Phytochemistry Letters 8 (2014) 69–72
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
40 -Prenyloxyderrone from the stem bark of Millettia oblata ssp. teitensis
and the antiplasmodial activities of isoflavones from some
Millettia species
Solomon Derese a, Leonard Barasa a, Hoseah M. Akala b, Amir O. Yusuf a, Edwin Kamau b,
Matthias Heydenreich c, Abiy Yenesew a,*
a
Department of Chemistry, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya
Global Emerging Infections Surveillance (GEIS) Program, United States Army Medical Research Unit-Kenya (USAMRU-K), Kenya Medical Research Institute
(KEMRI) – Walter Reed Project, P.O. Box 54 - 40100, Kisumu, Kenya
c
Institut für Chemie, Universität Potsdam, P.O. Box 60 15 53, D-14415 Potsdam, Germany
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 10 October 2013
Received in revised form 4 February 2014
Accepted 5 February 2014
Available online 22 February 2014
The CH2Cl2/MeOH (1:1) extract of the stem bark of Millettia oblata ssp. teitensis showed antiplasmodial
activity (IC50 = 10–12 mg/mL) against the chloroquine-sensitive (D6) and chloroquine-resistant (W2)
strains of Plasmodium falciparum. Chromatographic separation of the extract led to the isolation of a new
isoflavone, 40 -prenyloxyderrone (1), together with known isoflavones (8-O-methylretusin, durmillone,
maximaisoflavone B, maximaisoflavone H and maximaisoflavone J), a rotenoid (tephrosin) and a
triterpene (lupeol). Similar investigation of Millettia leucantha resulted in the identification of the
isoflavones afrormosin and wistin, and the flavone chrysin. The identification of these compounds was
based on their spectroscopic data. Five of the isoflavones isolated from these plants as well as 11
previously reported compounds from Millettia dura were tested and showed good to moderate
antiplasmodial activities (IC50 = 13–53 mM), with the new compound, 40 -prenyloxyderrone, being the
most active (IC50 = 13–15 mM).
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Millettia oblata ssp. teitensis
Millettia leucantha
Millettia dura
Isoflavone
40 -Prenyloxyderrone
Plasmodium falciparum
1. Introduction
Flavonoids and isoflavonoids are known for wide variety of
biological activities including antiplasmodial (Batista et al., 2009;
Bero et al., 2009; Kaur et al., 2009), antioxidant (Arredondo et al.,
2004), antimicrobial (Yenesew et al., 2005) and cancer chemoprevention (Walle, 2007). Whereas flavonoids are ubiquitous, the
distribution of isoflavonoids is rather restricted to the subfamily
Papilonoideae of the family Leguminoseae with sporadic occurrence in few other families (Lapčı́k, 2007).
In our interest on the antiplasmodial activities of Kenyan plants
belonging to the family Leguminoseae, we have identified several
prenylated flavonoids and isoflavonoids with antiplasmodial
activities from the genera Erythrina (Yenesew et al., 2012),
Tephrosia (Juma et al., 2011; Muiva et al., 2009) and Millettia
* Corresponding author. Tel.: +254 733 832576; fax: +254 20 4446138.
E-mail addresses: ayenesew@uonbi.ac.ke, abiyenesew@yahoo.com
(A. Yenesew).
(Yenesew et al., 2003b). The genus Millettia is a rich source of
isoflavonoids, especially isoflavones and rotenoids (Derese et al.,
2003; Yenesew et al., 1996, 1997, 1998, 2003a,b). In this study we
report the isolation of a new isoflavone along with known
compounds from an endemic Kenyan Millettia species, Millettia
oblata ssp. teitensis (J.B. Gillett). Similar investigation of Millettia
leucantha (Vatke) only gave known compounds. In addition, the
antiplasmodial activities of some of the compounds isolated from
these plants as well as those previously isolated from Millettia dura
(Dunn) (Derese et al., 2003; Yenesew et al., 1996, 1997) are
reported.
2. Results and discussion
The dried and ground stem bark of M. oblata ssp. teitensis was
extracted with CH2Cl2/MeOH (1:1). The extract showed antiplasmodial activity against the chloroquine-resistant Indochina 1 (W2)
and chloroquine-sensitive Sierra Leone 1 (D6) strains of Plasmodium falciparum (Table 1). Chromatographic separation of the extract
yielded a new isoflavone (1) along with seven known compounds.
http://dx.doi.org/10.1016/j.phytol.2014.02.001
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
70
S. Derese et al. / Phytochemistry Letters 8 (2014) 69–72
Table 1
In vitro antiplasmodial activities of (a) crude extracts and (b) isoflavones of Millettia species against the W2 and D6 strains of Plasmodium falciparum.
.
Extracts and isoflavones
IC50 in mM
W2
D6
10.0 2.3
21.4 2.9
18.5 2.6
25.4 1.9
12.0 1.2
23.6 4.1
18.8 2.5
22.5 0.9
a
(a) Crude extracts
Millettia oblata ssp. teitensis (stem bark extract)
M. dura (stem bark extract)
M. dura (seedpods extract)
M. dura (seeds extract)
(b) Isoflavones
7,20 -Dimethoxy-40 ,50 -methylenedioxyisoflavone
Maximaisoflavone B
Maximaisoflavone J
Maximaisoflavone H
7,30 -Dimethoxy-40 ,50 -methylendioxyisoflavone
Mildurone
Wistin
Nordurlettone
40 -Prenyloxyderrone (1)
Isoerythrin A 40 -(3-methylbut-2-enyl) ether
Calopogoniumisoflavone A
Durmillone
Jamaicin
Isojamaicin
Durallone
6-Methoxycalopogonium isoflavone A
R1
R2
R3
R4
OCH3
H
H
H
H
OCH3
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
OCH3
H
H
–OCH2O–
–OCH2O–
OCH3
H
OCH3
H
–OCH2O–
–OCH2O–
OCH3
H
O-pry
H
O-Pry
H
O-Pry
H
OCH3
H
–OCH2O–
–OCH2O–
–OCH2O–
OCH3
OCH3
OCH3
H
R5
R6
R7
R8
W2
D6
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
OCH3
H
H
OCH3
OCH3
OCH3
O-Pry
O-Pry
–OCH2O–
OCH3
OCH3
O-b-glu
OH
2,2-DMC
2,2-DMC
2,2-DMC
2,2-DMC
2,2-DMC
2,2-DMC
2,2-DMC
2,2-DMC
H
H
H
45.6 7.0
42.0 4.2
29.7 6.8
38.8 2.0
48.4 5.5
44.1 6.2
23.2 2.8
28.9 5.3
14.9 2.2
21.6 1.5
51.5 5.7
25.1 4.2
38.6 3.2
38.9 2.1
50.0 6.1
53.1 4.7
47.5 2.1
36.0 4.8
35.7 3.6
45.6 5.7
37.7 4.9
35.9 5.1
22.3 3.2
25.1 4.7
13.3 2.4
19.3 2.1
45.8 3.2
37.3 4.8
41.0 5.6
48.7 2.9
32.7 3.6
34.8 5.2
H
H
H
H
O-b-glu = O-b-glucopyranoside.
Standard drugs: chloroquine (IC50 = 0.09 0.01 m; for W2; 0.008 0.001 m; for D6), quinine (IC50 = 0.26 0.01 m; for W2; 0.051 0.001 m; for D6).
a
IC50 values for crude extracts are given in mg/mL.
The known compounds were identified as durmillone (Ollis et al.,
1967), 8-O-methylretusin (Jurd et al., 1972), maximaisoflavone B
(Dagne et al., 1991), maximaisoflavone H (Dagne et al., 1991;
Yenesew et al., 1996), maximaisoflavone J (Murthy and Rao, 1985),
tephrosin (Ollis et al., 1967; Luyengi et al., 1994) and lupeol
(Furukawa et al., 2002).
The new compound (1) was isolated as white crystals, m.p.
130–132 8C. The HRMS showed a [M]+ at m/z 404.1603 corresponding to the molecular formula C25H24O5. The 1H (d 7.93 for H2) and 13C (d 152.9 for C-2, 123.8 for C-3 and 181.3 for C-4) NMR
spectra (Table 2) indicated that compound 1 is an isoflavone
derivative (Yenesew et al., 1996). The presence of a chelated
hydroxyl (dH 12.94, OH-5), a 2,2-dimethylpyrano and a prenyloxy
groups were evident from the 1H and 13C NMR spectra (Table 2). In
the EIMS, the fragment ion at m/z 203 (1a, Fig. 1), formed through
the loss of methyl group and retro-diels-Alder (RDA) cleavage of
ring-C, showed that the hydroxyl (at C-5) and the 2,2-dimethylpyrano groups are placed in ring-A. In the HMBC spectrum
(Table 2), the singlet at dH 6.25 (H-6) and the chelated hydroxyl (dH
12.94), showed correlation with C-5 (dC 162.5), while H-400 (dH
6.71) correlated with C-7 (dC 159.4), C-8 (dC 101.5) and C-8a (dC
152.5), fixing the 2,2-dimethylpyrano ring at C-7/C-8.
In the 1H NMR spectrum, the presence of an AA0 XX0 spin system
centered at dH 7.44 and 6.96 (d, J = 9.0 Hz) indicated that ring-B is
Table 2
1
H (600 MHz) and
13
C (150 MHz) NMR data for compound 1 in CD2Cl2.
Position
dC
dH (m, J in Hz)
HMBC (2J, 3J)
2
3
4
4a
5
6
7
8
8a
10
20 /60
30 /50
40
200
200 -Me2
300
400
1000
2000
3000
4000 -Me
5000 -Me
5-OH
152.9
123.8
181.3
106.3
162.5
100.2
159.4
101.5
152.5
123.1
130.4
114.9
159.8
78.4
28.2
127.8
114.7
65.1
119.9
138.4
18.2
25.7
7.93 (s)
C-3, -8a, -4, -10
6.25 (s)
C-4a, -5, -7, -8
7.44 (d, 9.0)
6.96 (d, 9.0)
C-10 , -3, -40 , -20 /60
C-10 , -30 , -40
1.47
5.62
6.71
4.55
5.49
C-200
C-200 , -8, 200 -Me2
C-200 , -7, -8, -8a
C-2000 , -3000 , -40
(s)
(d,
(d,
(d,
(d,
10.2)
10.2)
6.6)
6.6)
1.80 (s)
1.76 (s)
12.94 (s)
5000 -Me
4000 -Me
C-4a, -5, -6
S. Derese et al. / Phytochemistry Letters 8 (2014) 69–72
71
micromass spectrometer (Micromass, Wythenshawe, Waters Inc.,
UK). 13C NMR (125 or 50 MHz) and 1H NMR (500 or 200 MHz) were
run on Bruker or Varian-Mercury spectrometers using residual
solvent signals as reference. COSY, NOESY, HMBC and HMQC
spectra were acquired using standard Bruker software.
3.2. Plant materials
Fig. 1. Structure of compound 1 and its RDA fragment ion (1a).
substituted at C-40 with the prenyloxy group, and its placement was
confirmed by the HMBC correlation of CH2-1000 with C-40 . Therefore,
the new compound (1) was characterized as 5-hydroxy-200 ,200 dimethylpyrano[500 ,600 :7,8]-40 -prenyloxyisoflavone for which the
trivial name 40 -prenyloxyderrone is suggested (see Fig. 1).
Similar investigation of the methanol extract of the root bark of
M. leucantha led to the isolation of the isoflavone afrormosin (Gong
et al., 2009), the 7-b-D-O-glucoside of afrormosin, wistin (Kaneko
et al., 1988), and the flavone chrysin (Wolfman et al., 1994). Wistin
and chrysin are reported here for the first time in the genus
Millettia.
The in vitro antiplasmodial activities of flavonoids and
isoflavonoids are well documented (Batista et al., 2009; Bero
et al., 2009; Kaur et al., 2009); with some chalcones (Ziegler et al.,
2004; Batista et al., 2009; Bero et al., 2009), flavones (Bero et al.,
2009; Juma et al., 2011), flavanones (Batista et al., 2009; Bero et al.,
2009), isoflav-3-enes (Yenesew et al., 2012), and biflavonoids
(Batista et al., 2009; Bero et al., 2009; Kaur et al., 2009) showing
IC50 values less than 10 mM. The chalcone lichochalcone A, besides
showing in vitro antiplasmodial activity (IC50 5.6 0.6 mM), it also
markedly decreased parasitemia in mice infected with Plasmodium
yoelii, and hence identified as a lead structure for antimalarial drug
development (Ziegler et al., 2004). Furthermore, licochalcone A (as
well as some other flavonoids) also shows synergistic effects with
artemisinin against P. falciparum, providing the other dimension
where flavonoids can be used in malaria control (Mishra et al., 2009;
Ferreira et al., 2010).
Among the isoflavonoids, the isoflavones are the most
abundant; however, the report on their antiplasmodial activities
is limited to few (Kraft et al., 2000; Andayi et al., 2006; Kaur et al.,
2009). In order to expand the information, five isoflavones isolated
in this study along with eleven previously reported from M. dura
(Derese et al., 2003; Yenesew et al., 1996, 1997) were tested for
their antiplasmodial activities against the chloroquine-resistant
(W2) and chloroquine-sensitive (D6) strains of Plasmodium
falciparum (Table 1). In this test the new compound, 40 prenyloxyderrone (1), showed good activity (IC50 = 13–15 mM),
while the rest of the isoflavones had IC50 values between 20 and
53 mM, which are classified as moderate, based on the criteria
proposed by Batista et al. (2009). Whether these isoflavones also
have in vivo antiplasmodial activities, or synergistic effects with
antimalarial drugs, is yet to be established.
The stem barks of M. oblata ssp. teitensis were collected from
Taita Hill forest, Coast province, Kenya in July 2009; M. leucantha
was collected at Kavingo shopping center, Makueni District, Kenya,
December 2008; M. dura was collected in January, 2000, from and
around Chiromo campus, University of Nairobi. The plants were
identified by Mr. Patrick C. Mutiso of the University Herbarium,
School of Biological Sciences, University of Nairobi, where voucher
specimen are deposited.
3.3. Extraction and isolation of compounds from the stem bark of M.
oblata ssp. teitensis
Air dried and ground stem bark of M. oblata ssp. teitensis (450 g)
was extracted with CH2Cl2/MeOH (1:1) by cold percolation. The
extract was evaporated under reduced pressure to yield a brown
extract (23 g). A 20 g portion of the extract was subjected to
column chromatography on silica gel (200 g) eluting with nhexane containing increasing amounts of ethyl acetate and some
23 fractions each ca 1 L were collected.
Crystallization (from dichloromethane/methanol; 1:1) of the
fraction eluted with 3% EtOAc in n-hexane gave 40 -prenyloxyderrone (1, 56 mg). Similarly crystallization of the combined fractions
eluted with 7–12% EtOAc gave durmillone (400 mg). Crystallization (from dichloromethane/methanol; 1:1) of the fraction eluted
with 15% EtOAc gave lupeol (40 mg). Similar treatment of the
fraction eluted with 20% EtOAc gave a mixture of maximaisoflavone B and maximaisoflavone J (180 mg). The fraction eluted
with 25% EtOAc was subjected to CC over Sephadex LH-20 (eluent:
CH2Cl2/CH3OH; 1:1) and gave maximaisoflavone H (10 mg). The
30% EtOAc eluent was subjected to CC on silica gel (27 g, eluting
with n-hexane containing increasing amounts of ethyl acetate) and
afforded tephrosin (78 mg) and 8-O-methylretusin (5 mg).
3.4. Extraction and isolation from the root bark of M. leucantha
Dried and ground root bark (500 g) of M. leucantha was extracted
with CH2Cl2/MeOH (1:1) to give 22 g of crude extract. Part of the
extract (10 g) was subjected to CC on silica gel (100 g) eluting with
dichloromethane containing increasing amounts of methanol and
some 15 fractions each ca 1 L were collected. The fraction eluted with
1.5% MeOH after purification using PTLC (2% MeOH in CH2Cl2) gave
afrormosin (6 mg); 4% MeOH gave crystals of chrysin (10 mg); and
8% MeOH was purified by CC over Sephadex LH-20 (eluent: CH2Cl2/
CH3OH; 1:1) yielding wistin (55 mg).
3.5. Extraction and isolation from M. dura
The isolation and identification of isoflavones from the stem
bark (Derese et al., 2003) and seed pods (Yenesew et al., 1996,
1997) of M. dura has already been described.
3.6. 40 -Prenyloxyderrone (1)
3. Experimental
3.1. General
UV/VIS spectra were recorded using a Pye-Unicam SPS-150
Spectophotometer. HR-EIMS was done on a Micromass GC-TOF
White crystals; m.p. 130–132 8C. UV lmax (MeOH) nm: 226,
264. 1H NMR (Table 2). 13C NMR (Table 2). EIMS m/z (rel. Int.) 404
(20, [M]+, C25H24O5), 336 (25, [C20H16O5]+), 321 (100, [C19H13O5]+),
203 (9, [C11H7O4]+), 69 (36, [C5H9]+). HR-EIMS [M]+: found m/z
404.1603 C25H24O5 (calcd. mass 404.1618).
72
S. Derese et al. / Phytochemistry Letters 8 (2014) 69–72
3.7. In vitro antiplasmodial activity
The crude extracts and pure compounds were assayed using a
non-radioactive assay technique as described by Smilkstein et al.
(2004) with modifications (Juma et al., 2011; Yenesew et al., 2012).
Acknowledgements
S.D. would like to thank IFS and OPCW; grant No. F/4581 for
funding. Mr. Patrick Mutiso is highly appreciated for the
identification of the plant materials. We are also grateful to
International Science Program (ISP, Sweden) for research grant
(KEN-02).
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