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Laboratoire de Pharmacognosie et des Huiles Essentielles. Facult des Sciences de la Sant (FSS). Facult
des Sciences Techniques (FAST). Universit dAbomey-Calavi (UAC), 01 BP: 188 Cotonou, Bnin.
b
CHAM unit, laboratoire de pharmacognosie. Universit Catholique de Louvain UCL 72 30-CHAM, Av
Mounier 72, B-1200 Bruxelles, Belgium.
* corresponding author. Tel.: (+ 229) 21 30 90 77; (+229) 97 44 88 87.
e-mail: ahokannou@yahoo.fr
Received November 2006; revised Februry2007, accepted Februry2007.
Abstract
Purification of the allelopathic ethanol extract of Justicia anselliana aerial part led to the isolation of three
known compounds: two sterols (stigmasterol and -sitosterol) and one triterpene (lupeol). Their structures
were determined by analysis of spectroscopic data and confirmed by GC/MS analysis. All isolated
compounds were tested for their allelopathic effects on cowpea (Vigna unguiculata) growth and for their
cytotoxic activity on human (HeLa, WI-38 and Mel-43) and mouse (J774) cells. All isolated compounds
showed an inhibitory effect on the three parameters measured on cowpea (Vigna unguiculata) germination
(rate of germination, shoot length and fresh weight). The highest rate of inhibition of cowpeas germination
was observed with 200 ppm of lupeol (-28.4 % 0.7%) whereas all three parameters were inhibited very
meaningfully by 200 ppm stigmasterol (-26.6% 0.5%; -24.2% 0.7% and -21.1% 0.7% respectively for
germination, shoot length and fresh weight of Vigna unguiculata (L.) Walp). None of the isolated
compounds was toxic on J774 (murine macrophages), WI-38 (human lung fibroblasts), human HeLa
(human cervix carcinoma cells) and melanoma Mel-43 cell. This is the first report of the allelopathic
activities of lupeol, stigmasterol and -sitosterol on cowpea (Vigna unguiculata).
Key Words: Justicia anselliana, purification, sterols, triterpene, allelopathy, Vigna unguiculata (cowpea),
cytotoxicity
Introduction
Vigna unguiculata (L) Walp (cowpea) is cultivated
over more than nine million hectares in all the
tropical area, in the mediteranean basin and in the
United States (Guigaz, 2002). Worldwide, it is
estimated that 37 million tones are annually
produced (Langyintuo et al., 2003). Cowpea is a
source of relatively low cost, high quality protein
which contained adequate levels of most essential
amino acids for pre-school children and all essential
amino acids for adults. Their digestibility is higher
than the one of other common legumes (Alessandra
et al., 2004).
It is the most consumed leguminous: its seeds
reduced into flour are used to make fritter,
particularly in Benin and in some of the West
African countries.
In south Benin, cowpea cultivation takes the
lead on the economical level because of the
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60
Germination
Shoot length
40
Fresh w eight
20
0
-20
-40
-60
*
*
-80
-100
*
*
* *
-120
Name of extract
Figure 1: Effects at 600 ppm of Justicia anselliana soxhlet fractions on cowpea (Vigna unguiculata). N =6, P* < 0.05. A = hexane; B =
CHCl3; C = EtOAc; D = MeOH; E = EtOH The upwards evolution corresponds to a stimulation of the parameters measured while the
downwards evolution indicates an inhibition.
29
20
30
12
25
11
26 13
1
2
14
10
17
16
241
21
22
22
18
28
1
27
11
9
10
HO
23
20
12
19
15
8
7
HO
19
24
18
242
21
17
13
14
26
23
24
25
27
16
15
7
242
241
21
18
12
19
1
2
10
11
9
HO
13
14
8
7
22
20
17
26
23
24
25
27
16
15
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Table 1: NMR data of 1, 2 and 3 (400.13 MHz for 1H and 100.62 MHz for 13C; in CDCl3 and using tetramethylsilane (TMS) as an internal
standard.)
1
Position
13
2
1
13
C(ppm)
38.7
27.5
79.0
H(ppm)
38.7m
0.90m
1.59m
1.67m
3.20dd
38.9
42.3
5
6
55.3
18.3
140.8
121.7
34.3
8
9
10
11
40.8
50.5
37.2
20.9
12
25.2
13
14
15
38.1
42.8
27.5
16
35.6
17
18
19
20
21
43.0
48.3
48.0
151.0
29.9
22
40.0
23
24
25
26
27
28
29
28.0
15.4
16.1
16.0
14.6
18.0
109.3
30
241
19.3
0.68m
1.40m
1.50m
1.32m
1.42m
_
1.29m
_
1.20m
1.40m
1.07m
1.68m
1.68m
_
1.00m
1.68m
1.37m
1.48m
_
1.37m
2.38ddd
_
1.37m
1.92m
1.37m
1.19m
0.97s
0.76s
0.83s
1.03s
0.94s
0.79s
4.54d
4.67d
1.68s
242
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C(ppm)
37.3
31.9
71.8
31.9
31.9
50.2
36.5
21.2
39.7
3
1
H(ppm)
1.84 m
1.08 m
1.83 m
1.51 m
3.51 m
2.30 m
2.23 m
_
5.33 m
1.97 m
1.50 m
1.46 m
0.94 m
_
1.50 m
13
C(ppm)
37.3
31.6
1.37 m
71.8
3.82 m
42.3
2.62 m
140.8
121.7
_
5.32 t
31.9
1.93 m
31.7
50.2
36.5
21.1
1.54 m
0.94 m
_
1.44 m
39.8
1.69 m
42.3
56.7
24.3
_
1.10 m
1.51 m
H(ppm)
1.01 m
28.3
4.61 m
56.8
12.3
19.4
40.5
21.2
2.00 m
1.18 m
__
1.01 m
1.58 m
1.06 m
1.72 m
1.28 m
1.75 q
0.70 s
1.01 s
2.06 m
1.03 d
56.1
11.9
19.4
36.2
18.8
1.74 m
0.67 s
0.98 s
1.90 m
0.92 d
138.3
5.17 dd
34.0
1.62 m
129.3
51.3
31.9
21.1
19.0
5.04 dd
1.54 m
1.55 m
0.85 dd
0.80 dd
26.1
45.9
29.2
19.0
19.8
1.65 m
1.58 m
1.58 m
0.82 d
0.80 d
25.4
1.43 m
1.18 m
0.81 t
23.1
1.52 m
12.3
0.84 t
42.2
57.0
24.4
28.9
12.1
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Table 2: Retention times and some characteristic fragment ions observed in the EI mass spectra after GC-MS of isolated or reference
compounds.
compound
Molecular
masse
426
426
412
412
414
414
1
lupeol
2
stigmasterol
3
sitosterol
a
[M]+
[M-CH3]+
426
426
412
412
414
414
[M-H2O]+
411
411
399
397
399
399
408
408
393
394
396
396
[M-CH3-H2O]+
393
393
379
379
381
381
TR
(min)
27.7
27.7
26.8
29.8
27.2
27.2
0
-5
-10
Germination
-15
Shoot length
-20
Fresh weight
-25
-30
-35
name of isolated compounds
Compound
s
a
d
Hexane (A)
CHCl3 (B)
EtOAc (C)
MeOH (D)
EtOH (E)
Lupeol (1)
Stigmasterol (2)
-Sitosterol (3)
2.2 1.1
> 100
> 100
> 100
3.91.0
> 100
> 100
> 100
IC50(g/mL)a
HeLa
WI-38d
37.61.2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
c
Mel-43e
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
IC50 (Concentration of drug causing 50% inhibition of mitochondrial activity); bmurine macrophages; chuman cervix carcinoma cells;
human lung fibroblasts; emelanoma.
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Acknowledgment
We wish to thank the members of the Vegetable Biology Laboratory of Faculty of Agricultural Sciences (FSA)
of the University of Abomey-Calavi (UAC) Bnin and C. KAPANDA. This work was supported by the CGRI
(Commissariat Gnral aux Relations Internationales), CIUF (Coopration Institutionnelle Universitaire
Francophone), ASBL (Association But non Lucratif) Centre Pharmaceutique de Louvain-la-Neuve. We also
thank the Belgian national Fund for Scientific research (FNRS) for a grant to JQL (nr 9.4553.04).
References
A. Ahanchede, G. Gbehounou, K. D. Kossou,
E. Yayi, E. Akpo, Contribution ltude de la
caractrisation du pouvoir alllopathique de
Justicia anselliana (nees) T. Anders: approche
de bioessais utilisant des extraits obtenus
partir du matriel vgtal sch. (2003)
Annales
de
la
FSA.
no
6.
http://www.annales-fsa.bj.refer.org
R. Alessandra, S. Karina, S. Patricia, S. N.
Marcelo, B. D. Gilberto, T. F. Sergio, P.
Cristiana, Biological evaluation of a protein
isolate from cowpea (Vigna unguiculata)
seeds. Food Chemistry (2004), 87, 491-499.
A. S. Langyintuo, J. Lowenbrg-Deboer, M.
Faye, D. Lambert, G. Ibro, B. Moussa, A.
Kergna, S. Kushwaha, S. Musa, G. Ntoukam,
Cowpea supply and demand in West and
Central Africa. Field Crops Research (2003),
82, 215-231.
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