Phytochemistry 71 (2010) 110116

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Phytochemistry
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Botryorhodines AD, antifungal and cytotoxic depsidones from Botryosphaeria rhodina, an endophyte of the medicinal plant Bidens pilosa
Randa Abdou a, Kirstin Scherlach a, Hans-Martin Dahse a, Isabel Sattler a, Christian Hertweck a,b,*
a b

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knll Institute (HKI), Beutenbergstr. 11a, 07745 Jena, Germany Friedrich Schiller University, Jena, Germany

a r t i c l e

i n f o

a b s t r a c t
An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidens pilosa (Asteraceae) that is known for its anti-inammatory, antiseptic and antifungal effects. The ethyl acetate extract of the fungal isolate exhibits signicant antifungal activity as well as potent cytotoxic and antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted in the isolation of a complex of four depsidones, botryorhodines AD and the auxin indole carboxylic acid. Botryorhodine A and B show moderate to weak cytotoxic activities against HeLa cell lines with a CC50 of 96.97 lM and 36.41 lM, respectively. In addition, they also show antifungal activity against a range of pathogenic fungi such as Aspergillus terreus (MIC 26.03 lM for botryorhodine A and 49.70 lM for B) and the plant pathogen Fusarium oxysporum (MIC 191.60 lM for botryorhodine A and 238.80 lM for B). A potential role of the endophyte in modulating fungal populations living within or attacking the host plant is discussed. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 17 July 2009 Received in revised form 23 September 2009 Available online 11 November 2009 Keywords: Antifungal agents Depsidones Botryosphaeria rhodina Endophytes Polyketides Symbiosis

1. Introduction Of the 300,000 plant species that exist on the earth, each individual plant is host to one or more endophytes, thus providing a rich reservoir of microorganisms (Ryan et al., 2008; Strobel, 2006; Strobel and Daisy, 2003; Strobel et al., 2004; Zhang et al., 2006). Various endophytic fungi interact in mutualism with their host plant taking advantage of nutrients provided by the host and in turn producing bioactive substances to enhance the growth and competitiveness of the host in nature (Strobel et al., 2004; Zhang et al., 2006). In many cases an improved resistance of the host plant to adversity is observed due to the production of bioactive secondary metabolites by its endophyte (Zhang et al., 2006). This benet is based on a ne-tuned balance between the demands of the invading endophyte and the plant response. If the interaction becomes unbalanced, disease symptoms appear or the fungus is excluded by induced host defence reactions (Kogel et al., 2006). Furthermore, endophytes have been recognized as a prolic source of a wide array of new pharmacologically active secondary metabolites that might prove suitable for specic medicinal or agrochemical applications (Strobel and Daisy, 2003).

* Corresponding author. Address: Leibniz Institute for Natural Product Research and Infection Biology, Hans Knll Institute (HKI), Beutenbergstr. 11a, 07745 Jena, Germany. Tel.: +49 3641 5321100; fax: +49 3641 5320804. E-mail address: Christian.Hertweck@hki-jena.de (C. Hertweck). 0031-9422/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.09.024

Bidens pilosa is a herbaceous plant widely distributed in Africa, America, China, and Japan that is used in traditional medicines for treatment of inammation and various diseases, including hepatitis and diabetes. The boiling water extract of the aerial parts of B. pilosa in Japan has anti-inammatory and anti-allergic properties (Horiuchi and Seyama, 2006). Furthermore, the ethanolic crude extract from the roots of B. pilosa contains polyacetylenes and avonoids that exert in vitro antimalarial activity against Plasmodium falciparum (Oliveira et al., 2004). More recently, a study was carried out to examine the possibility of using B. pilosa for weed and plant fungus control assuming that the wide distribution of the plant might be due to its antifungal activity against phytopathogens (Deba et al., 2007; Strobel, 2003). All studies performed so far focused on the phenolic constituents of the plant extract, and yet endophytes of B. pilosa and their potentially active metabolites have been neglected. Here, we report the rst isolation, structural elucidation and biological evaluation of novel depsidones from the fungal endophyte Botryosphaeria rhodina (also known as Lasiodiplodia theobromae) of this medicinally relevant plant. B. rhodina is known as a multiinfectious microorganism that causes considerable crop damage, particularly to tropical fruits. It spoils many farm products in tropical regions and is one of the main pathogens responsible for the decay of fruits (He et al., 2004). Therefore, the search for the toxins involved in this decay has been the focus of interest of some researchers and resulted in the identication of the toxin (3S, 4R)-3-carboxy-2-methyleneheptan-4-olide as well as decumbic acid from the culture ltrate

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of L. theobromae isolated from rotted mango branches in Miyako islands, Okinawa, Japan (He et al., 2004). The chemical prole of B. rhodina had been also found to include compounds such as jasmonic acid and its derivatives, some polyketides such as lasiodiplodin which has been found to inhibit photophosphorylation and electron transport chain in Thylakoids (Veiga et al., 2007) as well as the glucans botryosphaeran and laminarin with laminarin exerting a non-genotoxic activity (Giese et al., 2006). Furthermore, four exopolysaccharides were obtained from B. rhodina isolated from rotting tropical fruit and were shown to possess antimutagenic and immunomodulatory effects (Vasconcelos et al., 2008; Miranda et al., 2008). In addition, the macrophorins E, F and G were isolated from B. berengeriana and showed potent antifungal and antibacterial activity against phytopathogenic fungi as well as cytotoxic effects (Sassa et al., 1998).

2. Results and discussion Samples of B. pilosa were collected near Cairo, Egypt. After surface sterilization of the fresh aerial plant parts an endophytic fungal strain was isolated. The strain was identied as B. rhodina (L. theobromae) on the basis of the ITS sequence. To monitor the production of secondary metabolites of the isolate it was cultivated in four different culture media both as stationary and as shaken cultures. The resulting extracts were subjected to antimicrobial activity studies and cytotoxicity assays. We found that the extract from a medium 25 (M25) stationary culture exhibited the highest antimicrobial and antiproliferative activities. The ethyl acetate extract of the endophyte exhibited signicant antifungal activity against various test strains in agar diffusion assays (Sporobolomyces salmonicolor, Saccharomyces cerevisiae, Candida albicans, Penicillium notatum, Penicillium avellanea, and Aspergillus terreus). To identify and characterize the active compounds large scale fermentation (40 l) of the endophyte was carried out under the optimized culture condition. The crude extract was liberated from lipophilic components and subjected to ash chromatographic separation on silica gel, followed by open column chromatography with Sephadex LH-20. Activity-guided fractionation was performed by testing the resulting fractions against A. terreus, which proved to be the most sensitive test strain towards the crude extract.

Active fractions contained botryorhodine A and B (Fig. 1), which are chemically related as based on retention time and UV spectra, and seem to belong to a complex of four aromatic metabolites (1 4). These four compounds were isolated by open column chromatography and repeated preparative RP-HPLC using an acetonitrile/ water gradient, yielding 1 (8 mg), 2 (5 mg), 3 (2 mg) and 4 (3 mg). Compound 1 has a molecular formula of C16H12O6 as indicated by HRESIMS and 13C NMR data (Table 1). The 1H NMR spectrum exhibited signals corresponding to two aromatic methyl groups (d 2.26; 2.41), three aromatic protons (d 6.47; 6.54; 6.59), one hydroxyl proton (d 9.79) and one aldehyde proton (d 10.57). The 13C NMR spectrum revealed the presence of 16 carbons of which two were methyl groups (d 16.7; 21.4), one aldehyde carbonyl (d 191.9) one ester carbonyl group (d 164.5). From HSQC and HMBC data (Fig. 2 and 3) the presence of two aromatic phenyl groups was concluded. HMBC data indicated that the rst ring contained a methyl group (d 2.41), which was correlated with a methine carbon at d 116.9 (C-5) and an ester carbonyl carbon (C-7) at d 164.5. Furthermore, the aldehyde moiety was correlated to the same carbon C-5 and to an OH-bonded carbon (C-4, d 163.8). The second phenyl group is substituted with two meta coupled protons at d 6.54 (H-3) and d 6.47 (H-10 ) as well as a methyl group at d 16.7 which was correlated with a methine carbon (C-10 ) at d 114.2 and two quaternary carbons (C-6) at d 131.2 and (C-5) at d 141.2 on the basis of HMBC correlations. The presence of another oxygen-bound carbon was deduced from the correlations observed

Table 1 13 C NMR data for compounds 14. Position d 1 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 111.5, qC 161.8, qC 112.3, qC 163.8, qC 116.9, CH 152.0, qC 164.5, qC 191.9, CH 21.4, CH3 114.2, CH 155.1, qC 105.3, CH 144.0, qC 141.2, qC 131.2, qC 16.7, CH3
13

C 2 114.0, qC 163.4, qC 112.1, qC 165.4, qC 118.1, CH 155.6, qC 166.4, qC 194.6, CH 22.3, CH3 114.3, CH 155.0, qC 116.1, qC 145.3, qC 144.0, qC 128.3, qC 17.1, CH3 9.2, CH3 3 113.6, qC 162.5, qC 117.0, qC 163.0, qC 116.2, CH 145.9, qC 166.0, qC 54.8, CH2 21.4, CH3 114.1, CH 153.9, qC 115.3, qC 144.7, qC 144.1, qC 128.6, qC 16.9, CH3 9.2, CH3 4 113.7, qC 162.5, qC 117.0, qC 162.8, qC 116.2, CH 146.1, qC 165.7, qC 54.7, CH2 21.4, CH3 115.2, CH 155.9, qC 105.9, CH 145.8, qC 143.7, qC 132.8, qC 17.2, CH3

9 6 5

O
1 2 7

O O
4' 5' 6' 3'

O
2'

a
3 8

HO

O H 1 O

b
1'

OH

HO O H

O 2 O

OH
CH3 O H HO O O H H3C 1 CH3 O H HO O H3C 3 H O CH3 OH H HO HO 4 O H3C H H O H OH H HO O O H H3C 2 CH3 O O H OH H CH3 O O CH3 OH

O HO HO 3 H N O OH HO HO O 4

O OH

OH O

HO

Fig. 1. Chemical structures of compounds 15.

Fig. 2. Key HMBC correlations of compounds 14.

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R. Abdou et al. / Phytochemistry 71 (2010) 110116 Table 2 1 H NMR data of compounds 14.

CH3 O H HO O O H H3C 1 CH3 O H HO HO CH2 3 O H3C H O CH3 OH H HO HO H O H OH H HO O

CH3 O O O H H3C 2 CH3 O O O CH2 4 H3C H H OH H CH3 OH

Position

d 1H (J in Hz) 1 2 3 4

Fig. 3. HSQC correlations of compounds 14.

1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 OH

6.59, s

6.73, s

6.59, s

6.60, s

10.57, s 2.41, s 6.47, d (3.0) 6.54, d (3.0)

10.71, s 2.48, s 6.46, s

4.95, s 2.38, s 6.43, s

4.94, s 2.37, s 6.44, s 6.44, s

2.26, s 9.79, s

2.30, s 2.19, s

2.39, s 2.12, s

2.43, s

for the proton at d 6.54 (H-30 ) with two quaternary carbons, C-50 at d 141.2 and C-40 at d 144.0. This correlation together with a strong HMBC correlation between H-10 and C-50 and a COSY correlation between H-10 and the methyl protons H-70 led to the complete deduction of the substitution pattern of ring b. This was furthermore supported by comparisons with previously reported NMR data (Bezivin et al., 2004; Lohezic-Le Devehat et al., 2007; Pittayakhajonwut et al., 2006). Contrary to the downeld shift observed for the oxygen bonded carbons C-2, C-50 and C-40 an upeld shift was observed for C-1 (d 111.5), thus suggesting its connection to a carbonyl carbon. Consequently, both phenyl groups are connected by a seven-membered ring containing an ether linkage and an ester bridge, revealing compound 1 to be a new depsidone, for which the name botryorhodine A was proposed. Compound 2 (Fig. 1) was obtained as a second product with signicant antifungal activity. From HRESIMS and 13C NMR data the molecular formula C17H14O6 was deduced, thus suggesting that 2 is a homologue of 1 with an additional methyl group. The 13C NMR signal at d 194.6 and the 1H NMR signal at d 10.71 attached to it on the basis of HSQC correlations conrmed the presence of the aldehyde function. Its proton was correlated with an OH-bearing quaternary carbon at d 165.4 and a methine carbon (C-5, d 118.1) on the basis of HMBC correlations. This proton (H-5) is in turn correlated with its neighboring methyl carbon at d 22.3, as well as a quaternary carbon connected to the ester carbonyl group (C-1) at d 114.0 on the basis of HMBC (Fig. 2). The second aromatic ring bears two methyl groups, a hydroxyl group and a single proton, as deduced from the 13C NMR, HSQC and HMBC correlations. One of the methyl groups (C-80 ) is correlated with the OH-bearing carbon (C-20 , d 155.0) and the quaternary carbon bound to an oxygen atom (C-40 , d 145.3). The protons of the second methyl group on the other hand are correlated with the methine aromatic carbon (C-10 , d 114.3), the quaternary carbon bound to the same methyl group (C-60 d 128.3) and a carbon bound to an oxygen atom (C50 , d 144.0). This information fully established the substitution pattern of 2. By comparison of the deduced structure with literature data it was found that 2 is identical with a compound produced at rst in the context of the total synthesis of the depsidone dechloropannarin, a lichen (Psoroma sp.) metabolite (Elix et al., 1982). The structure of 2 was also proposed for a minor product in an extract of Erioderma chilense solely on the basis of its chromatographic properties (Elix et al., 1986). Here, we report the rst isolation and spectral assignment (Tables 1 and 2) as well as antifungal and cytostatic activities of nordechloropannarin (botryorhodine B).

Compound 3 (botryorhodine C, Fig. 1) appeared also as a major compound of the extract. It showed no antifungal activity but exhibited antibacterial activity against Bacillus subtilis in agar diffusion assays (MIC = 1265.80 lM). Its molecular formula was determined as C17H16O6 on the basis of HRESIMS and 13C NMR data. The spectral data of compound 3 (Tables 1 and 2) are similar to those of 1 indicating that both might have the same basic skeleton. However, the 13C NMR spectrum lacks an aldehyde signal, but shows a methylene signal (d 54.8, DEPT). The chemical shift suggested that this methylene is located in the periphery of a phenyl and is connected to a hydroxyl group. This was further supported by the HMBC correlations (Fig. 2) between the methylenic protons and the carbons C-2 (d 162.5), C-4 bearing an OH group at d 163.0 and the quaternary carbon (C-3) at d 117.0. Compound 3 also differs from 1 in the additional methyl group at C-30 , which is also present in 2 (Fig. 1). Compound 4 (botryorhodine D, Fig. 1), a minor product from the fraction of compound 3, has a molecular formula of C16H14O6 as indicated by HRESIMS and 13C NMR. The 1H NMR and 13C NMR spectral data (Tables 1 and 2) showed close similarity to those of compound 3 suggesting that both compounds have the same basic framework. In contrast to compound 3 the 1H NMR shows only two methyl proton signals (d 2.37, d 2.43) but three aromatic proton signals of which one appeared at d 6.60 and two at d 6.44. On the basis of HMBC correlations (Fig. 2) it was conrmed that the C-80 methyl group of 3 is lacking as in the b-ring of 1. Compounds 14 belong to the group of depsidones, which are typically found in lichens (Elix and Wardlaw, 1999; Elix et al., 2000, 1999, 1997; Rezanka and Guschina, 1999). Only a few depsidones have been isolated from non-lichen sources, such as auranticins A and B from the mangrove fungal isolate Preussia aurantiaca (Poch and Gloer, 1991), emeguisins from the ascomycete Emericella unguis (Pittayakhajonwut et al., 2006) and the depsidones garcinisidone BF from two species of Garcinia plants, Garcinia neglecta and Garcinia puat (Ito et al., 2001; Xu et al., 2000). Only recently, depsidones from endophytic fungi have been reported: excelsione from an endophytic fungus from the New Zealand endemic tree Knightia excelsa (Lang et al., 2007) and phomopsidone from an endophyte of the medicinal plant Eupatorium arnottianum (Meister et al., 2007) as well as the depsidones from the endophytic fungus BCC 8616 isolated from a leaf of the Hala-Bala evergreen forest in Thailand (Pittayakhajonwut et al., 2006). It was previously reported that lichen metabolites exert a variety of biological actions including antibiotic, antimycobacterial,

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antiviral, anti-inammatory, and analgesic, antipyretic, antiproliferative and cytotoxic effects (Mller, 2001). Although various pharmacologically relevant activities of lichen metabolites have been recognized, their therapeutic potential has not yet been fully explored. An improved access to depsidones may aid in identifying new leads with therapeutic potential (Mller, 2001). One may speculate on the role of these compounds in the microbial interaction. Depsidones have been reported to act as photoprotectors limiting the deleterious effects of UV through their antioxidant activity (Fernandez et al., 1998; Neamati et al., 1997). Furthermore, they may modulate microbial populations living within or attacking the plant. We thus evaluated the antimicrobial efcacy of compounds 12 against test strains A. terreus and B. subtilis using nystatin as a positive control for the antifungal activity, ciprooxacin for the antibacterial activity and methanol as a negative control. Compounds 1 and 2 are active against both microorganisms while 3 and 4 do not have antifungal activity (Table 4) but show weak antibacterial activity (MIC 1265.80 lM for 3 and 1582.20 lM). The minimum inhibitory concentration of compound 1 against A. terreus was found to be 26.03 lM, while the reference nystatin has a MIC-value of 13.50 lM. Compound 2 also showed antifungal activity against A. terreus (MIC 49.70 lM). The nding that 3 and 4, both lacking the aldehyde group of 1 and 2, showed no antifungal activity suggests the possible importance of this functional group for antifungal activity. To evaluate the cytotoxicity and antiproliferative effects of 1 and 2 they were subjected to cytotoxicity assays against HeLa, K562 and HUVEC cell lines. Compounds 1 and 2 exhibited potent antiproliferative activity against K-562 and HUVEC (Table 3 and Fig. 4). In both assays, the two compounds showed signicant antiproliferative activity over a wide concentration range with 2 covering the wider range (Fig. 4). As for the cytotoxic assay against HeLa cancer cell line CC50 values of 96.97 lM and 36.41 lM were obtained for compounds 1 and 2, respectively. Compounds 3 and 4 on the contrary did not show any antiproliferative activity. The observed antifungal activity of 1 and 2 is intriguing as endophytes may be producing bioactive substances that may be involved in a hostendophyte relationship and can have the capacity to control plant pathogens (Deba et al., 2007). To test whether the endophyte metabolites have the potential to protect the host plant from fungal infections, they were also tested against the phytopathogen Fusarium oxysporum. Indeed, 1 and 2 were active with a MIC of 191.60 lM and 238.80 lM observed for 1 and 2 respectively. Compounds 3 and 4 on the contrary were inactive (Table 4). It seems that the plantmicrobe system presented here belongs to those where the endophyte is aiding the plant as well as itself in its survival and tness. By producing two antifungal and two anti-

(a) HUVEC
100

Proliferation (% vs. control)

control botryorhodine A botryorhodine B

50

0 0.001

0.01

0.1

10

100

Concentration (g ml-1)

(b) K-562
100

Proliferation (% vs. control)

control botryorhodine A botryorhodine B

50

0 0.0001

0.001

0.01

0.1

10

100

Concentration (g ml-1)

(c) HeLa
100

Cytotoxicity (% vs. control)

control botryorhodine A botryorhodine B

50

0 0.001

0.01

0.1

10

100

Concentration (g ml-1)
Table 3 Antiproliferative (GI50) and cytotoxic (CC50) activities of botryorhodines A (1) and B (2). Compounds 1 2 Antiproliferative activity GI50 (K-562) GI50 (HUVEC) 1.67 lM 0.07 lM 0.84 lM 0.003 lM Cytotoxicity CC50 (HeLa) 96.97 lM 36.41 lM Fig. 4. Antiproliferative and cytotoxic activity of botryorhodines A (1) and B (2) against HUVEC, K-562 and HeLa cancer cell lines.

Table 4 Antifungal activities of compounds 14. Compounds MIC (lM) against A. terreus MIC (lM) against F. oxysporum 1 26.03 191.60 2 49.70 238.80 3 0 0 4 0 0

bacterial compounds the endophytic fungus thus provides broad spectrum antimicrobial activity to protect itself from competing invaders and/or the plant from phytopathogens. This is in agreement with previous reports of suppressed pathogenic attack, removed contaminants and promoted plant growth and yield by endophytic fungi (Rosenblueth and Martinez-Romero, 2006). In this context it may be interesting to note that we also identied indole-b-carboxylic acid (5) as an endophyte metabolite in the extract of its pure culture (by comparison with an authentic sample). Like the phytohormone indole acetic acid compound 5 belongs to the plant auxins, which are responsible for growth stimulation and elongation of plants. It has been previously reported that

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endophyte-infected plants often grow faster than non-infected ones (Cheplick et al., 1989; Gasoni and Stegman de Gurnkel, 1997). This effect is either due to the endophytes production of phytohormones such as indole-3-acetic acid, cytokines and other plant growth promoting substances and/or owing to the fact that endophytes could have enhanced the hosts uptake of nutritional elements such as nitrogen and phosphorus (Tan and Zou, 2001). Thus, the one meter height often observed for B. pilosa (Muchuweti et al., 2007) could be referred at least partly to the production of indole-b-carboxylic acid by its endophyte. In conclusion, we have isolated a fungal endophyte from the important plant B. pilosa and identied it as a B. rhodina strain. Through bioactivity-guided fractionation we succeeded in the isolation and full characterization of four depsidones, botryorhodines AD, which have not been isolated from a natural source before. Our nding that 1 and 2 are signicantly active against a variety of pathogenic fungi is not only relevant for a potential therapeutic application, but also suggests that endophytes might be involved in protecting the host plant from invasion of phytopathogens. Furthermore, we found that the fungus produces the plant auxin indole-b-carboxylic acid (5), which may play another important role in the plantfungal interaction. 3. Experimental 3.1. General NMR spectra were recorded on a Bruker DPX-300 and a Bruker DRX-500 at 300 MHz and 500 MHz for 1H, and 125 MHz for 13C NMR, respectively; chemical shifts are given in d values (ppm). IR spectra were recorded on a Bruker FT-IR (IFS 55) spectrometer. UV spectra were recorded on a Cary 1 Bio UVvis spectrophotometer (Variant). HPLC-MS measurements were recorded on an Agilent high performance 1100 series LC/MSD Trap module with an API - electrospray source, PC printer and LC/MSD chemstation software for data aquisition and data analysis. HRESIMS were recorded on a Finnigan TSQ Quantum Ultra AM Thermo Electron. Open column chromatography was performed on silica gel 60 (Merck, 0.04 0.063 mm, 230400 mesh ASTM) and Sephadex LH-20 (Pharmacia). TLC: silica gel plates (silica gel 60 F254 on aluminium foil or glass, Merck), spots were visualized by spraying with anisaldehyde/sulfuric acid followed by heating. Analytical HPLC was conducted on a Shimadzu HPLC system using a Nucleosil 100-5 C18 column (5 lm, 125 4.6 mm) with MeCN/0.1% TFAH2O as eluent (ow rate 1 ml min1, 15/85 to 100% MeCN in 30 min) and UV detection at 254 nm. Preparative HPLC was performed on a Shimadzu HPLC system using a Nucleosil 100-5 C18 column (5 lm, 250 16 mm, pore diameter 100 ) using a ow rate of 10 ml min1 starting elution with 25% MeCN and ending with 100% MeCN in 45 min with a UV detector. All solvents used were spectral grade or distilled prior to use. 3.2. Strain isolation and taxonomic classication The herb B. pilosa (Asteraceae) has been collected at the museum of agriculture in Cairo, Egypt. To obtain the endophyte from within the stem parts of the plant, surface sterilization was carried out with diluted formaldehyde (3740% for 1 min) to kill the epiphytic fungi from the stem parts. The surface sterilized segments were cut into small pieces using a sterile blade and were incubated at room temperature for 4 weeks on agar plates containing an antibiotic to suppress bacterial growth (composition of isolation medium: 15 g l1 malt extract, 15 g l1 agar and 0.2 g l1 chloramphen- icol in distilled water, pH 7.47.8, adjusted with 10% NaOH or 36.5% HCl). From the growing cultures a pure strain of B. rhodina

was isolated by repeated reinoculation on malt agar plates. Culture purity was determined from colony morphology. The endophytic fungus was identied on the basis of ITS sequence as B. rhodina at the Centraalbureau voor Schimmelcultures in the Netherlands. 3.3. Endophyte fermentation, extraction and isolation To monitor the production of secondary metabolites the fungus was cultured in four different media, a malt extract, caseineesh peptone, cornsteep and dextroseyeast medium both as a shaken and stationary cultures after which the antifungal activity of each extract was examined. Both the chemical and biological analyses showed that the antimicrobial activity of the fungal extract and the production of secondary metabolites were highest using a stationary culture (21 days) at 23 C of M25, a cornsteep medium composed of sucrose (20 g l1), soy bean starch (10 g l1), cornsteep (10 g l1) completed with distilled water to 1 l (pH 6.5). The fungus was grown on potato dextrose agar (PDA) at 23 C for 14 days and the mycelium of each plate was cut into 12 pieces each of which was used as an inoculum in a 1 l Erlenmeyer ask, containing 250 ml of M25. Incubation was carried out for 21 days (23 C) under static conditions. The extract from a 40 l culture (160 1 l Erlenmeyer asks) was prepared by homogenizing the culture ltrate and the mycelium and then macerating for 24 h in EtOAc, which was then collected by decantation. After evaporating to dryness and defatting with n-hexane 10 g of crude extract was obtained. The major products of the extract were compounds 13. Activity-guided isolation started by subjecting the extract to fractionation on a silica gel column using (hexane: EtOAc/1:1) as eluent which resulted in nine main fractions. The fractions exhibiting antimicrobial activity were successively puried on silica gel using CHCl3: MeOH/9:1; Sephadex LH-20 (MeOH) and nally RP-18 Silica on preparative HPLC starting gradient elution with 25% acetonitrile in H2O and ending with 100% acetonitrile after 45 min, to give compounds 1 (8 mg), 2 (5 mg), 3 (2 mg) and 4 (3 mg). The plant growth promoting auxin indole carboxylic acid was isolated using Sephadex LH-20 (MeOH) and identied by comparison of its chromatographic, UV, IR and MS data with an authentic sample. 3.3.1. Botryorhodine A (1) Yellowish-white amorphous powder; UV (MeOH) kmax (log) 203 (3.63), 220 (4.32), 326 (sh) (0.43) nm; IR (lm) mmax 3651, 2886, 2332, 1683, 1456, 1197, 1147, 1061, 850, 727, 669 cm1; HRESIMS m/z 299.0547 [MH] calc. m/z 299.0550 [MH] for C16H11O6; NMR data see Table 1. 3.3.2. Botryorhodine B (nordechloropannarin) (2) White amorphous powder; UV (MeOH) kmax (log) 207 (3.87), 227 (3.22), 328 (sh) (0.43); IR (lm) mmax 3442, 2888, 2346, 1683, 1446, 1207, 1143, 847, 726, 669 cm1; HRESIMS m/z 313.0722 [MH] calc. m/z 313.0707 [MH] for C17H13O6; NMR data see Table 1. 3.3.3. Botryorhodine C (3) White amorphous powder; UV (MeOH) kmax (log) 209 (3.72), 271 (1.16); IR (lm) mmax 3335, 2340, 1670, 1450, 1199, 1146, 849, 726, 666 cm1; HRESIMS m/z 315.0858 [MH] calc. m/z 315.0863 [MH] for C17H15O6; NMR data see Table 2. 3.3.4. Botryorhodine D (4) Yellowish-white amorphous powder; UV (MeOH) kmax (log) 217 (4.043); 269 (2.73); IR (lm) mmax 3306, 2889, 2340, 1678, 1460, 1200, 1147, 850, 727, 667 cm1; HRESIMS m/z 301.0705

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[MH] calc. m/z 301.0707 [MH] for C16H13O6; NMR data see Table 2. 3.4. Antimicrobial assay Antifungal activities were studied qualitatively by agar diffusion tests according to the literature (Afonin et al., 2003; Heinisch et al., 2002) and quantitatively by determination of minimal inhibitory concentration (MIC) according to the NCCLS guidelines using the broth microdilution method (Heinisch et al., 2002). Fifty microliters of the test compound solution in methanol were serially diluted by factor two with the culture medium (RPMI 1640 with Lglutamine, MOPS and without sodium bicarbonate, LONZA Verviers SPRL, Belgium). Then, the wells were inoculated with the test organism to give a nal concentration of 6 103 CFU ml1. After incubation of the microtiter plates at 37 C (A. terreus) for 24 h, the MIC-values were read with a Nepheloscan Ascent 1.4 automatic plate reader (Lab systems, Vantaa, Finland) as the lowest dilution of compound allowing no visible growth. The MIC for F. oxysporum and B. subtilis was determined by the agar diffusion method. Fifty microliters of each of the 12 serial twofold dilutions were lled in agar (malt extract agar from Roth, Karlsruhe, Germany; seeded with 0.5 ml of a pretested mycelial solution) holes of 9 mm in diameter. After incubation for 24 h the MIC was read as the lowest concentration giving an inhibition zone. 3.5. Antiproliferative and cytotoxic assays 3.5.1. Cells and culture conditions Cells of HUVEC (ATCC CRL-1730), K-562 (DSM ACC 10) and HeLa (DSM ACC 57) were cultured in DMEM (CAMBREX 12-614F), RPMI 1640 (CAMBREX 12-167F) and RPMI 1640 (CAMBREX 12-167F) respectively. All cells were grown in the appropriate cell culture medium supplemented with 10 ml l1 ultraglutamine 1 (Cambrex 17-605E/U1), 500 ll l1 gentamicin sulfate (CAMBREX 17-518Z), and 10% heat inactivated fetal bovine serum (PAA A15-144) at 37 C in high density polyethylene asks (NUNC 156340). 3.5.2. Antiproliferative assay The assay was carried out according to previously described method (Dolezal R. et al., 2009). The test substances were dissolved in DMSO before being diluted in DMEM. The adherent cells were harvested at the logarithmic growth phase after soft trypsinization, using 0.25% trypsin in PBS containing 0.02% EDTA (Biochrom KG L 2163). For each experiment, approximately 10,000 cells were seeded with 0.1 ml culture medium per well of the 96-well microplates (NUNC 167008). 3.5.3. Cytotoxic assay For the cytotoxic assay, HeLa cells were pre-incubated for 48 h without the test substances. The dilutions of the compounds were carried out carefully on the subconuent monolayers of HeLa cells after the pre-incubation time. Cells were incubated with dilutions of the test substances for 72 h at 37 C in a humidied atmosphere and 5% CO2. 3.5.4. Method of evaluation For estimating the inuence of chemical compounds on cell proliferation of K-562, the numbers of viable cells present in mul tiwell plates were determined via CellTiter-Blue assay. The indicator dye resazurin was used to measure the metabolic capacity of cells as an indicator of cell viability. Viable cells of untreated control retain the ability to reduce resazurin into resorun, which is highly uorescent. Nonviable cells rapidly lose metabolic capacity, do not reduce the indicator dye, and thus do not generate a uorescent signal. Under our experimental conditions, the signal from the

CellTiter-Blue reagent is proportional to the number of viable cells. The adherent HUVEC and HeLa cells were xed by glutaraldehyde and stained with a 0.05% solution of methylene blue for 15 min. After gentle washing the stain was eluted with 0.2 ml of 0.33 N HCl in the wells. The optical densities were measured at 660 nm in SUNRISE microplate reader (TECAN). The GI50 and CC50 values were dened as being where the dose response curve intersected the 50% line, compared to untreated control. The comparisons of the different values were performed with software Magellan (TECAN). Acknowledgements We are grateful to Dr. Grit Walther, Centraalbureau voor Schimmelcultures for the identication of the endophytic fungus and to Dr. Abdel Megid, museum of agriculture, Cairo, Egypt for the identication of the plant. We are also thankful to Maria-Gabriele Schwinger for performing the cultivation and agar diffusion assays, Franziska Rhein for NMR measurements, Andrea Perner for HRESIMS measurements and Heike Heinecke for dereplication with reference compound data. Financial support by the Ministry of Higher Education, Egypt (R.A.) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2009.09.024. References
Afonin, S., Glaser, R.W., Berditchevskaia, M., Wadhwani, P., Guhrs, K.H., Mollmann, U., Perner, A., Ulrich, A.S., 2003. 4-Fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides. Chem. Biol. Chem. 4, 11511163. Bezivin, C., Tomasi, S., Rouaud, I., Delcros, J.G., Boustie, J., 2004. Cytotoxic activity of compounds from the lichen: Cladonia convoluta. Planta Med. 70, 874877. Cheplick, G.P., Clay, K., Marks, S., 1989. Interactions between infection by endophytic fungi and nutrient limitation in the grasses Lolium perenne and Festuca arundinacea. New Phytol. 111, 8997. Deba, F., Xuan, T.D., Yasuda, M., Tawata, S., 2007. Herbicidal and fungicidal activities and identication of potential phytotoxins from Bidens pilosa L. var. radiata Scherff. Weed Biol. Manage. 7, 7783. Dolezal, R., Waisser, K., Petrlikova, E., Kunes, J., Kubicova, L., Machacek, M., Kaustova, J., Dahse, H.M., 2009. N-Benzylsalicylthioamides: highly active potential antituberculotics. Arch. Pharm. 342, 113119. Elix, J.A., Wardlaw, J.H., 1999. Four new beta-orcinol meta depsidones from Pertusaria and Siphula lichens. Aust. J. Chem. 52, 713716. Elix, J.A., Lajide, L., Galloway, D.J., 1982. Metabolites from the lichen genus Psoroma. Aust. J. Chem. 35, 23252333. Elix, J.A., Jenie, U.A., Arvidsson, L., Jrgennsen, P.M., James, P.W., 1986. New depsidones from the lichen genus Erioderma. Aust. J. Chem. 39, 719722. Elix, J.A., Wardlaw, J.H., Yashimura, I., 1997. Sublobaric acid and oxolobaric acid, two new depsidones from the lichen Anzia hypoleucoides. Aust. J. Chem. 50, 763 765. Elix, J.A., Wardlaw, J.H., Obermeyer, W., Archer, A.W., 1999. 2-Methoxypsoromic acid, a new lichen depsidone from Pertusaria and Sulcaria species. Aust. J. Chem. 52, 717719. Elix, J.A., Wardlaw, J.H., Obermeyer, W., 2000. 2-Hydroxyvirensic acid, a new depsidone from the lichen Sulcaria sulcata. Aust. J. Chem. 53, 233235. Fernandez, E., Reyes, A., Hidalgo, M.E., Quilhot, W., 1998. Photoprotector capacity of lichen metabolites assessed through the inhibition of the 8-methoxypsoralen photobinding to protein. J. Photochem. Photobiol. B 42, 195201. Gasoni, L., Stegman de Gurnkel, B., 1997. The endophyte Cladorrhinum foecundissimum in cotton roots: phosphorus uptake and host growth. Mycol. Res. 101, 867870. Giese, E.C., Covizzi, L.G., Dekker, R.F.H., Monteiro, N.K., Da Silva, M.D.C., Barbosa, A.M., 2006. Enzymatic hydrolysis of botryosphaeran and laminarin by beta-1,3glucanases produced by Botryosphaeria rhodina and Trichoderma harzianum Rifai. Proc. Biochem. 41, 12651271. He, G., Matsuura, H., Yoshihara, T., 2004. Isolation of an alpha-methylene-gammabutyrolactone derivative, a toxin from the plant pathogen Lasiodiplodia theobromae. Phytochem. 65, 28032807. Heinisch, L., Wittmann, S., Stoiber, T., Berg, A., Ankel-Fuchs, D., Moellmann, U., 2002. Highly antibacterial active aminoacyl penicillin conjugates with acylated biscatecholate siderophores based on secondary diamino acids and related compounds. J. Med. Chem. 45, 30323040.

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R. Abdou et al. / Phytochemistry 71 (2010) 110116 Rosenblueth, M., Martinez-Romero, E., 2006. Bacterial endophytes and their interactions with hosts. Mol. Plant Microbe Interact. 19, 827837. Ryan, R.P., Germaine, K., Franks, A., Ryan, D.J., Dowling, D.N., 2008. Bacterial endophytes: recent developments and applications. FEMS Microbiol. Lett. 278, 19. Sassa, T., Ishizaki, A., Nukina, M., Ikeda, M., Sugiyama, T., 1998. Isolation and identication of new antifungal macrophorins E, F and G as malonyl meroterpenes from Botryosphaeria berengeriana. Biosci. Biotechnol. Biochem. 62, 22602262. Strobel, G.A., 2003. Endophytes as sources of bioactive products. Microbes Infect. 5, 535544. Strobel, G., 2006. Harnessing endophytes for industrial microbiology. Curr. Opin. Microbiol. 9, 240244. Strobel, G., Daisy, B., 2003. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 67, 491502. Strobel, G., Daisy, B., Castillo, U., Harper, J., 2004. Natural products from endophytic microorganisms. J. Nat. Prod. 67, 257268. Tan, R.X., Zou, W.X., 2001. Endophytes: a rich source of functional metabolites. Nat. Prod. Rep. 18, 448459. Vasconcelos, A.F.D., Monteiro, N.K., Dekker, R.F.H., Barbosa, A.M., Carbonero, E.R., Silveira, J.L.M., Ssassaki, G.L., Da Silva, R., Da Silva, M.D.C., 2008. Three exopolysaccharides of the beta-(1-6)-D-glucan type and a beta -(1-3; 1-6)-Dglucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit. Carbohyd. Res. 343, 24812485. Veiga, T.A.M., Silva, S.C., Francisco, A.C., Filho, E.R., Vieira, P.C., Fernandes, J.O.B., Silva, M.F.G.F., Muller, M.W., Lotina-Hensen, B., 2007. Inhibition of photophosphorylation and electron transport chain in thylakoids by lasiodiplodin, a natural product from Botryosphaeria rhodina. J. Agric. Food Chem. 55, 42174221. Xu, Y.J., Chiang, P.Y., Lai, Y.H., Vittal, J.J., Wu, X.H., Tan, B.K., Imiyabir, Z., Goh, S.H., 2000. Cytotoxic prenylated depsidones from Garcinia parvifolia. J. Nat. Prod. 63, 13611363. Zhang, H.W., Song, Y.C., Tan, R.X., 2006. Biology and chemistry of endophytes. Nat. Prod. Rep. 23, 753771.

Horiuchi, M., Seyama, Y., 2006. Antiinammatory and antiallergic activity of Bidens pilosa L. var. radiata Scherff. J. Health Sci. 52, 711717. Ito, C., Itoigawa, M., Mishina, Y., Tomiyasu, H., Litaudon, M., Cosson, J.P., Mukainaka, T., Tokuda, H., Nishino, H., Furukawa, H., 2001. Cancer chemopreventive agents. New depsidones from Garcinia plants. J. Nat. Prod. 64, 147150. Kogel, K.H., Franken, P., Huckelhoven, R., 2006. Endophyte or parasite what decides? Curr. Opin. Plant Biol. 9, 358363. Lang, G., Cole, A.L., Blunt, J.W., Robinson, W.T., Munro, M.H., 2007. Excelsione, a depsidone from an endophytic fungus isolated from the New Zealand endemic tree Knightia excelsa. J. Nat. Prod. 70, 310311. Lohezic-Le Devehat, F., Tomasi, S., Elix, J.A., Bernard, A., Rouaud, I., Uriac, P., Boustie, J., 2007. Stictic acid derivatives from the lichen Usnea articulata and their antioxidant activities. J. Nat. Prod. 70, 12181220. Meister, J., Weber, D., Martino, V., Sterner, O., Anke, T., 2007. Phomopsidone, a novel depsidone from an endophyte of the medicinal plant Eupatorium arnottianum. Z. Naturforsch. C 62, 1115. Muchuweti, M., Mupure, C., Ndhlala, A., Murenje, T., Benhura, M.A.N., 2007. Screening of antioxidant and radical scavenging activity of Vigna ungiculata, Bidens pilosa and Cleome gynandra. Am. J. Food Technol. 2, 161168. Mller, K., 2001. Pharmaceutically relevant metabolites from lichens. Appl. Microbiol. Biotechnol. 56, 916. Neamati, N., Hong, H., Mazumder, A., Wang, S., Sunder, S., Nicklaus, M.C., Milne, G.W., Proksa, B., Pommier, Y., 1997. Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching. J. Med. Chem. 40, 942951. Oliveira, F.Q., Andrade-Neto, V., Krettli, A.U., Brandao, M.G., 2004. New evidences of antimalarial activity of Bidens pilosa roots extract correlated with polyacetylene and avonoids. J. Ethnopharmacol. 93, 3942. Pittayakhajonwut, P., Dramae, A., Madla, S., Lartpornmatulee, N., Boonyuen, N., Tanticharoen, M., 2006. Depsidones from the endophytic fungus BCC 8616. J. Nat. Prod. 69, 13611363. Poch, G.K., Gloer, J.B., 1991. Auranticins A and B: two new depsidones from a mangrove isolate of the fungus Preussia aurantiaca. J. Nat. Prod. 54, 213217. Rezanka, T., Guschina, I.A., 1999. Brominated depsidones from Acarospora gobiensis, a lichen of central Asia. J. Nat. Prod. 62, 16751677.