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). 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