Cl 1:1 at r.t. for 2-Hydroxy-4-[(4-hydroxy-2-methoxy-6-methylbenzoyl)oxy]-
C48H84NaO ; calc. 731.6313).
3
2
2
4
were subjected to CC over SiO 60 (80 g) and eluted with
8 h and MeOH for 8 h. The combined extracts (15 g)
6-methylbenzoic Acid (4). Brown amorphous powder. UV
(acetone): 209.7 (5248). IR (KBr): 3229.1 (H–O), 1714
2
1
PE/CHCl3 and CHCl /MeOH of increasing polarity.
3
(C=O), 1599.3, 1517.2, 1441.5 (C=C), 1239.2 (O–C=O). H-
3
Twenty fractions of 150 ml each were collected, concen-
trated, monitored by TLC and similar ones pooled to give (3 )); 6.38 (d, J(H,H) = 1.2, H–C(5 )); 6.54 (d, J(H,H) =
a total of five fractions (Frs. A – E). Fr. C (2.5 g, PE/
NMR (300 MHz, CD OD): 6.33 (d, J(H,H) = 1.2, H–C
3
0
3
0
3
3
2.1, H–C(4)); 6.59 (d, J(H,H) = 1.2, H–C(6)); 2.62 (s,
Me); 2.35(s, Me); 3.85 (s, MeO). C-NMR (75 MHz,
1
3
CHCl 40:60 – 0:100), was passed through Sephadex LH-
3
2
0 eluted with CHCl /MeOH 70:30 to give trilepisflavene
3
CD OD): 116.5 (s, C(1)); 161.6 (s, C(2)); 108.6 (d, C(3));
3
(1; 65 mg). Fr. E (2 g, CHCl /MeOH 95:5 to 85:15), was
passed through SephadexLH-20 eluted with CHCl3/ (7)OOH); 114.5 (s, C(1 )); 160.6 (s, C(2 )); 97.9 (d, C(3 ));
155.3 (s, C(4)); 114.9 (d, C(5)); 140.1 (s, C(6)); 168.1 (s, C
0
3
0
0
0
0
0
MeOH 70:30 to give methyl 2,4-dihydroxy-6-methylbenzo- 161.6 (s, C(4 )), 110.3 (d, C(5 )), 116.5 (s, C(6 )), 164.8 (s,
0
0
0
ate (9; 5 mg), 3 ,4 ,5,7-tetrahydroxyflavanone (7; 7 mg) C(7 )); 56.4 (s, MeO); 20.0 (s, Me); 23.6 (s, Me). HR-ESI-
+
þ
and naringenin (6; 14 mg).
MS: 355.0788 ([M + Na] , C17H16NaO ; calc. 355.0788).
7
ꢀ
ESI-MS (-ve): 331.1 ([M ꢀ H] ).
Acetylation of Eurothridiol Palmitate (11)
Antimicrobial Assay
Eurothridiol palmitate (11) (60 mg) was placed in a
round-bottomed flask equipped with a magnetic stirrer.
Microbial Strains and Culture Media. The studied
microorganisms included reference (ATCC) and multi-
Ac O (30 ml) and pyridine (30 ml) were added to the
2
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Helv. Chim. Acta 2016, 99, 1 – 8
7
drug-resistant strains of Providencia stuartii (ATCC299
6), Pseudomonas aeruginosa (PA01), Klebsiella pneu-
[3] G. N. Teke, J. R. Kuiate, V. Kuete, R. B. Teponno, L. A.
Tapondjou, G. Vilarem, ‘Antidiarrheal activity of extract and
compounds from Trilepisium madagascariense stem bark’,
Indian J. Pharm. 2010, 42, 157 – 163.
1
moniae (ATCC11296), Escherichia coli (ATCC 8739 and
AG100), Salmonella typhi ATCC6539, and Candida
albicans ATCC 9002 obtained from the American type
Culture Collection. They were maintained on agar slant
at 4 °C and subcultured on fresh appropriate agar plates
[
4] Y. P. Ango, G. D. W. F. Kapche, V. Kuete, B. T. Ngadjui, M.
Bezabih, B. M. Abegaz, ‘Chemical constituents of Trilepisium
madagascariense (Moraceae) and their antimicrobial activity’,
Phytochem. Lett. 2012, 5, 524 – 528.
[
[
5] J. A. Elix, V. K. Jayanthi, L. F. McCaffery, J. Yu, ‘Synthesis
of new lichen tridepside’, Aust. J. Chem. 1998, 51,
24 h prior to any antimicrobial test. Nutrient Agar and
Sabouraud Glucose Agar were used for the activation of
1
045 – 1052.
bacteria and fungi, resp. The Mueller–Hinton broth
(MHB) was used for the determination of the minimal
inhibitory concentrations (MIC) [31].
6] S. M. Zoran, T. M. Nedeljko, ‘Analytical characterization of
lichexanthone in lichen: HPLC, UV spectroscopic, and DFT
analysis of lichexanthone extracted from Laurera benguelensis
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[
[
[
7] K. N. Scott, ‘Carbon-13 nuclear magnetic resonance of biologi-
cally important aromatic acids’, J. Amer. Chem. Soc. 1972, 94,
Chemicals for Antimicrobial Assay
8
564 – 8568.
Chloramphenicol and nystatin (Sigma–Aldrich, St. Quen-
tin Fallavier, France) were used as reference antibiotics
8] I. Zakaria, N. Ahmat, R. Ahmad, M. F. Jaafar, N. A. Ghani, S.
Khamis, ‘Flavanones from the flower of Macaranga triloba’,
World Appl. Sci. J. 2010, 9, 1003 – 1007.
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(RA), resp., against bacteria and C. albicans. p-Iodonitro-
tetrazolium chloride (INT; Sigma–Aldrich) was used as
microbial growth indicator [32][33].
1
18 – 119.
[
10] X.-F. Li, H.-Z. Jin, M. Yang, G. Chen, W.-D. Zhang, ‘A New
methyl orsellinate glycoside from the aerial parts of Rhodo-
dendron primulaeflorum’, Chin. J. Nat. Med. 2008, 6,
336 – 338.
MIC Determinations
The MIC determinations on bacteria was conducted
using rapid INT colorimetric assay according to
described methods [32][33] with some modifications.
Briefly, the test sample was first of all dissolved in 10%
[
11] T. R u€ egg, A. I. Calder oꢀ n, E. F. Queiroz, P. N. Sol ꢀı s, A. Mar-
ston, F. Rivas, E. Ortega-Barr ꢀı a, K. Hostettmann, M. P. Gupta,
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(
1
v/v) DMSO/MHB to give a final concentration of
024 lg/ml and serially diluted twofold to obtain concen-
tration ranges. A quantity of 100 ll of each concentra-
tion was added in a well (96-well microplate) containing
9
4 – 97.
13] K. Tatjana, F. Nicolas, M. Prokopios, K. Nada, C. Ioanna,
Triterpenic derivatives of Achillea alexandri-regis Bornm. &
Rudski’, Chem. Pharm. Bull. 2004, 52, 1462 – 1465.
[
9
1
5 ll of MHB and 5 ll of inoculum (standardized at
.5 9 10 CFU/ml by adjusting the optical density to 0.1
‘
6
at 600 nm SHIMADZU UV-120-01 spectrophotometer)
34]. The final concentration of DMSO in the well was
[14] L. Jurd, ‘Anthocyanidins And Related Compounds-Xi Cate-
chin-Flavylium Salt Condensation Reactions’, Tetrahedron 1967,
[
2
3, 1057 – 1064.
15] P. Papadopoulou, O. Tzakou, C. Vagias, P. Kefalas, V. Roussis,
Orcinol metabolites from the lichen Hypotrachyna revolute’,
less than 3% (preliminary analyses with 3% (v/v) DMSO
do not alter the growth of the test organisms). The nega-
tive control well consisted of 195 ll of MHB and 5 ll of
the standard inoculum [35]. The plates were covered
with a sterile plate sealer, then agitated to mix the con-
tents of the wells using a plate shaker, and incubated at
[
‘
Molecules 2007, 12, 997 – 1005.
[
16] M. F. Talontsi, I. M. Tofazzal, F. Petrea, D. C. Meli, V. A.
Tiedemann, H. Laatch, ‘Depsidones and other constituents from
Phomopsis sp. CAFT69 and its host plant Endodesmia calophyl-
loides with potent inhibitory effect on motility of zoospores of
grapevine pathogen Plasmopara viticola’, Phytochem. Lett.
3
7 °C for 24 h. The assay was repeated three times in
triplicate. The MIC of samples was detected following
addition (40 ll) of 0.2 mg/ml INT and incubation at
2
012, 5, 657 – 664.
[
[
[
17] J. Meister, D. Weber, V. Martino, O. Sterner, T. Anke, ‘Pho-
mopsidone, a novel depsidone from an endophyte of the medic-
inal plant Eupatorium arnottianum’, Z. Naturforsch. 2007, 62c,
11 – 15.
18] G. Lang, A. L. J. Cole, J. W. Blunt, W. T. Robinson, M. H. G.
Munro, ‘Excelsione, a depsidone from an endophytic fungus
isolated from the New Zealand endemic tree Knightia excels’, J.
Nat. Prod. 2007, 70, 310 – 311.
3
7 °C for 30 min [32][33]. Viable microorganisms
reduced the yellow dye to a pink color. MIC was defined
as the lowest sample concentration that prevented this
change and exhibited complete inhibition of bacterial
growth [35].
19] C. Bezivin, S. Tomasi, I. Rouaud, J. G. Delcros, J. Boustie,
‘Cytotoxic activity of compounds from the lichen Cladonia con-
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Received April 6, 2016
Accepted May 26, 2016
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