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International Journal of Natural Products Research
Universal Research Publications. All rights reserved
ISSN: 2249-0353
Original Article
Anti-protozoal and structure-activity relationships of chemical constituents of
Acridocarpus chloropterus Oliver (Malpighiaceae) from Tanzania
Hamisi M. Malebo1,2,3,5,6*, Tanja Wenzler2, Monical Cal2, Sauda M. Swaleh3, Ahmed Hassanali3,
Alex K. Machocho3, Urs Séquin4, Daniel Häussinger4, Petur Dalsgaard5, Maurice O. Omolo6, Matthias Hamburger5,
Reto Brun2 and Isaiah O. Ndiege3
1
Department of Traditional Medicine Research, National Institute for Medical Research,
P.O. Box 9653, Dar es Salaam, Tanzania
2
Medical Parasitology and Infection Biology, Parasite Chemotherapy Unit,
Swiss Tropical and Public Health Institute, University of Basel, Socinstrasse 57, CH-4002, Basel, Switzerland.
3
Department of Chemistry, Kenyatta University, P.O. Box 43844, Nairobi, Kenya.
4
Institute of Organic Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056, Basel, Switzerland.
5
Institute of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, CH-4056, Basel, Switzerland.
6
Department of Pure and Applied Chemistry, MasindeMuliro University of Science & Technology, P. O. Box 190,
Kakamega, Kenya.
*Corresponding author’s e-mail address: hmalebo@nimr.or.tz
Received 16 July 2013; Accepted 08 August 2013
Abstract
Chromatographic separation of Acridocarpus chloropterus extract led to the isolation and identification of five triterpenes:
β-sitosterol (1), stigmasterol (2), friedelin (3), oleanolic acid (4), ursolic acid (5); and five flavonoids: apigenin (6), luteolin
(7), vitexin (8), kaempferol (9) and quercetin (10). Quercetin (10) exhibited moderate in vitro anti-plasmodial activity (IC50
2.6+0.05 μg/ml) while the rest of compounds were inactive. Mild to weak in vitro anti-trypanosomal activity was observed
in quercetin (10) (IC50 3.60+0.1 μg/ml), ursolic acid (5) (IC50 7.80+0.1 μg/ml) and apigenin (6) (IC50 9.0+0.1 μg/ml).
Ursolic acid (5) exhibited strong in vitro anti-leishmanial activity (IC50 0.80+0.001 μg/ml) while oleanolic acid (4),
apigenin (6), kaempferol (9) and quercetin (10) showed moderate to mild activity (2.10+0.1, 2.20+0.1, 5.90+ 0.1 and
3.5+0.2 μg/ml, respectively) whereas favorable selectivity was observed with all flavonoids. Structure-activity-relationship
(SAR) comparison of the isolated triterpenoids confirmed that the hydroxyl group at C-3 together with C-23, C-25, C-26
and C-30 methyl groups, C-12/C-13 double bond, the C-28 carboxylic acid group, and H-20 in ursolic acid (5) and related
compounds are all responsible for the strong anti-leishmanial activity. The 3-OH and 3′-OH in the apigenin (6) and related
compounds are responsible for the strong anti-protozoal activity observed in the isolated flavonoids. The strong to
moderate anti-leishmanial activity of the isolated triterpenes and flavonoids make them good candidates or templates for
new anti-protozoal drug development.
© 2013 Universal Research Publications. All rights reserved
Keywords: Acridocarpus chloropterus; antiprotozoal, cytotoxicity, SAR, triterpenes, flavonoids
in treatment of pemphigus (Dalziel, 1937). A decoction of
Introduction
Ethnobotanical information from many tropical countries the roots of Acridocarpus plagiopterus is used in Conakry,
indicates the use of several plant species in the genus Guinea as a vermifuge and as a remedy for sleeping
Acridocarpus as remedies for African trypanosomiasis, sickness (Ghazanfar & Al-Sabahi, 1993). Literature search
gastro-intestinal disorders, paralysis and pemphigus for previous phytochemical investigations on the genus
(Bennet & Alarcon, 1994; Dalziel, 1937; Watt & Breyer- Acridocarpus
only
revealed
phytochemical
and
Brandwijk, 1962; Ghazanfar & Al-Sabahi, 1993). The pharmacological information on Acridocarpus vivy J. Ar.
leaves of Acridocarpus orientalis are used in Oman as a collected from the rain forest in Madagascar. Bioassaytopical application for the treatment of paralysis (Bennet & guided fractionation of the cytotoxic methanolic stem bark
Alarcon, 1994) whereas the Tsonga and Shangana of South extract from A. vivy led to the isolation of eight cytotoxic
Africa use the powdered root of Acridocarpus natalitius as triterpenoids: acridocarpusic acid A-E, moronic acid,
a purgative for colic pains, constipation and as an ointment ursolic acid and oleanolic acid; together with two nonInternational Journal of Natural Products Research 2013; 3(4): 74-81
74
cytotoxic flavonoids: 4',5-dihydroxy-7-methoxyflavone
(genkwanin)
and
4',5-dihydroxy-3',7-methoxyflavone
(diosmetin) (Cao et al., 2004).
In East Africa, there is only two representative of this
genus: Acridocarpus chloropterus and Acridocarpus
zanzibaricus both being rare lianas occurring only in
Tanzania. Literature search revealed that there were no
previous phytochemical or pharmacological investigations
on A. chloropterus and A. zanzibaricus. Due to the use of
other plants in the genus as anti-protozoal agents, we
decided to investigate initially the A. chloropterus for its
anti-plasmodial, anti-trypanosomal, anti-leishmanial and
cytotoxic activity.
Methods and Materials
Plant Materials
The plant materials of Acridocarpus chloropterus Oliv. was
located in the Pugu Forest Reserve in Coast Region,
Tanzania by Mr. Leonard B. Mwasumbi, a qualified and
experienced Plant Taxonomist. The plant was collected and
then re-identified by Mr. Simon Mathenge, a Plant
Taxonomist at the University of Nairobi (Kenya) where
voucher specimen (HM 2004/01) was deposited in the
University Herbarium in the Department of Botany. The
plant materials (leaves, root-bark and stem-bark) were dried
under shade for 14 days and powdered.
Extraction, isolation and identification
Extraction
The plant powder (1.1 kg) was extracted sequentially with
solvents of increasing polarity (petroleum ether,
75
dichloromethane and methanol). The extraction was done
at room temperature for 48 hours with intermittent shaking;
the solvent extract was then filtered off. After filtration, the
solvent was removed under reduced pressure at 30 °C and
the resulting extracts were dried further under a stream of
nitrogen for 24 hours before being weighed and used for
biological assays.
Isolation
The dichloromethane (DCM) extract of dried stem bark of
A. chloropterus collected in Tanzania was fractionated by
medium pressure liquid chromatography (MPLC) (SiO2, nC6H14:EtOAc). MPLC was performed with a Büchi B-680
system, consisting of a Büchi B-688 chromatograpic pump,
a Büchi B-687 gradient former, a Knauer variable
wavelength monitor, a Knauer strip chart recorder and a
Büchi B-684 fraction collector. MPLC, column
chromatography (CC) (SiO2, n-C6H14-EtOAc, EtOAc–
MeOH) Sephadex LH-20 CC (CHCl3:MeOH), Preparative
TLC (SiO2, C6H5CH3:MeOH) was done using normal
phase silica gel 60 F254 (Merck) precoated on glass plates
(20 x 20 cm), with varying thickness (0.25, 0.5, 1.0 or
2.0 mm) leading to the isolation of ten known compounds:
5 triterpenes: β-sitosterol (1), stigmasterol (2), friedelin (3),
oleanolic acid (4), ursolic acid (5); and 5 flavonoids:
apigenin (6), luteolin (7), vitexin (8), kaempferol (9) and
quercetin (10).
Structure elucidation
All the isolated compounds were identified by their
physical (mp) and spectroscopic ( IR, UV, 1D and 2D
International Journal of Natural Products Research 2013; 3(4): 74-81
NMR, MS) properties and comparison with reported data.
Analytical TLC was performed on both aluminium and
plastic sheets precoated with silica gel 60 F254 (Merck) with
a 0.2 mm layer thickness. Melting points of re-crystallized
solids were measured on a Büchi B-540 apparatus and are
uncorrected. IR spectra were recorded on a Perkin Elmer
1600 FT-IR spectrophotometer using potassium bromide
pellets. Mass spectra were recorded on mass spectrometer
VG 70S (EIMS) and a Finnigan MAT 312 (FABMS). The
m/z values are reported in amu with the corresponding
relative intensities in parentheses. NMR spectra were
measured on Bruker Avance 400 (1H NMR 400 MHz; 13C
NMR 101 MHz), Bruker VRX 500 (1H NMR 500 MHz;
13
C NMR 125 MHz) and Bruker DRX 600 ( 1H NMR 600
MHz; 13C NMR 150.9 MHz). Deuterated solvents: CDCl3,
D2O, (CD3)2CO, DMSO-d6 and CD3OD were used in NMR
experiments. Chemical shifts were given in δ (ppm) values
with TMS (δ 0) as an internal standard. Unambiguous
assignment of 1H NMR and 13C NMR and chemical
connectivity of the molecules was confirmed through
HMQC, HMBC, COSY and NOESY experiments.
β-Sitosterol (1): white amorphous solid (252.6 mg), m.p.
138-140 °C, 1H NMR (CDCl3, 500 MHz) δ 1.08 (1H, m,
J=14.5, 3.6, 3.6, H-1ax), 1.84 (1H, m, J=14.5, 4.7, H-1eq),
1.51 (1H, m, J=15.4, 4.6, H-2ax), 1.83 (1H, m, J=11.2, 4.8,
H-2eq), 3.52 (1H, m, J=11.2, 4.8, H-3), 2.23 (1H, m,
J=13.2, 2.1, H-4ax), 2.30 (1H, m, J=1.9, 2.6, 2.6, H-4eq),
5.34 (1H, m, J=5.3, 1.9, H-6), 1.50 (1H, m, J=14.5, 2.1, H7ax), 1.97 (1H, m, J=14.3, H-7eq), 1.46 (1H, m, J=11.3,
11.2, H-8eq), 0.94 (1H, m, J=11.4, 4.9, H-9ax), 1.50 (1H,
m, J=13.2, 3.4, H-11ax), 1.60 (1H, m, J=13.2, 3.4, H-11eq),
1.18 (1H, m, J=3.1, H-12ax), 2.0 (1H, m, J=13.2, 3.2, H12eq), 1.01 (1H, m, J=10.6, 8.3, H-14ax), 1.06 (1H, m,
J=11.1, H-15ax), 1.56 (1H, m, H-15eq), 1.28 (1H, m,
J=10.8, 8.7, H-16ax), 1.72 (1H, m, J=10.7, H-16eq), 1.15
(1H, m, J=9.9, H-17ax), 0.70 (3H, s, H-18eq), 1.01 (3H, s,
H-19eq), 2.06 (1H, m, H-20), 0.92 (1H, d, H-21), 1.34 (1H,
m, H-22ax), 1.34 (1H, m, H-22eq), 1.31 (1H, m, H-23ax),
1.31(1H, m, H-23eq), 1.54(1H, m, H-24), 1.55(1H, m,
J=6.4, 6.4,H-25), 0.84(3H, d, J=7.0, H-26), 0.80(3H, d,
J=7.0, H-27), 1.18 (1H, m, H-28ax), 1.43 (1H, m, H-28eq),
0.85 (3H, t, J=7.0, H-29). 13C NMR (CDCl3, 500 MHz) δ
37.4 (t, C-1), 31.8 (t, C-2), 71.9 (d, C-3), 42.4 (s, C-4),
140.8 (s, C-5), 121.8 (d, C-6), 32.0 (t, C-7), 32.0 (d, C-8),
50.3 (d, C-9), 36.7 (s, C-10), 21.2 (t, C-11), 39.8 (t, C-12),
42.4 (s, C-13), 57.0 (d, C-14), 24.5 (t, C-15), 29.1 (t, C-16),
56.1 (d, C-17), 12.2 (q, C-18), 19.5 (q, C-19), 40.7 (d, C20), 21.4 (q, C-21), 33.91 (t, C-22), 26.02 (t, C-23), 51.4 (d,
C-24), 32.0 (d, C-25), 21.2 (q, C-26), 19.1 (q, C-27), 25.6
(t, C-28), 12.4 (q, C-29). MS: m/z 414 M+ (100%), 396
(48%), 381 (30%), 329 (35%), 303 (50%), 273 (25%), 255
(28%), 145 (38%), 107 (48%), 95 (43%), 81 (39%), 43
(52%). The molecular mass of 1 is m/z 414 amu which is
consistent with the formula C29H50O. All the data for
compound 1 were consistent with the reported values for βsitosterol (1) and was consistent with literature values
(Goad, 1991; Morales et al., 2003, De-Eknamkul &
Potduang, 2003). β-Sitosterol (1) has been reported to occur
in several plant species (Connolly & Hill, 1989).
Stigmasterol (2):white needles(320.3 mg), m.p.166-169 °C,
76
1
H NMR (CDCl3, 500 MHz) δ 1.05 (1H, m, J=14.5, 3.6,
3.6, H-1ax), 1.86 (1H, m, J=14.9, 4.7, H-1eq), 1.51 (1H, m,
J=15.4, 4.6, H-2ax), 1.84 (1H, m, J=11.2, H-2eq), 3.52 (1H,
m, J=11.2, 4.8, H-3), 2.24 (1H, m, J=13.2, 2.1, H-4ax), 2.27
(1H, m, J=1.9, 2.6, 2.6, H-4eq), 5.35 (1H, m, J=5.3, 1.9, H6), 1.49 (1H, m, J=14.5, 2.1, H-7ax), 1.96 (1H, m, J=14.3,
H-7eq), 1.45 (1H, m, J=11.3, 11.2, H-8eq), 0.95 (1H, m,
J=11.4, 4.9, H-9ax), 1.50 (1H, m, J=13.2, 3.4, H-11ax), 1.60
(1H, m, J=13.2, 3.4, H-11eq), 1.20 (1H, m, J=3.1, H-12ax),
2.00 (1H, m, J=13.2, 3.2, H-12eq), 1.01 (1H, m, J=10.6, 8.3,
H-14ax), 1.04 (1H, m, J=11.1, H-15ax), 1.55 (1H, m, H15eq), 1.27 (1H, m, J=10.8, 8.7, H-16ax), 1.71 (1H, m,
J=10.7, H-16eq), 1.15 (1H, m, J=9.9, H-17ax), 0.69 (3H, s,
H-18eq), 1.01 (3H, s, H-19eq), 2.04 (1H, m, H-20), 1.04 (3H,
d, J=6.6, H-21), 5.18 (1H, dd, J=8.8, 15.3, H-22), 5.03 (1H,
dd, J=8.3, 11.8, H-23), 1.51 (1H, m, H-24), 1.56 (1H, m,
J=6.4, 6.4, H-25), 0.87 (3H, d, J=6.5, H-26), 0.79 (3H, m,
J=6.5, H-27), 1.43 (1H, m, J=13.4, 3.6, H-28ax), 1.15 (1H,
m, J=7.3, 5.7, H-28eq), 0.81 (3H, t, J=7.5, H-29). 13C NMR
(CDCl3, 500 MHz) δ 37.4 (t, C-1), 31.8 (t, C-2), 71.9 (d, C3), 42.4 (t, C-4), 140.9 (s, C-5), 121.9 (d, C-6), 32.0 (t, C7), 32.0 (d, C-8), 50.3 (d, C-9), 36.7 (s, C-10), 21.2 (t, C11), 39.8 (t, C-12), 42.4 (s, C-13), 57.0 (d, C-14), 24.5 (t,
C-15), 29.1 (t, C-16), 56.1 (d, C-17), 12.2 (q, C-18), 19.5
(q, C-19), 40.7 (d, C-20), 21.4 (q, C-21), 138.5 (d, C-22),
129.4 (d, C-23), 51.4 (d, C-24), 32.0 (d, C-25), 21.2 (q, C26), 19.1 (q, C-27), 25.6 (t, C-28), 12.4 (q, C-29). The
molecular mass of 2 is m/z 412 amu which is consistent
with the formula C29H48O. MS: m/z 412 M+ (87%), 351
(35%), 255 (70%), 213 (27%), 159 (50%), 83 (70%),
55(100%). The spectroscopical data of 2 were consistent
with literature values (Goad, 1991; Morales et al., 2003,
De-Eknamkul & Potduang, 2003). Stigmasterol (2) has
been reported to occur in several plant species (Connolly &
Hill, 1989).
Friedelin (3): white needles (365.1 mg), m.p. 257-259 °C,
1
H NMR (CDCl3, 500 MHz) δ 1.68 (1H, m, J=14.0, H-1ax),
1.96 (1H, m, J=13.9, H-1eq), 2.30 (1H, m, J=14.0, H-2ax),
2.39 (1H, m, H-2eq), 2.24 (1H, m, J=14.0, H-4eq), 1.28 (1H,
m, H-6ax), 1.75 (1H, m, H-6eq), 1.38 (1H, m, H-7ax), 1.48
(1H, m, H-7eq), 1.39 (1H, m, H-8), 1.54 (1H, m, H-10),
1.26 (1H, m, H-11ax), 1.46 (1H, m, H-11eq), 1.35 (1H, m,
H-12ax), 1.35 (1H, m, H-12eq), 1.27 (1H, m, H-15ax), 1.46
(1H, m, H-15eq), 1.36 (1H, m, H-16ax), 1.57 (1H, m, H16eq), 1.54 (1H, m, H-18ax), 1.20 (1H, m, H-19ax), 1.38 (1H,
m, H-19eq), 1.30 (1H, m, H-21ax), 1.51 (1H, m, H-21eq),
0.94 (1H, m, H-22ax), 1.50 (1H, m, H-22eq), 0.87 (3H, m,
H-23), 0.71 (3H, s, H-24), 0.86 (3H, s, H-25), 1.00 (3H, s,
H-26), 1.05 (3H, s, H-27), 1.18 (3H, s, H-28), 1.00 (3H, s,
H-29), 0.95 (3H, s, H-30). 13C NMR (CDCl3, 500 MHz) δ
22.3 (t, C-1), 41.5 (t, C-2), 213.3 (s, C-3), 58.2 (d, C-4),
42.2 (s, C-5), 41.3 (t, C-6), 18.2 (t, C-7), 53.1 (d, C-8), 37.4
(s, C-9), 59.5 (d, C-10), 35.6 (t, C-11), 30.5 (t, C-12), 39.7
(s, C-13), 38.3 (s, C-14), 32.8 (t, C-15), 36.0 (t, C-16), 30.0
(s, C-17), 42.8 (d, C-18), 35.3 (t, C-19), 28.2 (s, C-20), 32.4
(t, C-21), 39.3 (t, C-22), 6.8 (q, C-23), 14.7 (q, C-24), 18.0
(q, C-25), 20.3 (q, C-26), 18.7 (q, C-27), 32.1 (q, C-28),
31.8 (q, C-29), 35.0 (q, C-30). The molecular mass of
friedelin (3) is m/z 426 amu which is consistent with the
formula C30H50O. MS: m/z 426 M+ (50%), 411 (25%), 341
International Journal of Natural Products Research 2013; 3(4): 74-81
(15%), 341 (15%), 302 (40%), 273 (70%), 246 (40%), 218
(50%), 205 (65%), 163 (55%), 125 (95%), 95 (100%), 69
(100%), 55 (50%). All the data confirmed the structure of 3
as friedelin and was consistent with literature values
(Budzikiewicz et al., 1963; Connolly & Hill, 1989; Klass et
al, 1992). Friedelin (3) has been previously obtained from
several plant species (Budzikiewicz et al., 1963; Connolly
& Hill, 1989; Klass et al, 1992).
Oleanolic acid (4): white needles (28.0 mg), m.p. 301-303
°C, 1H NMR (CDCl3, 500 MHz) δ 0.98 (1H, m, H-1ax),
1.64 (1H, m, H-1eq), 1.61 (1H, m, H-2ax), 1.63 (1H, m, H2eq), 3.22 (1H, dd, J=10.0, 4.8, H-3eq), 0.75 (1H, dd,
J=12.0, 2.0, H-5), 1.38 (1H, m, J=12.0, 2.0, H-6ax), 1.56
(1H, m, J=2.0, 3.0, 9.0, H-6eq), 1.30 (1H, m, J=2.0, 3.0, 9.0,
H-7ax), 1.55 (1H, m, J=9.0, H-7eq), 1.56 (1H, dd, J=6.0,
12.0, H-9), 1.61 (1H, m, J=4.0, 11.0, 12.0, H-11ax), 1.64
(1H, m, J=4.0, 6.0, 11.0, H-11eq), 5.28 (1H, m, J=4.0, H12), 1.08 (1H, m, J=4.0, 13.6, 14.0, H-15ax), 1.11 (1H, m,
J=4.0, 3.0, 14.0, H-15eq), 1.88 (1H, m, J=4.0, 13.0, 13.6, H16ax), 1.88 (1H, m, J=4.0, 13.0, 3.0, H-16eq), 2.82 (1H, dd,
J=11.0, H-18ax), 1.15 (1H, m, J=11.0 H-19ax), 1.18 (1H, m,
J=2.0 H-19eq), 1.22 (1H, m, J=2.2, 14.0, 13.0, H-21ax), 1.35
(1H, m, J=1.3, 2.9, 13.0, H-21eq), 1.43 (1H, m, J=1.3, 14.0,
13.0, H-22ax), 1.78 (1H, m, J=2.1, 3.0, 13.0, H-22eq), 0.99
(3H, s, H-23), 0.77 (3H, s, H-24), 0.92 (3H, s, H-25), 0.75
(3H, s, H-26), 1.14 (3H, s, H-27), 0.91 (3H, s, H-29), 0.93
(3H, s, H-30). 13C NMR (CDCl3, 500 MHz) δ 38.9 (t, C-1),
27.3 (t, C-2), 79.2 (d, C-3), 38.5 (s, C-4), 55.3 (d, C-5),
18.5 (t, C-6), 32.6 (t, C-7), 39.4 (s, C-8), 47.8 (d, C-9), 37.2
(s, C-10), 23.1 (t, C-11), 122.8 (d, C-12), 143.8 (s, C-13),
41.7 (s, C-14), 27.8 (t, C-15), 23.5 (t, C-16), 46.7 (s, C-17),
41.1 (d, C-18), 46.0 (t, C-19), 30.8 (s, C-20), 33.9 (t, C-21),
32.7 (t, C-22), 28.2 (q, C-23), 15.7 (q, C-24), 15.5 (q, C25), 17.3 (q, C-26), 26.1 (q, C-27), 183.3 (s, C-28), 33.2 (q,
C-29), 23.7 (q, C-30). Oleanolic acid (4) exhibited a
molecular ion peak at m/z 456 [M+] in LREIMS which
corresponded to the formula C30H48O3. MS: m/z 456 M+
(75%), 438 (70%), 423 (70%), 395 (50%), 300 (100%). All
the data confirmed the structure of 4 as oleanolic acid and
was consistent with literature values (Ikuta & Itokawa,
1988; Conrad et al., 1998; Klass et al, 1992; Mahato &
Kundu, 1994; Takahashi et al., 1999; Thanakijcharoenpath
& Theanphong, 2005). Oleanolic acid (4) has been
previously isolated from Acridocarpus vivy collected from
Madagascar rain forest (Cao et al., 2004) and from other
several plant species (Connolly & Hill, 1989; Ikuta &
Itokawa, 1988; Conrad et al., 1998; Klass et al, 1992;
Takahashi et al., 1999; Thanakijcharoenpath &
Theanphong, 2005).
Ursolic acid (5): white powder (12.0 mg), m.p. 268-270 °C.
1
H NMR (DMSO, 600 MHz) δ 1.00 (1H, m, J=2.8, 13.0,
H-1ax), 1.53 (1H, m, J=2.8, 3.4, 13.0, H-1eq), 1.80 (1H, m,
J=9.0, 2.8, 13.0, H-2ax), 1.80 (1H, m, J=6.0, 3.4, 13.0, H2eq), 3.14 (1H, dd, J=10.0, 4.8, H-3eq), 0.68 (1H, dd,
J=12.0, 2.0, H-5), 1.28 (1H, m, J=12.0, 10.0, H-6ax), 1.48
(1H, m, J=2.0, 3.0, 9.0, H-6eq), 1.26 (1H, m, J=2.0, 3.0, 9.0,
H-7ax), 1.45 (1H, m, J=9.0, H-7eq), 1.46 (1H, m, J=6.0,
12.0, H-9), 1.83 (1H, m, J=4.0, 11.0, 12.0, H-11ax), 1.86
(1H, m, J=4.0, 6.0, 11.0, H-11eq), 5.21 (1H, t, J=4.0, H-12),
1.00 (1H, m, J=4.0, 13.6, 14.0, H-15ax), 1.80 (1H, m, J=4.0,
77
3.0, 14.0, H-15eq), 1.53 (1H, m, J=4.0, 13.0, 13.6, H-16ax),
1.92 (1H, m, J=4.0, 13.0, 3.0, H-16eq), 2.10 (1H, dd, J=2.0,
11.0, H-18ax), 1.31 (1H, dd, J=6.6, 11.3, H-19eq), 1.31 (1H,
m, J=2.0, 6.3, H-20), 1.27 (1H, m, J=2.2, 14.0, H-21ax),
1.43 (1H, m, J=13.0, 1.4, 3.0, 13.0, H-21eq), 1.51 (1H, m,
J=1.4, 1.4, H-22ax), 1.58 (1H, m, J=13.0, 2.2, 3.0, 13.0, H22eq), 0.89 (3H, s, H-23), 0.67 (3H, s, H-24), 0.86 (3H, s,
H-25), 0.74 (3H, s, H-26), 1.04 (3H, s, H-27), 0.81 (3H, d,
J=6.6, H-29), 0.91 (3H, d, J=6.3, H-30). 13C NMR (DMSO,
600 MHz) δ 38.0 (t, C-1), 27.0 (t, C-2), 76.8 (d, C-3), 38.2
(s, C-4), 54.8 (d, C-5), 18.0 (t, C-6), 32.7 (t, C-7), 38.5 (s,
C-8), 47.0 (d, C-9), 36.5 (s, C-10), 22.8 (t, C-11), 124.6 (d,
C-12), 138.2 (s, C-13), 41.6 (s, C-14), 27.5 (t, C-15), 23.8
(t, C-16), 46.8 (s, C-17), 52.4 (d, C-18), 38.4 (d, C-19),
38.4 (d, C-20), 30.2 (t, C-21), 36.3 (t, C-22), 28.2 (q, C-23),
16.1 (q, C-24), 15.2 (q, C-25), 16.8 (q, C-26), 23.4 (q, C27), 178.3 (s, C-28), 18.0 (q, C-29), 21.1 (q, C-30).
Molecular ion peak was observed at m/z 456 in LREIMS
corresponding to the formula C30H48O3. MS: m/z 456 M+
(80%), 423 (72%), 395 (65%), 300 (100%), 250 (92%). All
the data confirmed the structure of 5 as ursolic acid and
was consistent with literature values (Connolly & Hill,
1989; Ikuta & Itokawa, 1988; Conrad et al., 1998; Klass et
al, 1992; Mahato & Kundu, 1994; Takahashi et al., 1999;
Thanakijcharoenpath & Theanphong, 2005). Ursolic acid
(5) has been previously isolated from Acridocarpus vivy
collected from the Madagascar rain forest (Cao et al., 2004)
and from several plant species (Connolly & Hill, 1989;
Ikuta & Itokawa, 1988; Conrad et al., 1998; Klass et al,
1992; Takahashi et al., 1999; Thanakijcharoenpath &
Theanphong, 2005).
4′,5,7-trihydroxyflavone (Apigenin) (6): yellow powder
(8.21 mg), m.p. 350-352 °C. 1H NMR (DMSO, 500 MHz)
δ 6.75 (1H, s, H-3), 6.15 (1H, d, J=2.08, H-6), 6.44 (1H, d,
J=2.08, H-8), 7.91 (1H, d, J=8.90, H-2′), 6.92 (1H, d,
J=8.90, H-3′), 6.92 (1H, d, J=8.90, H-5′), 7.91 (1H, d,
J=8.90, H-6′), 12.96 (1H, s, 5-OH). 13C NMR (DMSO, 500
MHz) δ 165.2 (s, C-2), 102.7 (d, C-3), 181.6 (s, C-4), 103.3
(s, C-4a), 161.4 (s, C-5), 99.1 (d, C-6), 163.6 (s, C-7), 94.1
(d, C-8), 157.4 (s, C-9), 121.1 (s, C-1′), 128.5 (d, C-2′),
116.0 (d, C-3′), 161.3 (s, C-4′), 116.0 (d, C-5′), 128.5 (d, C6′). LREIMS gave the molecular ion M+ at m/z 270,
corresponding to C15H10O5 as the formula consistent with
the structure 4′, 5, 7-trihydroxyflavone (apigenin) (6). MS:
m/z 270 M+ (100%), 242 (20%), 153 (18%), 121 (12%), 44
(14%). Comparison of the observed and reported data
confirmed the structure of 6 as 4′, 5, 7-trihydroxyflavone
(apigenin) (Agrawal, 1989; Shen et al., 1993). Apigenin (6)
has also been isolated from many other plant species
including Acacia spp., Alnus spp., Barleria christata,
Betula spp. and Combretum spp (Wollenweber & Jay,
1988).
3′,4′, 5,7-tetrahydroxyflavone (Luteolin) (7): yellow
crystals (6.0 mg), m.p. 323-325 °C. 1H NMR (DMSO, 500
MHz) δ 6.67 (1H, s, H-3), 6.18 (1H, d, J=2.1, H-6), 6.44
(1H, d, J=2.1, H-8), 7.39 (1H, d, J=8.3, H-2′), 6.88 (1H, d,
J=8.3, H-5′), 7.41 (1H, d, J=8.3, 2.3, H-6′), 12.98 (1H, s, 5OH). 13C NMR (DMSO, 500 MHz) δ 164.3 (s, C-2), 102.8
(d, C-3), 181.7 (s, C-4), 103.6 (s, C-4a), 161.5 (s, C-5),
98.9 (d, C-6), 163.9 (s, C-7), 93.9 (d, C-8), 157.3 (s, C-9),
International Journal of Natural Products Research 2013; 3(4): 74-81
121.4 (s, C-1′), 113.3 (d, C-2′), 145.8 (s, C-3′), 149.8 (s, C4′), 116.0 (d, C-5′), 119.0 (d, C-6′). LREIMS revealed the
molecular ion M+ at m/z 286, which corresponds to the
formula C15H10O6. MS: m/z 286 M+ (100%), 258 (22%),
229 (6%), 153 (27%), 69.1 (9%), 57.1 (12%). Luteolin (7)
is one of several flavonoids widely distributed in the plant
kingdom and it has aready been isolated from many other
plant species including Acacia spp., Alnus spp., Barleria
christata, Betula spp. and Combretum spp (Wollenweber &
Jay, 1988).
8-glucopyranosyl-4′,5,7-trihydroxyflavone (Vitexin) (8):
pale yellow crystals (4.2 mg), m.p. 269-270 °C. 1H NMR
(CDCl3, 500 MHz) δ 6.78 (1H, s, H-3), 6.25 (1H, s, H-6),
8.02 (1H, d, J=8.40, H-2′), 6.89 (1H, d, J=8.40, H-3′), 6.89
(1H, d, J=8.40, H-5′), 8.02 (1H, d, J=8.40, H-6′), 4.69 (1H,
d, J=10.2, H-1″), 3.85 (1H, dd, J=10.2, 10.2, H-2″), 3.28
(1H, d, J=10.2, H-3″), 3.36 (1H, d, J=10.2, H-4″), 3.24
(1H, m, H-5″), 3.76 (1H, dd, J=12.1, 6.8, H-6ax″), 3.52
(1H, dd, J=12.1, 6.2, H-6eq″), 4.58 (1H, br s, OH), 4.95
(1H, br s, OH), 4.95 (1H, br s, OH), 13.17 (1H, br s, 5OH). 13C NMR (CDCl3, 500 MHz) δ 163.9 (s, C-2), 102.4
(d, C-3), 182.1 (s, C-4), 103.9 (s, C-4a), 160.4 (s, C-5),
98.2 (d, C-6), 156.0 (s, C-7), 104.6 (s, C-8), 161.2 (s, C-9),
121.6 (s, C-1′), 129.0 (d, C-2′), 115.8 (d, C-3′), 160.4 (s, C4′), 115.8 (s, C-5′), 129.0 (s, C-6′), 73.4 (d, C-1″), 70.9 (d,
C-2″), 78.7 (d, C-3″), 70.5 (d, C-4″), 81.9 (d, C-5″), 61.3 (t,
C-6″). LREIMS gave a peak [M+2]+ at m/z 434
corresponding to the formula C21H20O10 consistent with the
structure 8-glucopyranosyl-4′, 5, 7-trihydroxyflavone
(vitexin) (8). MS: m/z 434 [M+2]+ (100%), 414 (10%), 396
(10%), 378 (30%), 342 (40%), 324 (87%), 312 (100%), 283
(100%), 254 (50%), 165 (100%), 121 (38%), 43 (55%).
Vitexin (8) is one of several flavonoids widely distributed
in the plant kingdom and it has aready been isolated from
many other plant species including Acacia spp., Alnus spp.,
Barleria christata, Betula spp. and Combretum spp
(Wollenweber & Jay, 1988).
3,4′,5,7-tetrahydroxyflavone (Kaempferol) (9): pale yellow
needles (3.8 mg), m.p. 276-278 °C. 1H NMR (DMSO, 500
MHz) δ 6.19 (1H, d, J=1.9, H-6), 6.45 (1H, d, J=1.9, H-8),
8.04 (1H, d, J=8.9, H-2′), 6.93 (1H, d, J=8.9, H-3′), 6.93
(1H, d, J=8.9, H-5′), 8.04 (1H, d, J=8.9, H-6′), 9.35 (1H,
br s, 3-OH), 10.10 (1H, br s, 4-OH), 12.48 (1H, br s, 5OH), 10.85 (1H, s, 7-OH). 13C NMR (DMSO, 500 MHz)
δ 146.8 (s, C-2), 135.6 (s, C-3), 175.9 (s, C-4), 103.1 (s, C4a), 160.7 (s, C-5), 98.2 (d, C-6), 163.8 (s, C-7), 93.5 (d, C8), 156.2 (s, C-9), 121.7 (s, C-1′), 129.5 (d, C-2′), 115.4 (d,
C-3′), 159.1 (s, C-4′), 115.5 (d, C-5′), 129.5 (d, C-6′).
LREIMS exhibited the molecular ion M+ at m/z 286, which
corresponds to the formula C15H10O6. MS: m/z 286 M+
(100%), 229 (9%), 121 (15%). Kaempferol (9) is one of
several flavonoids widely distributed in the plant kingdom
and it has already been isolated from many other plant
species including Acacia spp., Alnus spp., Barleria
christata, Betula spp. and Combretum spp (Wollenweber &
Jay, 1988; Marbry et al., 1970; Markham, 1982; Lin et al.,
2001; Vega et al., 2007).
3,3′,4′,5,7-pentahydroxyflavone (Quercetin) (10): dark
yellow powder (5.8 mg), m.p. 313-315 °C. 1H NMR
(DMSO, 500 MHz) δ 6.19 (1H, d, J=2.0, H-6), 6.41 (1H, d,
J=2.0, H-8), 7.69 (1H, d, J=2.2, H-2′), 6.89 (1H, d, J=8.5,
H-5′), 7.55 (1H, d, J=8.5, 2.2, H-6′), 12.98 (1H, s, 5-OH).
13
C NMR (DMSO, 500 MHz) δ 145.1 (s, C-2), 135.8 (s, C3), 175.9 (s, C-4), 103.1 (s, C-4a), 160.8 (s, C-5), 98.3 (d,
C-6), 164.0 (s, C-7), 93.41 (d, C-8), 156.2 (s, C-9), 122.0
(s, C-1′), 115.10 (d, C-2′), 146.9 (s, C-3′), 147.8 (s, C-4′),
115.7 (d, C-51′), 120.1 (d, C-6′). LREIMS gave the
molecular ion M+ at m/z 302, which corresponds to the
formula C15H10O7. MS: m/z 302 M+ (100%), 137 (9%), 69
(7%). Quercetin (10) is one of several flavonoids widely
distributed in the plant kingdom and it has aready been
isolated from many other plant species including Acacia
spp., Alnus spp., Barleria christata, Betula spp. and
Combretum spp (Wollenweber & Jay, 1988; Marbry et al.,
1970; Markham, 1982; Lin et al., 2001).
Table 1: Anti-protozoal activity (IC50)and cytotoxicity (CC50)data for compounds isolated from Acridocarpuschloropterus
P. falciparum K1
T. b. rhodesiense
L. donovani
L-6 cells
IC50
IC50
IC50
CC50
Compound
SI
SI
SI
S.E(μg/ml)
S.E(μg/ml)
S.E(μg/ml)
S.E(μg/ml)
β-Sitosterol (1)
>5.0
>6.6
33.5+0.01
1.0
>30.0
>1.1
32.8+0.00
Stigmasterol (2)
>5.0
>18.0
6.4+0.00
>14.1
>30.0
>3.0
>90
Friedelin (3)
>5.0
>18.0
56.6+0.00
>1.6
>30.0
>1.1
>90
Oleanolic acid (4)
>5.0
>8.5
16.3+0.01
2.6
3.5+0.00
12.1
42.3+0.00
Ursolic acid (5)
>5.0
>1.4
7.8+0.00
0.9
0.8+0.00
8.9
7.1+0.00
Apigenin (6)
>5.0
>7.9
9.0+0.00
4.4
5.9+0.00
6.7
39.4+0.00
Luteolin (7)
>5.0
>18
35.4+0.00
2.5
11.8+0.00
7.6
>90
Vitexin (8)
>5.0
>18
14.9+0.01
6.0
>30.0
>3.0
>90
Kaempferol (9)
>5.0
>5.2
16.4+0.00
1.6
2.2+0.00
11.9
26.1+0.00
Quercetin (10)
2.6+0.05
>8.0
3.6+0.00
5.9
2.1+0.00
11.9
21.2+0.01
P. falciparum – K1 strain,T.b. rhodesiense– STIB 900 strain, L. donovani - MHOM-ET-67/L82, L-6 - rat skeletal myoblast
cells, IC50 – inhibitory concentration for 50% of tested parasites, CC50 – cytotoxic concentration for 50% of tested cells,
chloroquine IC50 0.063+0.03, artemisinin IC50 0.002+0.000, melarsoprol IC50 0.002+0.000, militefosine IC50 0.11+0.001,
Pdx - podophyllotoxin IC50 0.009+0.000
In vitro anti-plasmodial assays
The anti-plasmodial activity was evaluated against the
multi-drug resistant Plasmodium falciparum K1 strain
78
(resistant to chloroquine and pyrimethamine), using the
method of Trager and Jensen (1976) with slight
modifications (Desjardins et al.,1979; Matile & Pink, 1990)
International Journal of Natural Products Research 2013; 3(4): 74-81
In vitro anti-trypanosomal assay
The in vitro antitrypanosomal activity was evaluated
against Trypanosoma brucei rhodesiense STIB 900 strain,
using the method of Räz et al. (1997).
In vitro anti-leishmanial assay
The in vitro anti-leishmanial assay was carried out against
Leishmania donovani MHOM-ET-67/82 strain according to
the procedure of Räz et al. (1997).
Cytotoxicity assay and drug selectivity index
The in vitro cytotoxicity assay was carried out against rat
skeletal myoblast (L-6) cells according to standard
procedure of Räz et al. (1997).
Results and Discussion
The anti-parasitic activity and cytotoxicity of the isolated
compounds were tested using Plasmodium falciparum,
Trypanosoma brucei rhodesiense, Leishmania donovani
and rat myoblast L-6 cells, respectively, in vitro. The
results are summarized in table 1.
Quercetin (10) exhibited moderate anti-plasmodial activity
(IC50 2.6+0.05 µg/ml) and moderate selectivity index (SI
>8). The remaining 9 compounds showed mild activity
(IC50 >5.0 µg/ml). Similarly, quercetin (10) was moderately
active against T. b. rhodesiense STIB 900 strain (IC50 3.6 +
0.00 µg/ml) in vitro with narrow selectivity (SI 5.9).
Ursolic acid (5), β-stigmasterol (2) and apigenin (6)
showed mild anti-trypanosomal activity (IC50 6.4+0.059.0+0.00 µg/ml) whereas the remaining six compounds
were inactive (IC50 16.3+0.01-56.6+0.01 µg/ml) with poor
selectivity (SI 1.6 – 6.0).
Ursolic acid (5) exhibited strong anti-leishmanial activity
(IC50 0.8+0.000 µg/ml) in vitro and moderate selectivity (SI
8.9). Quercetin (10), kaempferol (9) and oleanolic acid (4)
exhibited moderate activity (IC50 2.1 + 0.00 – 3.5 + 0.00
µg/ml) and moderate selectivity (SI 11.1). Apigenin (6)
(IC50 5.9+0.00 µg/ml) showed mild activity while the
remainingfourcompoundswereinactive(IC5011.8+0.01-30.0
µg/ml) with very narrow or no selectivity (SI >1.1 - >3.0).
Based on the molecular framework of isolated triterpenes
and flavonoids, the relationship between in vitro antiprotozoal, cytotoxicity activity and chemical structures
were examined with respect to different functional groups
therein. Structural comparison between β-sitosterol (1) and
stigmasterol (2) revealed the presence of an olefinic bond at
C-22/C-23 in the Δ5-sterol (Forgo & Köver, 2004) as the
only difference (Connolly & Hill, 1991; Budzikiewicz et
al., 1963; De-Eknamkul & Potduang, 2003). The presence
of an olefinic bond at C-22/C-23 in stigmasterol (2) had no
effect on anti-plasmodial and anti-leishmanial activity, but
significantly increased the anti-trypanosomal activity 5.2
fold (IC50 33.5+0.01 to 6.4+0.00 µg/ml) compared to βsitosterol (1). However, the presence of the olefinic bond at
C-22/C-23 in stigmasterol (2) resulted in decrease in
cytotoxicity >2.7 fold (CC50 32.8+0.00 µg/ml to >90 µg/ml)
confirming that the observed differences in bioactivity is
due the replacement of the single-bond with the doublebond at C-22/C-23. A corresponding increase in selectivity
from 1.1 to >14.1 was also observed.
The relationships between the in vitro anti-protozoal
activity, cytotoxicity and chemical structure of friedelin (3)
and oleanolic acid (4) and ursolic acid (5) were
79
investigated. Structural comparison of friedelin (3) and
oleanolic acid (4) revealed the replacement of several
functional groups: carbonyl with OH, H-4 with 24-CH3, H10 with 25-CH3, H-8 with 26-CH3, single bond at C-12/C13 with double bond and 28-CH3 with CO2H (Ikuta &
Itokawa, 1988; Klass et al., 1992; Mahato & Kundu, 1994).
Although the replacements had no effect on anti-plasmodial
activity, they significantly increased anti-trypanosomal
(IC50 56.6+0.001 to 16.3+0.001 µg/ml) and anti-leishmanial
activity (IC50 >30.0 to 3.5+0.00 µg/ml). However, the
replacements also resulted in a 2.1 fold increase in
cytotoxicity (CC50 >90.0 to 42.3+0.00 µg/ml), suggesting
that the observed differences in bioactivity may be partly
due to cytotoxicity. Similarly, the structural comparison of
friedelin (3) and ursolic acid (5) revealed the replacement
of several functional groups: the carbonyl group with OH,
H-4 with 24-CH3, H-10 with 25-CH3, H-8 with 26-CH3,
single bond at C-12/C-13 with double bond, H-19 with 30CH3, 24-CH3 with H-5, 28-CH3 with CO2H, and 30CH3with H-20. Although the changes in structure had no
effect on anti-plasmodial activity, they significantly
increased the anti-trypanosomal (IC50 56.6+0.001 to
7.8+0.00 µg/ml) and anti-leishmanial activity (IC50 >30.0 to
0.8+0.00 µg/ml). Furthermore, the structural changes
resulted in a 12.7 fold increase in cytotoxicity (CC50 >90.0
to 7.1+0.00 µg/ml), suggesting that the observed
differences in bioactivity may partly be due to cytotoxicity.
Further structural comparison of oleanolic acid (4) and
ursolic acid (5) revealed only the replacement of two
functional groups: H-19 with 30-CH3 and 30-CH3 with H20. Although the structural changes had no effect on antiplasmodial activity of ursolic acid (5), they significantly
increased the anti-trypanosomal (IC50 16.3+0.01 to
7.8+0.00 µg/ml) and anti-leishmanial (IC50 3.5 + 0.00 to 0.8
+ 0.00 µg/ml) activity corresponding to 2.1 and 4.4 fold,
respectively. The changes also resulted in a 6 fold increase
in cytotoxicity (CC50 42.3 + 0.00 to 7.1 + 0.00 µg/ml),
suggesting that the observed differences in bioactivity of
the two compounds may be partly due to cytotoxicity. The
strong to moderate anti-leishmanial activity observed in the
isolated triterpenes and flavonoids render them good
candidates as molecular templates for new anti-protozoal
drug development.
Apigenin (6) was used as a template to examine the
relationships between in vitro anti-protozoal activity,
cytotoxicity and chemical structures of the isolated
flavonoids. The structural comparison of apigenin (6) and
luteolin (7) revealed only the replacement of H-3′ with OH
(Marbry et al., 1970; Lin et al., 2001; Burns et al., 2007).
Although the changes had no effect on anti-plasmodial
activity, they significantly reduced the anti-trypanosomal
(IC50 9.0+0.00 to 35.4+0.00 µg/ml) and anti-leishmanial
activity (IC50 5.9+0.00 to 11.8+0.00 µg/ml). They also
resulted in the >2.3 fold decrease in cytotoxicity (CC50 39.4
+ 0.00 to >90.0 µg/ml), suggesting that the observed
differences in bioactivity of the two compounds may only
be due to the corresponding change in cytotoxicity.
Similarly, the structural comparison of apigenin (6) and
vitexin (8) revealed the replacement of H-8 with glycoside
unit (Agrawal, 1989; Lin et al., 2001; Burns et al., 2007).
International Journal of Natural Products Research 2013; 3(4): 74-81
The structural change did not significant affect the antiplasmodial activity but remarkably reduced the antitrypanosomal (IC50 9.0+0.00 to 14.9+0.01 µg/ml), antileishmanial (IC50 5.9+0.00 to >30.0 µg/ml) and cytotoxic
activity (CC50 39.4+0.00 to >90.0 µg/ml). The observed
differences in the bioactivity of the two compounds may be
due to their cytotoxicity levels.
The replacement of H-3 in apigenin (6) with OH gave
kaempferol (9) (Agrawal, 1989; Burns et al., 2007). The
change and had no effect on the anti-plasmodial activity but
remarkably reduced the anti-trypanosomal (IC50 9.0+0.00 to
16.4+0.01 µg/ml) activity. However, there was an increase
in anti-leishmanial activity (IC50 5.9+0.00 to 2.2+0.00
µg/ml) and cytotoxicity (CC50 39.4+0.00 to 26.1+0.00
µg/ml) suggesting that the observed change in activity is
due to cytoxicity. The replacement of both H-3′ and H-3 in
apigenin (6) with OH gave quercetin (10) (Markham, 1982;
Agrawal, 1989) with improved anti-protozoal activity: antiplasmodial (IC50 >5.0 to 2.6+0.05 µg/ml), and antitrypanosomal (IC50 9.0+0.00 to 3.6+0.00 µg/ml). However,
cytotoxicity also increased significantly (CC50 39.4+0.00 to
21.2+0.01 µg/ml) indicating that the observed
improvements in anti-protozoal activity may be partly due
to cytotoxicity. Quercetin (10) exhibited the strongest antiprotozoal activity among the isolated flavonoids indicating
that, the 3-OH and 3′-OH in the apigenin (6) molecular
framework contribute substantially to the strong antiprotozoal activity. The presence of the phenolic group at
the C-3 of apigenin (6) as in kaempferol (9) increased
cytotoxicity which is enhanced further by addition of
phenolic group at the C-3′ as in quercetin (10).
Conclusion
The present phytochemical and pharmacological results
indicate that A. chloropterus exhibits a wider array of
biological activities, which could be attributed to its content
of triterpenes and flavonoids. Structure-activityrelationship (SAR) comparison of the isolated triterpenoids
confirmed that the hydroxyl group at C-3 together with C23, C-25, C-26 and C-30 methyl groups, C-12/C-13 double
bond, the C-28 carboxylic acid group, and H-20 in ursolic
acid (5) and related compounds are all responsible for the
strong anti-leishmanial activity. The 3-OH and 3′-OH in the
apigenin (6) and related flavonoids are responsible for the
strong anti-protozoal activity. The strong to moderate antileishmanial activity of the isolated triterpenes and
flavonoids make them good candidates or templates for
new anti-protozoal drug development.
Acknowledgements
The German Academic Exchange Program (DAAD) is
appreciated for supporting this work through a scholarship
grant A/03/44009 to H.M.M. The Basel Canton Stipend
Commission for the research visit grant to H.M.M,
Kenyatta University, the National Institute for Medical
Research (NIMR), The Institute of Organic Chemistry
(UNIBAS), Institute of Pharmaceutical Biology
(PharmBio, UNIBAS) and the Swiss Tropical Institute
(STI) are thanked for supporting the research project. Mr.
Leonard B. Mwasumbi of the Botany Department of the
University of Dar es Salaam in Tanzania and Mr. Simon
Mathenge of the Botany Department at the University of
80
Nairobi in Kenya, are thanked for taxonomical
identification of Acridocarpus chloropterus.
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Source of support: The German Academic Exchange Program (DAAD),
Grant No: A/03/44009; Conflict of interest: None declared
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