Antimicrobial Isoflavonoids from Erythrina crista galli Infected with Phomopsis sp. Flavia Redkoa, Marı´a L. Clavina, Daniela Weberb, Fernando Raneac, Timm Ankeb, and Virginia Martinoa,*

a Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA) (UBA-CONICET), Ca´tedra de Farmacognosia, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina. Fax: 54 (11)-45083642. E-mail: [email protected] b Institut für Biotechnologie und Wirkstoff-Forschung (IBWF), Erwin-Schrödinger-Straße 56, D-67663, Kaiserslautern, Germany c Museo de Farmacobota´nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina * Author for correspondence and reprint requests Z. Naturforsch. 62c, 164Ð168 (2007); received October 4/November 7, 2006 The isoflavonoids coumestrol, genistein and daidzein have been isolated and identified by bioassay-guided fractionation from the acetone extract of Erythrina crista galli young twigs infected with Phomopsis sp. These compounds showed antimicrobial activity against Bacillus brevis (MIC values 16.3, 64.8 and 137.8 μm, respectively). This is the first time that coumes- trol, besides lutein and n-nonacosane, are reported in this species. Key words: Erythrina crista galli, Phomopsis sp., Isoflavonoids

Introduction (Mitscher et al., 1988). Besides, antinociceptive and antiinflammatory activities (Min˜ o et al., 2002) The isolation of Phomopsis sp., an endophytic as well as crown gall tumour inhibition and anti- fungi, from different collections of young and old fungal activity (Mongelli et al., 2000; Portillo et al., twigs of Erythrina crista galli has already been re- 2001) have been reported for this species. ported. Phomol, a compound with antibacterial, The discovery of a taxol-producing endophytic antifungal and in vivo antiinflammatory activities, fungus from the yew (Stierle and Strobel, 1995) has been isolated and identified from the fermen- brought the attention about the ecological and tation media of this fungus. Furthermore, eight economic importance of this discovery, since the new compounds have been identified from the production of a determinate metabolite from a same endophyte (Weber et al., 2004, 2005). fungus is a much more interesting source of a drug As part of an ongoing project in the search for than the plant material (Strobel et al., 2005). Many bioactive metabolites from Argentine medicinal medicinal plants have been investigated in recent plants and their endophytic fungi, the isolation of years for endophytic fungi and attention has been compounds from young twigs of E. crista galli, in- paid to their possible influence on the biological fected with Phomopsis sp., is now described. properties of the plants they live in. These findings Erythrina crista galli L. (Leguminosae) is a tree have encouraged us to investigate the presence of that grows in South America and is used in folk active metabolites in Argentine medicinal plants medicine for wound healing, as astringent, nar- infected with these microorganisms. cotic and analgesic (Toursarkissian, 1980). Alka- loids, pterocarpans, cinnamoylphenols and triter- penoids have been reported as the major com- Experimental pounds in bark and leaflets (Ingham and Mark- General procedures ham, 1980; Iinuma et al., 1994; Tanaka et al., 1997). Erycristin, sandwisencin and erythrabyssin II, Thin layer chromatography (TLC) was per- pterocarpans from the EtOH extract of its bark, formed on Silicagel 60F254 plates (Merck), column have shown antimicrobial activity against Myco- chromatography (CC) on Sephadex LH20 (Amer- bacterium smegmatis and Staphylococcus aureus sham Biosciences) and Kieselgel MN 60 (0.063Ð

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0.2 mm/70Ð230 mesh, ATSM). Culture media From this fraction three compounds, named 1, 2, were: Difco Bacto Nutrient Broth dehydrated and and 3, were isolated. Britania Nutrient Broth dehydrated (Buenos Aires, Argentina) Antimicrobial assay Preparative HPLC was performed using a Wa- Tested microorganisms ters equipment with photodiode array detector Bacillus subtilis ATCC 6633; Bacillus brevis (Waters 2996), pump (Waters Delta 600), Waters ATCC 9999; Enterobacter dissolvens LMG 2683; 600 controller and in-line degasser; HPLC-MS was Paecilomyces variotti ETH 114646; Micrococcus done using an Agilent 1100 equipment with a bi- luteus ATCC 381; Nematospora coryli ATCC10647; nary pump, photodiode array detector, mass spec- Penicillium notatum IBWF collection were used in trometer detector, autosampler and column ther- the screening. Bioassay-guided fractionation and mostat. GC analysis was performed on a Varian bioautography were carried out using Bacillus sub- Star 3400 CX and GC-MS on a Hewlett Packard tilis, Bacillus brevis and Sarcina lutea. 5890 Series II MSD 5971a instrument. Acetone and MeOH extracts, F ÐF ,F sub- 1 I VIII IV H NMR, MS and UV spectra were recorded fractions and isolated compounds 1, 2, 3 dissolved using a Bruker AM 500, a Shimadzu QP 5000, and in MeOH were assayed in the disc diffusion test a Shimadzu 2101 PC spectrophotometer, respec- (Kupka et al., 1979) at 100 μg/6 mm disc. Petri tively. dishes were incubated at 37 ∞C in 2% agar in cul- ture medium with the microorganisms. Positive Plant material control: penicillin 2.5 μg/6 mm disc. A vehicle con- Young twigs of E. crista galli were collected be- trol was also performed. Inhibition zone diameter tween December 2002 and March 2003 in Buenos was measured after 24 h. Aires surroundings, identified by Ing. G. Giberti Bioautography and voucher specimens are kept at the Herbarium Chromatography of F was performed on of the Museo de Farmacobota´nica, Facultad de IV(35Ð39) Silicagel plates developed with cyclohexane/ Farmacia y Bioquı´mica, Universidad de Buenos EtOAc (3:7). Chromatograms were dried and Aires, Argentina. placed on Petri dishes containing 2% agar in the culture medium and incubated at 37 ∞C for 24 h. Extraction and chromatography 1.240 g of the powdered dry material were ex- Determination of minimum inhibitory concentration (MIC) tracted at room temperature for 24 h (three times) Bacillus brevis was cultured in nutrient medium. with acetone and MeOH successively. Yields of The optical density (OD) of the bacteria was ad- the acetone and MeOH extracts were 12.5 and justed to the standard of McFarland N∞ 0.5 with 4.4 g w/w, respectively. The acetone extract was fresh medium to achieve a concentration of ap- submitted to CC on Silicagel eluted with cyclohex- prox. 1 ¥ 108 CFU/ml. A final concentration of ane, EtOAc, acetone, MeOH and their mixtures. bacteria of approx. 5 ¥ 105 CFU/ml was obtained From this fractionation fractions F ÐF were I VIII by diluting 200 times with fresh medium. Suspen- obtained. TLC analysis of the fractions was per- sion of bacteria and serial two-fold dilution of the formed on Silicagel plates with cyclohexane/ test compounds in fresh medium (280 to 0.5 μg/ml) EtOAc (1:1 v/v). FI afforded a white precipitate which was submitted to GC-MS analysis. F was were dispensed at 0.1 ml/well in 96-well microtiter IV ∞ submitted to CC on Sephadex LH20 eluted with plates. Plates were incubated during 15 h at 35 C. CH Cl and MeOH: 80 fractions were obtained. Minimum inhibitory concentration (MIC) was de- 2 2 termined in triplicate and is defined as the concen- FIV(6Ð8) was analyzed by HPLC-MS. FIV(35Ð39) was submitted to preparative HPLC using a SPC18 tration of the test compound that completely in- column (250 mm ¥ 10 mm, Nucleosil100Ð7, Ma- hibits cell growth. cherey-Nagel) with a gradient of H2O/MeOH (70:30 v/v) up to 100% MeOH in 20 min and UV HPLC-MS and GC-MS analysis detection at 210, 280 and 330 nm; flow rate was FIV(6Ð8) was analyzed in a column thermostat at μ 4 ml/min. Three fractions were obtained (FAÐFC), 40 ∞C (LiChroCART 125Ð2, 4 m Supersphere one of which (FB) presented antimicrobial activity. 100 RP-18, Merck) with a gradient of H2O/ace- 166 F. Redko et al. · Antimicrobial Isoflavonoids from Erythrina crista galli tone (50:50 v/v) up to 100% acetone in 15 min. The flow rate was 0.5 ml/min and the sample vol- ume 20.0 μl. UV detection was at 450 nm and MS detection was with the following conditions: oven temperature, 350 ∞C (isothermic); drying gas, 6 ml/ min; injector temperature, 400 ∞C; detector tem- perature, 250 ∞C; fragmentor, 140 V (G1946D). GC was performed using: a split/splitless in- jector; fused silica capillary column 5% phenyl 95% methylpolysiloxane (DB-5 J&W Scientific, Folsom, CA, USA) (60 m ¥ 0.25 mm id, film thick- ness 0.25 μm); oven temperature, 230 ∞C (isother- mic); N2 flow, 0.8 ml/min; injector temperature, Fig. 1. Antimicrobial compounds from Erythrina crista 240 ∞C; split, 1:90; FID detector temperature, galli: coumestrol (1), genistein (2), and daidzein (3). 270 ∞C. GC-MS analysis was performed using the same column as for the analytical procedure but with the following conditions: oven temperature, tivity against different microorganisms using the 230 ∞C (isothermic); He flow, 1 ml/min; injector disc diffusion assay. Results are shown in Table I. temperature, 250 ∞C; split, 1:60; detector tempera- Bioassay-guided fractionation of the acetone ex- ture, 250 ∞C. tract was carried out on Bacillus subtilis and B. brevis in order to isolate the antimicrobial com- pounds. Bioautography of the most active fraction, Results and Discussion FIV(35Ð39), on B. brevis evidenced three active Chemical defense agents against pathogenic mi- bands with Rf values between 0.3Ð0.4, corre- croorganisms in the Leguminosae include alka- sponding to compounds 1, 2 and 3, isolated by suc- loids, coumarins and mainly isoflavonoid deriva- cessive CC and preparative HPLC from the ace- tives, such as coumestans and pterocarpans, some tone extract. These were identified as coumestrol of them acting as phytoalexins as a consequence (1), genistein (2) and daidzein (3), respectively of microorganisms’ attack. (Fig. 1) by comparison of their spectral data (UV, In spite of the numerous compounds isolated MS and 1H NMR) with literature references from E. crista galli bark, seeds and leaves, nothing (Kinjo et al., 1987) and with authentic samples. about the chemical composition and biological ac- Compounds 1, 2 and 3 inhibited the growth of B. tivities of twigs has already been reported nor brevis and its MIC values were calculated (Ta- about the presence of endophytic fungi in this spe- ble II) being coumestrol the most active com- cies. pound. In this investigation, E. crista galli acetone and Daidzein and genistein, biosynthethic precur- MeOH extracts from young twigs infected with sors of coumestans and pterocarpans, have been Phomopsis sp. were screened for antimicrobial ac- reported in some Erythrina species (Yenesew et al., 2003; Yu et al., 2000; Nkengfack et al., 2000, 2001) including E. crista galli bark (Imamura Table I. Screening of antimicrobial activity on Erythrina et al., 1981). crista galli. Besides acting as phytoalexins, daidzein and ge- Extract/ Microorganism (inhibition zone in mm) nistein have been reported having in vitro antibac- fraction Bacillus Bacillus Sarcina Penicillium brevis subtilis lutea notatum Table II. MIC of the compounds isolated from Erythrina crista galli. Acetone 12 14 ÐÐ Methanol ÐÐÐ Ð Compound MIC [μg/ml] MIC [μm] FIII 711 ÐÐ FIV 71111 Ð Coumestrol (1) 4.4 16.33 FV 7 ÐÐ 7 Genistein (2) 17.5 64.81 FIV(35Ð39) 15 13 10 Ð Daidzein (3) 35.0 137.81 FIV[(35Ð39) B] 12 10 11 Ð Penicillin < 0.5 < 3.07 F. Redko et al. · Antimicrobial Isoflavonoids from Erythrina crista galli 167 terial activity by Verdrengh et al. (2004) and Ulan- fied from the same extract. This is the first time owska et al. (2006). These last authors pointed out that coumestrol and these compounds are re- that genistein, which exhibited a more pronounced ported in this species. effect than daidzein, is a bacteriostatic agent in- The close relation between endophytes and its hibiting DNA, RNA and protein synthesis. Coum- plant hosts involves evolutionary processes that estrol is active against S. aureus, B. megaterium are able to influence physiological mechanisms of and E. coli, and its activity is increased in the pres- plants (Araujo et al., 2001). Based on this evi- ence of multidrug pump inhibitors (Tegos et al., dence, work is in progress in order to evaluate if 2002); it inhibits membrane-associated transport coumestrol, genistein and daidzein are constitutive processes in E. coli (Weinstein and Albersheim, compounds in E. crista galli or if their production 1983). is induced by the presence of Phomopsis sp. and if they influence the biological activities of this Phytochemical analysis of FIV(6Ð8) showed the presence of lutein which was identified by HPLC/ medicinal plant. MS. n-Nonacosane was isolated from FI and iden- tified by GC analysis through its Kovats retention Acknowledgements index and analysis of its MS spectrum (Mc Laf- We gratefully acknowledge M. T. Argerich for ferty and Stauffer, 2000). processing the plant material, A. Mejfert and In conclusion, three compounds antimicrobial Pharmacist C. van Baren for performing the against B. brevis have been isolated by bioassay- HPLC-MS and GC-MS analyses, respectively. This guided fractionation from the acetone extract of investigation was supported by VW foundation, Erythrina crista galli, infected with Phomopsis sp.: grant I/78249, and is part of the collaborative coumestrol, genistein and daidzein. Besides, lutein project ALPA BIO 3 between BMBF (Germany) and n-nonacosane have been isolated and identi- and SETCIP (Argentina).

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