Cytotoxic, anti-proliferative and antimicrobial furanoditerpenoids from Stuhlmania moavi

Article abstract of DOI:10.1016/j.phytochem.2009.08.017

The furanoditerpenoids voucapane, voucapane-6α,7α-diol, voucapane-18,19-diol and 18-hydroxyvoucapan-19-al were isolated from the cytotoxic stem and root bark extracts of Stuhlmania moavi Verdc. (Ceasalpiniaceae) and their structures established based on a

Full text of DOI:10.1016/j.phytochem.2009.08.017

Phytochemistry 70 (2009) 2047–2052
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cytotoxic, anti-proliferative and antimicrobial furanoditerpenoids
from Stuhlmania moavi
a,
b
b
Josiah O. Odalo a, Cosam C. Joseph a, , Mayunga H.H. Nkunya , Isabel Sattler , Corina Lange ,
*
*
Hans-Martin Dahse b, Ute Möllman b
a Department of Chemistry, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania
b Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstrasse 11a, 07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2009
Received in revised form 19 August 2009
Available online 19 September 2009
The furanoditerpenoids voucapane, voucapane-6a,7a-diol, voucapane-18,19-diol and 18-hydroxyvouca-
pan-19-al were isolated from the cytotoxic stem and root bark extracts of Stuhlmania moavi Verdc.
(Ceasalpiniaceae) and their structures established based on analysis of spectroscopic data. The com-
pounds exhibited anti-proliferative, cytotoxic, antibacterial and antifungal activities, 18-hydroxyvouca-
pan-19-al showing the highest anti-proliferative and cytotoxic properties. Voucapane-18,19-diol was
only mildly active but the activity was enhanced for its 18,19-di-(4-methyl)-benzenesulphonate. Some
of these results thus corroborate the traditional medicinal uses of the crude extracts for the treatment
of skin infections.
Keywords:
Stuhlmania moavi
Ceasalpiniaceae
Furanoditerpenoids
Anti-proliferative
Antimicrobial
Cytotoxic
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
In East Africa several plant species of the family Caesalpiniaceae
are used as sources of traditional medicines for the treatment of
malaria (Kokwaro, 1976; Chin et al., 2006), sleeping sickness (Frei-
burghaus et al., 1998) and inflammatory conditions (Dalziel, 1936).
Among such plant species is Stuhlmania moavi that grows in Tanza-
nia, and whose extracts are used for the treatment of skin infec-
tions. This, and the recent isolation of antitrypanosomal,
antimosquito and other compounds from some Caesalpiniaceae
species (Freiburghaus et al., 1998; Chin et al., 2006; Kihampa,
2008) prompted us to analyze the crude S. moavi root and stem
bark extracts for anti-proliferative, cytotoxic and antimicrobial
natural products, as part of our ongoing investigations for bioactive
and other constituents of the less common Tanzanian indigenous
plant species (Baraza et al., 2008), some of which are considered
to be threatened by extinction. We now hereby report the
isolation, structural determination, and bioactivities of the furano-
Repeated chromatography of the root and stem bark extracts
yielded the metabolites 1–4 whose 1H and 13C NMR spectra (Tables
1 and 2), and other spectroscopic data compared well with those
reported for 1 and other related furanoditerpenoids (Mendoza
et al., 2003; Cheenpracha et al., 2006). Furthermore, the 1H and
13C NMR spectra of 2 indicated two of the six methylene groups
in compound 1 to be hydroxylated in 2 (dH 3.45, dd, J = 10.8,
3.3 Hz, and 4.31, dd, J = 3.3, 1.5 Hz; dC 73.9 and 70.6; IR,
m
O–H = 3433 cmꢀ1), whose C-6 and C-7 positions were established
based on the fact that apart from the mutual coupling of 3.3 Hz,
each of the two carbinol methine protons also coupled with only
one other vicinal proton. This could be achieved only if C-6 and
C-7 were the carbinol methines, as in structure 2, where the ob-
3
3
served J5,6,
3J6,7 and J7,8 values of 10.8, 3.3 and 1.5 Hz indicated
trans (diaxial) and cis (equatorial/axial) H-5/H-6 and H-7/H-8,
respectively, and hence H-6/H-7 being cis (axial/equatorial). This
led to H-5/H-6, H-6/H-7 and H-7/H-8 dihedral angle to be ca.
160o, 75o and 90o, respectively (Becker, 1980), which was consis-
tent with the observed coupling constants. For similar compounds
diterpenoids voucapane (1), voucapane-6a,7a-diol (2), voucapane-
18,19-diol (3) and 18-hydroxyvoucapan-19-al (4).
where the 6-OH and 7-OH relative stereochemistry is
a and b the
J5,6, J6,7 and J7,8 values would be 11.1, 8.7 and 10.5 Hz, respectively
(Amtonio and Luiz, 1996). The 6-OH and 7-OH relative stereo-
chemistry in 2 was further indicated from the observed H-6/20-
CH3 and H-7/H-8 NOE interactions (Fig. 1).
* Corresponding authors. Tel.: +255 22 2772657; fax: +255 22 2772891.
E-mail addresses: cosam@chem.udsm.ac.tz (C.C. Joseph), Nkunya@chem.udsm.
ac.tz, mnkunya@tcu.go.tz (M.H.H. Nkunya).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.08.017

2048
J.O. Odalo et al. / Phytochemistry 70 (2009) 2047–2052
Table 1
1H NMR spectral data for the furanoditerpenoids 2–4.
H
1
d (2)
J (Hz)
d (3)
J (Hz)
d (4)
J (Hz)
1.02–2.01
m
0.97
1.55
1.40
1.46
1.30
1.59
1.37
1.18
1.59
1.27
1.62
1.65
1.42
2.25
2.45
2.53
6.23
7.35
0.89
3.32
3.24
3.60
3.28
0.82
4.28
4.17
m
m
m
m
m
m
1.05
1.76
1.52
1.60
1.41
1.76
1.42
1.26
2.32
1.62
1.90
1.80
1.52
2.33
2.59
2.61
6.16
7.20
0.96
3.87
3.49
9.90
m
m
m
m
m
m
2
3
1.02–2.01
1.02–2.01
m
m
5
6
1.46
3.45
d, 11.4
dd, 10.8, 3.3
dd, 11.7, 2.3
m
m
m
m
m
m
dd, 17.1,9.0
dd, 17.1,7.8
m
d, 1.8
d, 1.8
dd, 12.0, 2.5
m
m
m
m
m
m
dd, 16.7, 10.5
dd, 16.7, 6.7
m
d, 2.0
d, 2.0
7
4.31
dd, 3.3, 1.5
8
9
11
1.69
1.69
2.46
2.59
3.04
6.20
7.21
1.07
1.27
m
m
dd, 17.7, 10.0
dd, 17.7, 7.0
m
d, 2.0
d, 2.0
d, 6.9
s
14
15
16
17
18
d, 6.9
d, 7.5
dd, 10.8,5.6
dd, 10.8,5.6
dd, 11.1,5.1
dd, 11.1,5.1
s
d, 10.8
d, 10.8
s
19
1.20
0.99
s
s
20
18-OH
19-OH
0.79
s
d, 5.6
d, 5.1
Table 2
13C NMR chemical shifts (d values) for the furanoditerpenoids 2–4.
both C-18 and C-19 were the hydroxylated carbons, as in structure
3, which was further confirmed upon analysis of HMBC and NOE
interactions (Figs. 2 and 3).
C
2
3
4
C
2
3
4
The spectroscopic data for compound 4 compared well with
those observed for 3, except that one of the two hydroxymethylene
groups had been oxidized to an aldehyde unit (dH 9.90, 1H, s, CH@O
and dC@O 205.8; IR, mC@O 1700 cmꢀ1), whose C-19 position was
1
2
3
4
5
6
7
8
9
10
42.5
18.8
43.5
34.1
54.6
73.9
70.6
37.9
44.3
37.3
38.8
17.8
31.3
41.9
47.4
28.8
20.8
35.1
45.2
36.6
38.6
18.0
31.3
53.7
51.0
29.1
21.4
35.5
44.4
37.4
11
12
13
14
15
16
17
18
19
20
21.7
21.9
22.7
149.2
121.6
33.8
109.6
140.5
17.8
27.3
24.7
17.3
148.9
122.2
31.0
109.7
140.6
17.6
66.5
61.7
15.1
149.3
122.3
31.4
109.5
140.5
17.4
67.3
205.8
15.0
O
H
H
CH3
H
H
H
CH3
H
H
H
H
H
H
H
O
H
OH
H3C
HO
H
CH3
H
H
CH3
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H3C
HO
CH3
Fig. 2. Important H/C HMBC correlations for voucapane-18,19-diol (3).
H
H
OH
Fig. 1. Important NOE correlations for voucapane-6a,7a-diol (2).
O
H
H
H
CH3
H
H
H
O
H
The spectral features of 3 were closely related to those observed
for 1 and 2, except that instead of signals due to three tertiary
methyl groups, both the 1H and 13C NMR spectra exhibited
resonances for only one such group, and new signals due to
H
H
H
H
H
H
H
H
H
CH3
hydroxymethylene groups appeared (Tables
m
1 and 2; IR,
H
H
H
H
O–H = 3295 cmꢀ1). This indicated that two of the tertiary methyl
OH
groups in 1 were hydroxylated in 3. HMBC C/H interactions
(Fig. 2) indicated that both the hydroxymethylene units were
substituted at the same carbon atom. This was achievable only if
Fig. 3. Important NOE correlations for voucapane-18,19-diol (3).

J.O. Odalo et al. / Phytochemistry 70 (2009) 2047–2052
2049
deduced based on the observed H-19/H-10 and H-5/H-18 NOE and
HMBC interactions (Figs. 4 and 5), which also further confirmed the
structure.
O
Although furanoditerpenoids similar to 1–4 have previously
been reported from various sources including the genus Caesalpinia
(Cheenpracha et al., 2006; Ragasa et al., 2002; Lyder et al., 1998;
Peter and Tinto, 1997), the co-existence of a series of such com-
pounds as C-18 and C-19 variously oxidized metabolites like com-
pounds 1–4 is unprecedented.
H
The crude extracts when evaluated for possible cytotoxicity in
the brine shrimp test (Meyer et al., 1982) showed potent activity
(Table 3), the efficacy being comparable to that shown by the stan-
dard cytotoxic agent Cyclophosphamide. This prompted us to ana-
lyze the isolated furanoditerpenoids for possible cytotoxicity and
anti-proliferative activity. The results given in Table 4 from the
in vitro anti-proliferative and cytotoxic assay of the furanoditerpe-
noids 3 and 4 against L-929, K-562, and HeLa cell lines respectively
showed different levels of mild activity as compared to the stan-
dard anticancer agents TaxolÒ, Colchicine and Camptothecin. The
difference in anti-proliferative and cytotoxic potency of the furano-
diterpenoids 3 and 4 suggested the effect of the aldehyde group in
enhancing the activity. The 18,19-diacetoxy and 18,19-dibenzoyl-
oxy derivatives 5 and 6 of 3 showed diminished activity compared
to 3, while the activity of the 18,19-di-(4-methyl)-benzenesulpho-
nate 7 was higher than that of 3 for all the cell lines, indicating the
sulphonate group to be a better activity enhancer as compared to
the acetoxy and benzoyloxy moieties. However, overall all com-
pounds 3–7 were less active than any of the anticancer agents used
as standards, the most active compounds being 100 times less ac-
tive than the standards, hence making the isolated compounds and
their derivatives to be unsuitable as candidates for anticancer
agents development.
H
H
H
O
OH
Fig. 5. Important HMBC correlations for 18-hydroxyvoucapan-19-al (4).
Table 3
Toxicity (LC50 in lg/ml) of S. moavi crude extracts in the brine shrimp test.
Extract
Root bark
Stem bark
Pet ether
34.88 (23.39, 48.47)
14.42 (9.43, 19.52)
16.34 (6.02, 27.25)
18.28 (5.60, 32.36)
19.84 (13.19, 26.78)
61.93 (38.26, 99.11)
Dichloromethane
Methanol
Cyclophosphamide used as standard toxic agent [LC50 = 16.33 (10.60, 25.15)
ml]; values in parentheses represent 95% lower and upper confidence limits.
lg/
Table 4
Anti-proliferative and cytotoxic activity of compounds 3–7 against L-929, K-562, and
HeLa cell lines.
These results corroborate the previously reported moderate
cytotoxicity of furanoditerpeneoids similar to 1–4 against a series
of cell line panels (Liu et al., 2006; Kanokmedhakul et al., 2006),
suggesting that the furanoditerpenoid skeleton is not a suitable
pharmacophore in anticancer drug research.
Compound
Anti-proliferative activity (
l
g/ml)
Cytotoxicity (lg/ml)
L-929 (GI50
)
K-562 (GI50
)
HeLa (CC50)
3
4
5
6
14.7
5.4
34.2
>50
10.2
0.1
13.9
9
38.7
>50
8.4
0.01
0.02
0.002
20.3
10.8
36.2
>50
12.3
0.01
0.006
0.2
In order to establish the possible antibacterial and antifungal
activity of the isolated metabolites that would corroborate the tra-
ditional use of the crude extracts for the treatment of skin infec-
tions, compounds 1–4 were assayed for these activities in vitro
against the standard bacteria species Bacillus subtilis [ATTC 6633
(IMET NA)], Staphylococcus aureus [SG511 (IMET 10760)], Esche-
richia coli (SG 458), Pseudomonas aeruginosa (K 799/61) and Myco-
bacterium vaccae (IMET 10670), and against the fungal species
Sporobolomyces salmonicolor (SBUG 549), Candida albicans (BMSY
212) and Penicillium notatum. Generally, all the furanoditerpenoids
showed better activity against the bacteria than the fungal strains
(Table 5). Compound 2 exhibited the highest activity against M.
vaccae, while 4 was highly potent against B. subtilis. The furanodit-
7
TaxolÒ
Colchicine
Camptothecin
0.9
0.02
erpenoid 3 showed both antibacterial and antifungal activity. Com-
pared with the standard antibacterial and antifungal agents
Ciprofloxacin and Amphotericin respectively, the natural products
1–4 were less active, except 2 that showed a slightly higher activity
against M. vaccae compared to Amphotericin.
The results indicated that the functional groups of the parent
furanoditerpenoids were important for the exerted antibacterial
activity against B. subtilis, as the least functionalized diterpenoid
1 was also the least active. Furthermore, the diterpene diols 2
and 3 exhibited moderate activity, which indicated that the pres-
ence and appropriate position of the hydroxyl groups was essential
for enhanced activity. The furanoditerpenoid 4, which is a struc-
tural analogue of 3 but having one of the hydroxyl groups oxidized
to an aldehyde moiety, was more active against B. subtilis than the
other furanoditerpenoids 1–3. This suggested the importance of an
electrophilic site (aldehyde carbon) for enhanced B. subtilis activity.
In order to evaluate the effect of the hydroxyl as well as other
substituents on the activity of the voucapanoids 1–4, the 18,19-
diacetoxy, 18,19-dibenzoyloxy and 18,19-di-(4-methyl)benze-
nesulphonate derivatives 5–7 of 3 were assayed against the
bacteria and fungal strains. All the compounds were inactive
O
H
H
H
CH3
H
H
H
O
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
OH
Fig. 4. Important NOE correlations for 18-hydroxyvoucapan-19-al (4).

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J.O. Odalo et al. / Phytochemistry 70 (2009) 2047–2052
Table 5
Zones of inhibition (mm) for the furanoditerpenoids 1–4 and derivatives 5–7 of 2 against bacterial and fungal strains.
Compound
Bacterial and fungal strains, and zones of inhibition
B1
B3
B4
B9
M4
H4
H8
P1
1
2
3
4
5
6
7
12.0 0.6
13.0 0.3
16.0p 1.0
20.5 1.1
13.0 0.8
0
11.0 0.3
12.0 0.6
14.5p 1.1
0
12.0 0.2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
16.0p 1.0
24.0p 1.2
14.5 1.0
17.0p 1.3
0
0
0
0
0
0
0
0
0
13.0p 0.8
0
0
0
0
–
12.0p 0.7
0
0
0
0
–
14.0 1.0
0
14.0 0.6
13.0 0.8
13.0 0.4
–
0
Ciprofloxacin (5 mg/ml)
Amphotericin (10 mg/ml)
28.0 1.0
–
18.0 1.0
–
23.0/32.0p 1.0
–
22.0/270p 1.0
–
22.0p 1.0
–
14.0 1.0
20.0 1.0
18.0 1.0
B1 = Bacillus subtilis [ATTC 6633 (IMET NA)]; B3 = Staphylococcus aureus [SG511 (IMET 10760)]; B4 = Escherichia coli (SG 458); B9 = Pseudomonas aeruginosa (K 799/61);
M4 = Mycobacterium vaccae (IMET 10670); H4 = Sporobolomyces salmonicolor (SBUG 549); H8 = Candida albicans (BMSY 212); P1 = Penicilium notatum; each value represents
mean SD (n = 3); p = partial inhibition.
3.2. Plant materials
Table 6
MIC (mg/ml) values for furanoditerpenoid 2 against some bacterial strains.
The root and stem barks were collected in October 2004 from
Compound
Bacteria strains
Kwedijela forest in Handeni District in Tanzania. The plant species
was authenticated at the Herbarium of the Department of Botany,
University of Dar es Salaam where a voucher specimen No. FMM
3326 is preserved.
B1
B3
M2
M3
M4
M5
2
>100
50
25
6.25
6.25
12.5
<0.05
Ciprofloxacin
<0.05
0.2
1.6
<0.05
<0.05
3.3. Extraction and isolation
against B. subtilis, S. aureus, S. salmonicolor, C. albicans and M. vac-
cae, strains that were originally susceptible to the parent com-
pound 3 (Table 5). However, derivative 5 exhibited the same
level of moderate activity as 3 against B. subtilis as well as against
S. aureus. The level of activity of 5–7 against the fungus P. notatum
was similar to that observed for 3, hence indicating no influence on
the antifungal activity on replacement of the hydroxyl groups in 3
to form 5–7.
The air-dried and powdered plant materials were extracted
sequentially at room temperature with pet ether, CH2Cl2 and
MeOH (each 2 ꢁ 72 h). The pet ether extract when fractionated
by vacuum liquid chromatography (VLC, pet ether/EtOAc gradient
elution) and then separated by repeated CC yielded compounds 1
and 2. The CH2Cl2 and MeOH extracts on similar work up gave
the furanoditerpenoids 3 and 4.
The minimum (bacterial) inhibitory concentration (MIC) was
determined for the more active compound 2. The results (Table
6) indicated the compound to be weakly active compared to the
standard antibacterial agent Ciprofloxacin.
Despite the weak antibacterial and antifungal activities, the re-
sults tend to corroborate the traditional use of the crude extracts
for the treatment of skin infections. However, since the bioactivi-
ties exerted by the crude plant extracts as used in traditional med-
icines could be influence by many factors, including synergism of
the constituents, it would be important to carry out further bioas-
says of combination formulations of the isolated furanoditerpe-
noids, which could not be undertaken in these studies due to
paucity of the available samples.
3.3.1. Voucapane-6
a,7a-diol (2)
White crystals; m.p. 95–98 °C; yield, 7.0 mg; spray – pink;
½
a 2D0
+ 53.5° (c, 0.22, MeOH); IR, mmax 3433, 1459, 1366, 1205,
ꢂ
1118, 1060 and 1040 cmꢀ1; HRESIMS, m/z 317.2137{[MꢀH]+, calc.
for C20H30O3: 318.2117}; ESIMS, m/z (% rel. int.) 319 ([M+H]+, 92),
301 (71), 283 (100), 267 (59), 239 (29), 237 (23), 219 (25), 209
(24), 199 (21), 173 (26), 171 (16), 159 (26), 157 (18), 143 (10),
133 (19), 119 (11) and 109 (9); 1H and 13C NMR: Tables 1 and 2.
3.3.2. Voucapane-18,19-diol (3)
White crystals; m.p. 206–207 °C; yield, 484 mg; spray – pink;
a 2D0
m
ꢂ
+ 49.0° (c, 0.22, MeOH); UV, k
(log e) 213 (3.79) nm; IR,
MeOH
max
½
max 3295, 1595, 1506, 1194, 1139, 1085 and 1059 cmꢀ1; HRESIMS,
m/z 317.2133 {[MꢀH]+, calcd for C20H29O3: 317.2117}; ESIMS, m/z
(% rel. int.) 319 ([M+H]+, 20), 301 (54), 283 (100), 271 (38), 265
(78), 255 (44), 253 (36), 239 (36), 237 (27), 201 (26), 199 (47),
189 (70), 185 (31), 173 (24), 159 (22), 147 (34), 145 (30), 133
(27), 121 (13) and 109 (7); 1H and 13C NMR: Tables 1 and 2.
3. Experimental
3.1. General remarks
CC: Silica gel 60 (Merck 230–400 mesh, pet ether/EtOAc gradi-
ent elution) and SephadexÒ LH-20 (Pharmacia, MeOH/CHCl3); TLC:
Kieselgel 60 F254 (Merck) pre-coated on plastic plates (0.20 mm),
visualization by UV/VIS and anisaldehyde spray (Stahl, 1969);
m.p. (uncorrected): Electrothermal 9100; UV and IR spectra: Hit-
achi 200-20, and JASCO FT/IR-4100 spectrophotometers respec-
tively; optical rotation: JASCO P-1020 polarimeter; 1H NMR (300
and 500 MHz) and 13C NMR (75 MHz): Bruker DRX-300 spectrom-
eter in CDCl3, internal standard TMS (1H NMR) and solvent signal
3.3.3. 18-Hydroxyvoucapan-19-al (4)
White crystals; m.p. 185–187 °C; yield, 8 mg; spray – pink;
½
a 2D0
+ 37.1° (c, 0.22, MeOH); IR, mmax 3491, 2872, 1700, 1652,
ꢂ
1507, 1194, 1134, 1098, 1069, 1043 and 1033 cmꢀ1; HRESIMS, m/
z 339.1933 {[M+Na]+, calcd for C20H28O3Na: 339.1936}; ESIMS m/
z (% rel. int.) 316 ([M+H]+), 298 (50), 283 (100), 267 (17), 255
(42) and 240 (30); 1H and 13C NMR: Tables 1 and 2.
(
13C NMR); electron spray ionization mass spectra (ESIMS): HP
5990/5988A mass spectrometer at 70 eV; high resolution electron
spray ionization mass spectra (HRESIMS): JEOL JMS-HX 110 mass
spectrometer.
3.3.4. 18,19-Dibenzoyloxyvoucapane (5)
A solution of compound 3 (60 mg, 0.15 mmol), benzoyl chloride
(75 mL, 0.72 mmol), and a catalytic amount of DMAP in CH2Cl2

J.O. Odalo et al. / Phytochemistry 70 (2009) 2047–2052
2051
(40 mL) was stirred at room temperature for 24 h and the resulting
mixture diluted with CH2Cl2 (60 mL), then washed sequentially
with 2 M HCl (2 ꢁ 30 mL), saturated NaHCO3 (2 ꢁ 20 mL), and sat-
urated NaCl (30 mL). Workup of the organic layer and then CC gave
for changes in the diameter distribution curve were expressed as
diagrams. The monolayer of the adherent L-929 and HeLa cells
were fixed by glutaraldehyde and stained with a solution of meth-
ylene blue. After washing gently, the stain was eluted by 0.2 ml of
0.33 M HCl into the wells. The optical densities were measured at
660 nm in a microplate reader.
5 (50.1 mg, 83.5%); m.p. 163–165 °C, ½a D20
ꢂ
+ 13.6° (c 0.212, CDCl3);
spectral properties in agreement with the structure.
3.3.5. Voucapane-18,19-di-(4-methyl)benzenesulphonate (6)
Compound 6 was synthesized from 3 and 4-methylbenzenesul-
phonyl chloride as described for 5. Yield, 52.5 mg (87.5%), m.p.
3.6. Antibacterial and antifungal assay
The agar diffusion method (Jorgensen et al., 1999; Joseph et al.,
2006) was used in the assays against microorganisms obtained
from the Hans Knöll Institute for Natural Product Research and
Infection Biology (HKI), Jena in Germany, Ciprofloxacin (5 mg/mL)
and Amphotericin (10 mg/mL) being used as the standard antibac-
terial and antifungal agents. Antibacterial and antifungal activity
was expressed as the average diameter of inhibition zones.
The minimum inhibitory concentration (MIC) was determined
for compound 2 in a serial microplate dilution assay against each
test bacterial species (Elloff, 1998), with twofold serial dilution of
the compound dissolved in DMSO, beyond the level where no inhi-
bition of growth of the bacterial strainsB. subtilis [ATTC 6633 (IMET
NA)], S. aureus [SG511 (IMET 10760)], Mycobacterium smegmatis
(SG987), M. aurum (SB66), M. vaccae (IMET 10670) and M. fortuitum
was observed. Ciprofloxacin at a concentration of 100 mg/ml was
used as the reference antibiotic and two wells were used as steril-
ity and growth control, respectively, with the sterility control con-
taining only Oxoid MH broth, while the negative growth control
contained both MH broth as well as the test organism. The micro-
plates were sealed and incubated at 37 °C and 100% relative
humidity for 18 h. As an indicator of bacterial growth, 40 mL ali-
quots of a 0.2 mg/mL solution of p-iodonitrotetrazolium violet
(INT) dissolved in water were added to the microplate wells and
incubated at 37 °C for 30 min.
169–171 °C; ½a 2D0
ꢂ
+ 25.5° (c 0.201, CDCl3); spectral properties in
agreement with the structure.
3.3.6. 18,19-Di-acetoxyvoucapane (7)
Compound 7 was synthesized from 3 and acetic anhydride as
described for 5. Yield, 54 mg (90%); ½a D20
ꢂ
+ 30.6° (c 0.212, CDCl3);
spectral properties in agreement with the structure.
3.4. Brine shrimp test
The crude extracts were assayed in the brine shrimp test (BST)
in artificial seawater and DMSO according to standard procedures
(Meyer et al., 1982) and Cyclophosphamide was used as the stan-
dard toxic agent. LC50 values (the concentration required to kill 50%
of the shrimp larvae) were determined using Probit analysis (Fin-
ney, 1971).
3.5. Anti-proliferative and cytotoxicity assays
The anti-proliferative assay was carried out as described in the
literature (Dahse et al., 2001) using the cell lines K-562 (human
chronic myeloid leukemia) and L-929 (mouse fibroblast) and the
activity was expressed as GI50 values (concentration which inhib-
ited cell growth by 50%). Cytotoxicity assay was carried out against
HeLa cells and the activity expressed as GC50 values (concentration
at which cells are destroyed by 50%; used partially in referring to
the lysis of cells). For both the assays TaxolÒ, Colchicine and Cam-
ptothecin were used as the standard anticancer agents. The cells
were incubated at 10 different concentrations of each of the target
compounds. Cells of established suspended cell lines K-562 (DSM
ACC 10) and adherent L-929 were cultured in the RPMI medium
(Dahse et al., 2001). The adherent cells of L-929 were harvested
at the logarithmic growth phase after trypsinization, using 0.25%
trypsin in PBS containing 0.02% EDTA (Biochrom KG L2163). For
each experiment with K-562, L-929, and HeLa cells approximately
10,000 cells were seeded with 0.1 mL RPMI 1640 (GIBCO BRL
21875-034) containing 25 mg/ml Gentamicin sulfate (BioWhittak-
er 17-528Z but without HEPES) per well of the 96-well microplates
(K-562: NUNC 163320, L-929, HeLa: NUNC 167008). For the cyto-
toxicity assay, the HeLa cells were pre-inoculated for 48 h without
the test substances.
The dilution of the compounds was carefully made on the mon-
olayers of HeLa cells after the pre-incubation time. Cells of L-929,
K-562 and HeLa in the presence of the respective test compounds
were incubated for 72 h at 37 °C in a humidified atmosphere and
5% CO2. Suspension cultures of K-562 in microplates were analyzed
by an electronic cell analyzer system (CASY 1, SCHÄRFE, Reutlin-
gen, Germany) using an aperture of 150 mm. The contents of each
well (0.2 ml) in the microplate were diluted 1:50 with CAYSTON
(SCHÄRFE). Every count/ml was automatically calculated from
the arithmetic mean of three successive counts of 0.4 ml each.
From the dose response curves the GI50 values were calculated
using the computer program CASYSTAT. The GI50 value was defined
as the 50% intersection line of the concentration–response curve,
determined by the cell count/ml as compared to the control. The
essential parameters for the estimation of growth inhibition and
Acknowledgements
JOO thanks the Germany Academic Exchange Services (DAAD)
and NAPRECA for funding these studies through the DAAD/NAPRE-
CA Fellowship Scheme, which also supported the collaborative re-
search engagement with the Leibniz Institute for Natural Product
Research and Infection Biology, Hans Knöll-Institute in Jena, Ger-
many. Part of the research was funded through Sida/SAREC support
to the Faculty of Science, University of Dar es Salaam. We thank Mr.
F. Mbago of the Herbarium, Department of Botany at the University
of Dar es Salaam for identification of the investigated plant species.
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