Letter
15-Lipoxygenase Inhibitory Effects of
Prenylated Flavonoids from Erythrina
senegalensis
Adiaratou Togola 1, 2, Berte Hedding 1, Anette Theis 1,
Helle Wangensteen 1, Frode Rise 3, Berit Smestad Paulsen 1,
Drissa Diallo 2, Karl Egil Malterud 1
1
Section of Pharmacognosy, Department of Pharmaceutical
Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway
2
Département de Médecine Traditionnelle, Institut National de
Recherche en Santé Publique, Bamako, Mali
3
Department of Chemistry, University of Oslo, Oslo, Norway
Abstract
Fig. 1
Structure of compound 1 (relative stereochemistry 6aR*, 11aR*).
!
One new pterocarpan (named erybraedin F) along with seven
known prenylated flavonoids were isolated from the stem and
root bark of the medicinal plant Erythrina senegalensis. The structures were elucidated by 1D and 2D NMR and MS. The pterocarpans were found to be strong inhibitors of 15-lipoxygenase.
Table 1
Key words
Erythrina senegalensis (Fabaceae) · pterocarpans · flavonoid ·
DPPH · 15‑lipoxygenase
Erythrina senegalensis DC (Fabaceae) is a tree, up to 15 m high,
with large, red flowers [1]. Extracts of the leaves, bark, and roots
are used in traditional medicine. The sap from the crushed leaves
is applied to wounds for two or three days to promote healing;
the pounded bark and leaves are taken by women in a soup
against barrenness; a macerate of the trunk bark is taken internally for amenorrhea and externally against headaches and eye
troubles; and the wood is chewed as an aphrodisiac [1]. In Mali
the decoction of leaves is used to provoke diuretic activity and is
taken against amenorrhea, urinary bilharzias, and sterility [2]. In
addition to these traditional uses, biological activities such as
anti-plasmodial, analgesic, and anti-inflammatory are also reported for this plant [3]. A large number of chemical compounds,
particularly isoflavones and flavanones, have been reported from
E. senegalensis [4–8]. Several of the usages in traditional medicine
and the biological properties reported for this plant may be related to antioxidant activity. In addition to investigating the
chemistry of the plant, we also studied the antioxidant properties of extracts and pure substances in two different assay systems: radical scavenging and 15-lipoxygenase inhibition.
A total of 8 compounds were identified from the root and stem
" Fig. 1) was isolated from
bark of E. senegalensis. Compound 1 (l
the DCM extract of the root bark. The EI‑MS gave the molecular
ion peak at m/z = 352.1, consistent with the molecular formula
" Table 1) showed characteristic
C21H20O5. The NMR spectra of 1 (l
patterns of a pterocarpan similar to compound 7, only with small
differences in the D-ring. The assignment of the hydroxy group at
C-9 was based on the HMBC cross-peak between OH and C-10a.
The HMBC spectrum showed that the signals assigned to H-7, H10, 8-OCH3, and 9-OH came from atoms placed close to each other. The substitution pattern of the D-ring obtained is similar to
compound 5, and the chemical shift values of 1 and 5 are comparable [9]. Additionally, the substitution pattern of the A ring of 1
Togola A et al. 15-Lipoxygenase Inhibitory Effects …
1
H‑NMR and 13C‑NMR data of 1 (CDCl3).
Position
δH Multiplicity (J, Hz)
δC
1
2
3
4
4a
6 (eq)
6 (ax)
6a
6b
7
8
9
10
10a
11a
11b
4′
5′
6′
8-OCH3
9-OH
6′-CH3
6′-CH3
7.26 d (8.2)
6.53 d (8.4)
130.8
110.5
153.9
110.2
151.1
66.6
4.28 dd (11.0, 5.1)
3.65 t-like (11.0, 11.0)
3.49 m
6.80 s
6.51 s
5.43 d (6.9)
6.63 d (9.9)
5.57 d (9.9)
3.87 s
5.67 s
1.42 s
1.42 s
40.2
116.9
107.7
141.0
146.6
98.1
154.0
78.4
112.3
116.5
129.2
76.1
56.9
27.8
27.7
and 7 is identical, and the comparison of their NMR signals
confirmed the suggested structure [10]. Thus the structure of 1
was established as 9-hydroxy-8-methoxy-6′,6′-dimethylpyrano
[2′,3′:3,4]pterocarpan, and the trivial name erybraedin F was suggested. The measured coupling constant between 6a-H and 11aH (6.9 Hz) indicates a relative stereochemistry of 6aR*, 11aR* (cis
configuration) for this substance [11]. In addition to the new
compound 1, the pterocarpans erybraedin A (2) [12, 13], erybraedin D (3) [13, 14], erybraedin C (4) [12, 13], eryvarin K (5) [9],
phaseollin (6) [15], and shinpterocarpin (7) [10] and the flavone
carpachromene (8) [16, 17] were identified by comparison of
spectroscopic data with literature values. These compounds have
been identified for the first time in E. senegalensis. However, they
have been previously reported from the genus Erythrina.
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Table 2 Inhibition of 15-lipoxygenase by flavonoids and extracts from E. senegalensis.
Extract
15-Lipoxygenase IC50 ± SD
2
3
4
5
8
Root bark DCM extract
Stem bark DCM extract
Quercetin (pos. control)
38 ± 6 µM
< 32 µM
41 ± 5 µM
> 70 µM
82 ± 7 µM
14 ± 1 µg/mL
50 ± 7 µg/mL
30 ± 2 µM
Among the tested compounds, the most effective 15-lipoxygenase inhibitor appeared to be compound 3, which showed a
stronger inhibition than the positive control quercetin [61 % in" Table
hibition at 32 µM (12.5 µg/mL), quercetin: IC50 = 30 µM] (l
2). The root bark extract showed stronger 15-lipoxygenase inhib" Table
itory activity than the corresponding stem bark extract (l
2). One interesting finding is that the concentration of root bark
extract leading to 50% inhibition of 15-lipoxygenase (14 µg/mL)
is almost the same as that of 2 and 4 (IC50 values of 15 and
16 µg/mL, respectively). Because a large amount of the extract
contains 15-lipoxygenase−inactive components, there are probably more unidentified compounds in the DCM root bark extract
with strong 15-lipoxygenase inhibitory potential, or there might
be synergistic effects between pterocarpans or other constituents. Further work on the bioactive compounds of E. senegalensis
will be reported elsewhere. The DPPH radical-scavenging activity
of the root and stem bark extracts was low (IC50 > 83 µg/mL).
Moderate toxicity against brine shrimps was shown for the root
bark extract (73% dead larvae at 50 µg/mL), compound 4 (63 %
dead larvae at 64 µM), and compound 8 (64 % dead larvae at
149 µM). The phytochemical screening showed a richer and more
varied content of lipophilic compounds in the root bark compared with the stem bark. The high content of pterocarpans, as
shown in the NMR spectra, may partially explain the reported effects of the plant.
Materials and Methods
!
The stem and root bark of Erythrina senegalensis was collected in
Blendio, Mali, West Africa, in April 2005 and identified by Prof.
Drissa Diallo, Department of Traditional Medicine, Bamako, Mali.
A voucher specimen (0636 DMT) is deposited at this department.
The 1D and 2D NMR spectra were recorded on a Varian Gemini
200, a Bruker DPX 300, or a Bruker AVII600 instrument. EI‑MS
was accomplished on a Micromass Prospeq Q instrument. VersaPak C18 and VersaPak silica cartridges (40 × 150 mm) (Sigma-Aldrich) were used for VersaFlash column chromatography; silica
gel 60 PF254 (with gypsum; 2-mm layer; Merck) was used for centrifugally accelerated TLC (CA‑TLC) (Chromatotron type 7924T;
Harrison Research); and silica gel 60 F254 was used for preparative TLC. Analytical and preparative HPLC were carried out on a
ProStar Polaris system, with a Microsorb mv 100–5 C18 (250 ×
4.6 mm) or a Microsorb 60–8 C18 (250 × 21.4 mm) column, respectively, with absorbance at 280 nm used for detection. For UV
spectroscopy, a Shimadzu UV-160A equipped with a Shimadzu
CPS-240A thermostatted cell changer was employed.
The dried root bark of E. senegalensis (1055 g) yielded 34 g dichloromethane (DCM) extract after exhaustive Soxhlet extrac-
tion. Ten grams of the DCM extract was chromatographed repeatedly on silica gel with benzene, benzene−CHCl3 mixtures,
CHCl3, and EtOAc, yielding substances 6 and 7 (mixture;
185 mg). Another portion of the DCM extract (7.0 g) was chromatographed over silica gel (Versaflash; DCM−CHCl3−EtOAc mixtures) followed by CA‑TLC (DCM−acetone mixtures), yielding 2
(20 mg). Preparative TLC (hexane/EtOAc 17 : 3) and/or preparative HPLC (MeOH/H2O 17 : 3) of CA‑TLC fractions yielded substances 1 (4.7 mg), 3 (6.3 mg), and 4 (41.1 mg). One of the fractions
from the Versaflash column was rechromatographed over RP silica gel (Versaflash; MeOH−H2O gradient) to yield 5 (5.1 mg). The
dried stem bark (628 g) yielded 7.5 g DCM extract after Soxhlet
extraction. The extract (5.5 g) was applied to a silica gel column
(Versaflash; EtOAc-CHCl3 mixtures) followed by CA‑TLC (DCM
−EtOAc mixtures). Substance 8 (20 mg) was obtained after purification by preparative HPLC (MeOH−H2O gradient).
9-Hydroxy-8-methoxy-6′,6′-dimethylpyrano[2′,3′:3,4]pterocarpan
" Table 1; 13C‑NMR (CDCl ,
(1): 1H‑NMR (CDCl3, 600 MHz): see l
3
"
150 MHz): see l Table 1; EI‑MS: m/z (%) = 352 [M+] (81), 337 [M
– CH3] (100), 322 [337 – CH3] (14), 307 [322 – CH3] (13).
DPPH scavenging and 15-lipoxygenase inhibition assays
Test substances were dissolved in DMSO, and the assays were
carried out as reported previously [18]. Soybean 15-lipoxygenase
(Sigma-Aldrich) was employed. Quercetin (Sigma-Aldrich; minimum 98 % purity) was used as positive control, with an IC50 of
23 ± 8 µM (DPPH) and 30 ± 2 µM (15-lipoxygenase).
Brine shrimp toxicity assay
The assay was carried out as previously described [19]. Podophyllotoxin (50 µg/mL) (Sigma-Aldrich; approx. 98%) was used as the
positive control.
Statistics
Samples were analyzed in triplicate. Values are given as averages ± SD.
Acknowledgements
!
The NMR and MS laboratories at the Chemistry Department, University of Oslo, are acknowledged for the spectrometer facilities.
A. T. is grateful to the Quota grant for financial support. This work
is part of the NUFU Project PRO 22/2002 and NUFUSM-10154.
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received
revised
accepted
December 17, 2008
January 28, 2009
February 3, 2009
Bibliography
DOI 10.1055/s-0029-1185449
Published online March 16, 2009
Planta Med 2009; 75: 1168–1170
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Karl Egil Malterud
Department of Pharmaceutical Chemistry
Division of Pharmacognosy
School of Pharmacy
University of Oslo
P. O. Box 1068
0316 Oslo
Norway
Phone: + 47 22 85 65 63
k.e.malterud@farmasi.uio.no
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