Phytochemistry Letters 12 (2015) 328–331
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Terpenoids from the stem bark of Neoboutonia macrocalyx
(Euphorbiaceae)
Timoleon Maffo a , Pascal Wafo b, ** , Ramsay Soup Teoua Kamdem a,e, * , Raduis Melong a ,
Philip F. Uzor d, Pierre Mkounga a , Zulfiqar Ali c , Bonaventure Tchaleu Ngadjui a
a
Department of Organic Chemistry, Faculty of Science, University of Yaoundé I P.O. Box 812, Yaoundé, Cameroon
Higher Teachers’ Training College, University of Yaoundé I P.O. Box 47, Yaoundé, Cameroon
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
d
Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria, Nsukka 410001, Nigeria
e
Institute für Pharmaceutische Biologie und Biotechnologie HEINRICH HEINE Universität Düsseldorf Universitat StraBe 1, Gebäude 26.23, 40225 Düsseldorf,
Germany
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 8 February 2015
Received in revised form 23 April 2015
Accepted 29 April 2015
Available online 11 May 2015
Neoboutomannin A (1), a new degraded diterpenoid monomer, and 3a-acetyl-14a-hydroxytaraxan19,28-olide (2), a new triterpenoid derivative, have been isolated from the stem bark of Neoboutonia
macrocalyx Benth (Euphorbiaceae), together with the known compounds, 3-acetyl aleuritolic acid (3),
3b-acetoxy oleanolic acid (4), oleanolic acid (5), 3,3,4-tri-O-methylellagic acid (6), sitosterol 3-O-b-Dglucopyranoside (7) and sitosterol (8). Their structures were elucidated on the basis of spectral data and
comparison with those present in the literature. The methanol extract of the stem bark of N. macrocalyx
showed high toxicity to brine shrimp nauplii (LC50 = 0.6 0.05 mg/mL) and low antifungal activity on
Candida albicans and Mucus miehei. Compound 3 exhibited moderate toxicity to brine shrimp
(LC50 = 10.0 0.9 mg/mL) and compound 2 showed low antifungal activity.
ã2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Euphorbiaceae
Neoboutonia macrocalyx
Terpenoids
1. Introduction
Euphorbiaceae is a large family of flowering plants with
300 genera and around 7500 species. Most are herbs, but some,
especially in the tropics, are also shrubs or trees (Troupin,1982). This
family occurs mainly in the tropics, with the majority of the species in
the Indo-Malayan region and tropical America region. There is a large
variety in tropical Africa, but it is not as abundant or varied as in these
two other tropical regions. Euphorbiaceae also has many species in
non-tropical areas such as the Mediterranean Basin, the Middle East,
South Africa, and southern USA (Troupin, 1982). The genus
Neoboutonia is widely distributed in tropical West Africa and
Central Africa and is represented by the species, glabrescens,
melleriprain, manii and macrocalyx. The phytochemistry of the genus
Neoboutonia has not been extensively studied. However, diterpenes
and sterols have been reported in Neoboutonia macrocalyx; tigliane
derivatives and triterpenoids were also reported from the leaves of
Neoboutonia melleri (Kirira et al., 2007; Tchinda et al., 2003; Zhao
* Corresponding author at: Institute für Pharmaceutische Biologie und Biotechnologie HEINRICH HEINE Universität Düsseldorf Universitat StraBe 1, Gebäude
26.23, 40225 Düsseldorf, Germany. Tel.: +491 5213040618.
** Corresponding author.
E-mail addresses: wafopascal@yahoo.fr (P. Wafo), ramsay_kamdem@yahoo.fr
(R.S.T. Kamdem).
et al., 1998). N. macrocalyx is a plant used by traditional healers
among the Meru community in Kenya. The stem bark of
N. macrocalyx is used traditionally to treat headache and fever.
Previous investigations indicate that the methanol extract of the
stem bark of N. macrocalyx shows a high toxicity to brine shrimp
nauplii (LC50 = 21.04 1.8 mg/mL). The aqueous extract of
N. macrocalyx also exhibits a mild brine shrimp toxicity
(LC50 = 41.69 0.9 mg/mL) (Kirira et al., 2006).
In the course of our on-going research on Cameroonian
medicinal plants used traditionally to treat human microbial
infections, the methylene chloride–methanol (1:1) extract of the
stem bark of N. macrocalyx was examined for its antifungal and
cytotoxicity properties and also for its chemical constituents. This
study led to the isolation and structural characterization of
neoboutomannin A (1), a new degraded diterpenoid monomer
and 3a-acetyl-14a-hydroxytaraxan-19,28-olide (2), a new triterpenoid derivative, along with the six known compounds (3–8). The
cytotoxity and antimicrobial activities of the crude extract and
compounds 2 and 3 were evaluated.
2. Results and discussion
The extract of the stem bark of N. macrocalyx was subjected to
repeated column chromatography to give several fractions which
http://dx.doi.org/10.1016/j.phytol.2015.04.026
1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
T. Maffo et al. / Phytochemistry Letters 12 (2015) 328–331
329
CH3-29, and CH3-30 respectively), two carbinolic protons at dH/dC
4.55 (1H, m, Hb-3)/81.7 and at dH/dC 4.56 (1H, d, J = 12.2 Hz, Hb-19)
/79.7 attributed to C-3 and C-19 respectively, one methyl at d 2.13
(3H, s) corresponding to the methyl of an acetyl group. Its position
at C-3 was determined through an HMBC experiment in which the
oxymethine proton signal at d 4.55 (Hb-3) showed 3JC H
correlations with C-10 (d 171.0). The configuration of the acetyl
group at C-3 was assigned as a on the basis of the interactions of
H-3 with CH3-23 and CH3-25 in the NOESY experiment (Fig. 2). The
relative configurations of C-14, C-19 and C-17 were determined in a
similar manner. The hydroxyl at C-14 is probably from hydroxylation of the olefinic carbons of the D14–15 functionality of
taraxerane skeleton. From the HMBC spectrum, the proton at
H-19 showed correlations to carbons at d 43.0 (C-17), 45.4 (C-18),
31.2 (C-21), 181.6 (C-28); these correlations infer the presence of a
lactone group. The HMBC spectrum also showed that the lactone
system is in the ring E, between the 19-hydroxyl and 28-carbonyl
group. The lactone ring was deduced as b based on NOESY
interactions of H-19 (d 4.56) with CH3-30. The 1H and 13C NMR
chemical shifts were assigned using 1H–1H COSY, HSQC, HMBC, and
TOCSY spectra (Fig. 2). The following key unambiguous HMBC
correlations were observed: H-19 with C-28 and C-17; H-26 with
C-14 and C-9; H-16 with C-28 and C-17. The relative configuration
of 2 was deduced from the NOESY correlations (Fig. 2). The EI-MS of
2 corroborated the above structure with important fragments at m/
z 43.3 [CH3CO]; 439.3[M-CH3COOH-CH3], 454.3 [M-CH3COOH] and
411.3 [M-CH3COOH-CH3-CO]. Based on the above mentioned data,
the structure of compound 2 was deduced as 3a-acetyl-14ahydroxytaraxan-19,28-olide (2).
The known compounds were identified as 3-acetyl aleuritolic
acid (3) (Woo and Hildebert, 1977), 3b-acetoxyoleanolic acid (4)
(Maillard and Adewunmi, 1992), oleanolic acid (5) (Maillard and
Adewunmi, 1992), 3,30 ,4-tri-O-methylellagic acid (6) (Khac et al.,
1990), sitosterol 3-O-b-D-glucopyranoside (7) and sitosterol (8)
(Lawson et al., 1988).
The known compounds were identified as 3-acetyl aleuritolic
acid (3) (Woo and Hildebert, 1977), 3b-acetoxyoleanolic acid (4)
(Maillard and Adewunmi, 1992), oleanolic acid (5) (Maillard and
Adewunmi, 1992), 3,30 ,4-tri-O-methylellagic acid (6) (Khac et al.,
1990), sitosterol 3-O-b-D-glucopyranoside (7) and sitosterol (8)
(Lawson et al., 1988).
Results of the antimicrobial test showed that the extract was
active against the microbes used but was more active against the
fungi, Candida albicans and Mucor miehei (inhibition zone of 10 mm
each). Compounds 2 and 3 also showed moderate antifungal
were further purified to yield two new compounds 1 and 2,
together with six known compounds.
Compound 1 was obtained as a yellow powder from hexane/
EtOAc (60:40) fraction. The 1H and 13C NMR data (Table 1)
indicated the presence of 14 non-exchangeable protons and
16 carbon atoms corresponding to the molecular formula
C16H14O3 (10 of unsaturation) which was deduced from its mass,
1
H and 13C NMR spectra and comparison with literature data of
neoboutomannin (1b). The 1H NMR data of 1 revealed the presence
of three methyl singlet resonances at d 1.20, 1.20 and 2.22; two
vinylic proton singlet resonances at d 7.08 and 6.49; two aromatic
proton singlet resonances at d 7.40 and 7.82, and a hydroxyl group
resonance at d 10.62. The HMBC spectroscopic data were used to
construct the skeleton of 1. Cross-peaks were observed in HMBC
spectra between: H-1 (d 7.08) and C-3 (d 207.9), C-9 (d 129.5), C-5
(d 161.3), C-4 (d 45.3); H-6 (d 6.49) and C-10 (d 154.3), C-4 (d 45.3),
and C-7 (d 183.3); H-11 (d 7.40) and C-12 (d 160.0), C-10 (d 154.3),
C-9 (d 129.5) and C-8 (d 123.3); H-14 (d 7.82) and C-12 (d 160.0),
C-15 (d 16.4) and C-7 (d 183.3); H-15 (d 2.22) and C-12 (d 160.0),
C-13 (d 130.0), C-14 (d 129.3). The numbering system used for 1
(Figs. 1 and 2) is the same as neoboutomannin (1b) and reflects its
putative diterpenoid biogenetic origin (Tene et al., 2008). Thus
compound 1 is a new degraded diterpenoid monomer to which a
trivial name, neoboutomannin A, is given.
Compound 2 was an optically active white amorphous powder
from n-hexane/EtOAc (37:3) fraction. It was assigned the molecular
formula C32H50O5 from the 1H and broad band-decoupled 13C NMR
spectra and ESI-HRMS [M + Na]+ at m/z 537.3564 (calcd. 537.3550).
The IR spectrum showed absorptions for a hydroxyl group (3430.32
and 3300.92 cm 1), a lactone group (1732.00 cm 1), an ester group
(1684.00 cm 1), and a gem-dimethyl (1386.00 and 1375.00 cm 1)
(Lontsi et al., 1998). It gave a positive Liebermann–Burchard test for
triterpenes. The 13C NMR spectrum of compound 2 revealed
32 carbon signals which were sorted by DEPT 13C NMR as eight
methyls, ten methylenes, three methines, seven quaternary
carbons, two oxymethine and one quaternary alcohol (Table 2).
Further analysis of these spectra revealed resonances for two
carboxyl groups at d 171.0 and 181.6; two oxymethine groups at d
79.7 and 80.7, and eight methyl groups at d 16.6, 17.3, 21.3, 21.7,
22.9, 24.1, 27.9, and 33.5. A detailed analysis of the 1H NMR
spectrum of 2 (Table 2) confirmed the characteristic features for a
triterpenic taraxerane parent structure (Mahato and Kundu, 1994).
It was characterized by signals of seven tertiary methyls at dH/dC
0.84/16.6, 0.84/27.9, 0.95/17.3, 0.85/22.9, 1.12/21.7, 0.96/24.1, and
0.92/33.5 (3H each, s, CH3-24, CH3-23, CH3-25, CH3-26, CH3-27,
Table 1
1
H and 13C NMR spectroscopic data of compounds 1 and 1b (DMSO-d6)
C. No
Neoboutomanin A (1)
dC
1
3
4
5
6
7
8
9
10
11
12
13
14
15
18
19
12-OH
127.9
207.9
45.3
161.3
120.5
183.3
123.3
129.5
154.3
111.6
160.0
130.0
129.3
16.4
23.0
23.0
–
C. No
dH (mult.)
7.08 (s)
–
–
–
6.49 (s)
–
–
–
–
7.40 (s)
–
–
7.82 (s)
2.22 (s)
1.20 (s)
1.20 (s)
10.62(s)
0
1/1
3/30
4/40
5/50
6/60
7/70
8/80
9/90
10/100
11/110
12/120
13/130
14/140
15/150
18/180
19/190
12/12-OH
Neoboutomanin (1b) (Tene et al., 2008)
dC
dH (mult.)
133.5
204.6
45.7
160.3
121.4
183.4
124.0
130.0
151.8
112.1
160.6
130.0
130.3
16.5
22.8
23.9
–
–
–
–
–
6.76 (s)
–
–
–
–
7.14 (s)
–
–
7.94 (s)
2.21 (s)
1.44 (s)
1.33 (s)
10.53 (s)
330
T. Maffo et al. / Phytochemistry Letters 12 (2015) 328–331
Fig. 1. Structures of compounds 1, 1b and 2.
Fig. 2. Key correlations from HMBC 1 and 2, and NOESY of 2.
activity (inhibition zone of 10 mm each). The crude extract
showed a higher toxicity against brine shrimp nauplii (LC50 = 0.6
0.05 mg/mL) than compounds 2 (LC50 > 100.00 mg/mL) and 3
(LC50 = 10.0 0.9 mg/mL). In the present study, the extract and
compounds 2 and 3 showed LC50 of less than 1000 mg/mL
indicating their potential as cytotoxic and antitumor agents.
However, further studies are needed to determine their possible
role in cancer.
filtration, the solution was evaporated in vacuum to yield 150.3 g of
crude extract. A portion (140 g) of this extract was subjected to
flash chromatography over silica gel (300 g, 80 5 cm) eluting with
n-hexane/EtOAc in order of increasing polarity to give four
Table 2
1
H and 13C NMR data of compound 2 [CDCl3; d ppm (mult., J = Hz)]
C. No.
3.1. General experimental procedure
The optical rotation was measured on a PerkinElmer polarimeter 241 at the sodium D line. The NMR spectra were recorded on a
Varian Inova-500 spectrophotometer. The chemical shifts are given
in d values with TMS as internal reference, and coupling constants
are given in Hz. The ESI and ESI-HR mass spectra were recorded on
an APEX IV FTICR mass spectrometer Bruker Daltonik, at 7 T.
Column chromatography was performed on silica gel (type 60,
70–230 mesh, E. Merck). TLC experiments were carried out on
silica gel pre-coated plates (E. Merck, 0.25 mm), and detection was
achieved by UV light (254 or 366 nm) and spraying with 10% H2SO4
followed by heating. All solvents were distilled before use.
3.2. Plant material
The plant material was collected at Manjo, Littoral region of
Cameroon in May 2009 and identified by Mr. Victor Nana, the
botanist at the National Herbarium. The voucher specimen
(Ref 50111 HNC) has been deposited in the National Herbarium,
Yaounde, Cameroon.
3.3. Extraction and isolation
The air-dried and powdered stem bark (3 kg) was extracted
with MeOH/CH2Cl2 (1:1, 6 L 2, 48 h) at room temperature. After
2
13
3. Material and methods
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
20
C
33.7, CH2
23.5, CH2
80.7, CH
37.7, C
56.1, CH
18.3, CH2
44.3, CH2
40.4, C
47.9, CH
37.8, C
18.0, CH2
38.6, CH2
44.9, C
76.1, C
34.2, CH2
32.3, CH2
43.0, C
45.4, CH
79.7, CH
28.9, C
31.2, CH2
35.0, CH2
27.9, CH3
16.6, CH3
17.3, CH3
22.9, CH3
21.7, CH3
181.6, C
24.1, CH3
33.5, CH3
171.0, C¼O
21.3, CH3
1
H
1.20; 1.97 (m)
1.65; 1.61 (t, 2.3)
4.55 (m)
–
1.19 (m)
1.54; 1.49 (m)
1.30; 1.56 (m)
–
1.47 (m)
–
1.57; 1.45 (m)
1.30; 1.07 (m)
–
–
1.30; 1.07 (m)
1.64; 1.71(m)
–
2.74 (d, 12.2)
4.56 (d, 12.2)
–
1.37; 1.63 (m)
1.99; 1.26 (m)
0.84 (s)
0.84 (s)
0.95 (s)
0.85 (s)
1.12 (s)
–
0.96 (s)
0.92 (s)
–
2.13 (s)
T. Maffo et al. / Phytochemistry Letters 12 (2015) 328–331
fractions (A–D). Fraction B (23.2 g, eluted with hexane/EtOAc
(39:1–9:1), was applied to multiple column chromatography (CC)
[silica gel (50 g), column (50 3 cm)], to afford 3a-acetyl-14ahydroxyoleana-19,28-olide (2) (5.3 mg), 3-acetyl aleuritolic acid
(3) (10.0 mg), sitosterol (8) (3 g), 3-acetylolean-12-enoic acid (4)
(15 mg) and olean-12-enoic acid (5) (15 mg). Fraction C (148.3 mg,
eluted with hexane/EtOAc, 4:1–3:2) was subjected to CC [silica gel
(70 g), column (60 3 cm) eluting with mixtures of hexane/EtOAc]
to purify neoboutomannin A (1) (4.7 mg) and 3,30 ,4-tri-Omethylellagic acid (6) (25.3 mg). Sitosterol 3-O-b-D-glucopyranoside (7) (75 mg) was obtained as MeOH insoluble material from
fraction D.
3.4. Antimicrobial screening
Microbial strains of Staphylococcus aureus, Bacillus subtilis,
Escherichia coli, Streptomyces viridochromogenes, C. albicans and M.
miehei were used for the antimicrobial testing following the disk
diffusion method (Bauer et al., 1966). The compounds (2 and 3) as
well as the crude extract were tested against these bacterial and
fungal species, at concentrations varying from 200 to 0.78 mg/mL.
Compound 1, obtained in small amount, was not tested. An
inhibition zone of 14 mm or greater was considered as high
antimicrobial activity.
3.5. Brine shrimp lethality assay
The assay was done according to the method described by
Meyer et al. (1982) with some modifications. The crude extract
together with compounds 2 and 3 was evaluated. Brine shrimps
(Artemia salina) were hatched from brine shrimp eggs in a conical
shaped vessel (1 L), filled with sterile artificial sea water (prepared
using sea salt 38 g/L and adjusted to pH 8.5 with 1 N NaOH) under
constant aeration for 48 h. After hatching, active nauplii, free from
egg shells were collected from brighter portion of the hatching
chamber and used for the assay. To determine the LC50, several
concentrations (100, 50, 25, 12.5, 6.25 and 3.25 mg/mL) of the crude
extract or compound in DMSO were prepared. Then 10 mL of each
concentration was added to 990 mL of artificial sea water
containing more than 20 brine shrimps in each of the 24-well
tissue culture plate. After incubation for 24 h, the mean percentage
lethality per test concentration of the crude extract or compound
was determined. The percentage was plotted against the logarithm
of concentration. The concentration, at which 50% of the brine
shrimps died (LC50), was determined from the graph. The highly
cytotoxic actinomycine D was used as positive control while DMSO
was used as negative control. Brine Shrimp (Artemia sp.) Lethality
Assay (BSLA) is a general bioassay that appears capable of detecting
a broad spectrum of bioactivity present in plant crude extracts
(Pisutthanan et al., 2004). According to Meyer et al. (1982), it is
used as an indicator for general toxicity and also as a guide for the
detection of antitumor and pesticidal compounds. Crude
plant extract is toxic (active) if it has an LC50 value of less
than 1000 mg/mL while non-toxic (inactive) if it is greater than
1000 mg/mL.
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3.6. Spectroscopic data
3.6.1. Neoboutomanin A (1)
Yellow solid (hexane-EtOAc); m.p. > 310 C; IR nmax cm 1:
3411.00, 2966.00, 1681.00 1645.00, 1579.00, 1479.00, 1382.00,
1360.00; 1H NMR (DMSO-d6, 400.13 MHz) and 13C NMR (DMSO-d6,
100.6 MHz) data: see Table 1. HR-ESI-MS spectra gave the positive
ion at m/z 255.1015 [M + H]+ Analysis found: C 75.87, H 5.18%;
C16H14O3 requires C 75.88, H 5.17%;
3.6.2. 3a-acetyl-14a-hydroxytaraxan-19,28-olide (2)
White amorphous powder (hexane-EtOAc); ½a20
D = 9.9
(c = 0.10 in MeOH); IR nmax cm 1: 3430.32, 3300.92, 1730.73,
1680.52, 1469.49, 1450.21, 1386.57; EI-MS: m/z: 45 (100%); 55
(25%); 189 (42%); [M + Na]+ peak at m/z 537.3564 (calcd. for
[M + Na]+ 514.3550); 1H NMR (CDCl3, 300 MHz) and 13C NMR
(CDCl3, 75 MHz) data: see Table 2.
Acknowledgements
The authors are thankful to Mr. Nana, the botanist at the
National Herbarium Cameroon for identification of plant material.
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