an endophyte of the medicinal plant

7/29/2019 An Endophyte of the Medicinal Plant

1/7

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

a r t i c l e i n f o

Article history:

Received 17 July 2009Received in revised form 23 September2009Available online 11 November 2009

Keywords:

Antifungal agentsDepsidonesBotryosphaeria rhodina

EndophytesPolyketidesSymbiosis

a b s t r a c t

An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidenspilosa (Asteraceae) that is known for its anti-inflammatory, antiseptic and antifungal effects. The ethylacetate extract of the fungal isolate exhibits significant antifungal activity as well as potent cytotoxicand antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted inthe 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 CC 50 of96.97lM and 36.41 lM, respectively. In addition, they also show antifungal activity against a range ofpathogenic 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 forB). A potential role of the endophyte in modulating fungal populations living within or attacking the hostplant is discussed.

2009 Elsevier Ltd. All rights reserved.

1. Introduction

Of the 300,000 plant species that exist on the earth, each indi-vidual plant is host to one or more endophytes, thus providing arich 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 theirhost plant taking advantage of nutrients provided by the hostand in turn producing bioactive substances to enhance the growthand competitiveness of the host in nature (Strobel et al., 2004;Zhang et al., 2006). In many cases an improved resistance of thehost plant to adversity is observed due to the production of bioac-tive secondary metabolites by its endophyte (Zhang et al., 2006).This benefit is based on a fine-tuned balance between the demands

of the invading endophyte and the plant response. If the interac-tion becomes unbalanced, disease symptoms appear or the fungusis excluded by induced host defence reactions (Kogel et al., 2006).Furthermore, endophytes have been recognized as a prolific sourceof a wide array of new pharmacologically active secondary metab-olites that might prove suitable for specific medicinal or agrochem-ical applications (Strobel and Daisy, 2003).

Bidens pilosa is a herbaceous plant widely distributed in Africa,America, China, and Japan that is used in traditional medicines fortreatment of inflammation and various diseases, including hepati-tis and diabetes. The boiling water extract of the aerial parts of B.pilosa in Japan has anti-inflammatory and anti-allergic properties(Horiuchi and Seyama, 2006). Furthermore, the ethanolic crude ex-tract from the roots ofB. pilosa contains polyacetylenes and flavo-noids that exert in vitro antimalarial activity against Plasmodiumfalciparum (Oliveira et al., 2004). More recently, a study was carriedout to examine the possibility of using B. pilosa for weed and plantfungus control assuming that the wide distribution of the plantmight be due to its antifungal activity against phytopathogens(Deba et al., 2007; Strobel, 2003). All studies performed so far fo-cused on the phenolic constituents of the plant extract, and yet

endophytes of B. pilosa and their potentially active metaboliteshave been neglected. Here, we report the first isolation, structuralelucidation and biological evaluation of novel depsidones from thefungal endophyte Botryosphaeria rhodina (also known as Lasiodiplo-dia theobromae) of this medicinally relevant plant.

B. rhodina is known as a multiinfectious microorganism thatcauses considerable crop damage, particularly to tropical fruits. Itspoils many farm products in tropical regions and is one of themain pathogens responsible for the decay of fruits (He et al.,2004). Therefore, the search for the toxins involved in this decayhas been the focus of interest of some researchers and resultedin the identification of the toxin (3S, 4R)-3-carboxy-2-methylene-heptan-4-olide as well as decumbic acid from the culture filtrate

0031-9422/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.phytochem.2009.09.024

* Corresponding author. Address: Leibniz Institute for Natural Product Researchand Infection Biology, Hans Knll Institute (HKI), Beutenbergstr. 11a, 07745 Jena,Germany. Tel.: +49 3641 5321100; fax: +49 3641 5320804.

E-mail address: [email protected](C. Hertweck).

Phytochemistry 71 (2010) 110116

Contents lists available at ScienceDirect

Phytochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p h y t o c h e m
http://dx.doi.org/10.1016/j.phytochem.2009.09.024mailto:[email protected]://www.sciencedirect.com/science/journal/00319422http://www.elsevier.com/locate/phytochemhttp://www.elsevier.com/locate/phytochemhttp://www.sciencedirect.com/science/journal/00319422mailto:[email protected]://dx.doi.org/10.1016/j.phytochem.2009.09.024


2/7

ofL. theobromae isolated from rotted mango branches in Miyako is-lands, Okinawa, Japan (He et al., 2004).

The chemical profile ofB. rhodina had been also found to includecompounds such as jasmonic acid and its derivatives, some polyke-tides such as lasiodiplodin which has been found to inhibit photo-phosphorylation and electron transport chain in Thylakoids (Veigaet al., 2007) as well as the glucans botryosphaeran and laminarinwith 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 pos-sess antimutagenic and immunomodulatory effects (Vasconceloset al., 2008; Miranda et al., 2008). In addition, the macrophorinsE, F and G were isolated from B. berengeriana and showed potentantifungal and antibacterial activity against phytopathogenic fungias well as cytotoxic effects (Sassa et al., 1998).

2. Results and discussion

Samples ofB. pilosa were collected near Cairo, Egypt. After sur-

face sterilization of the fresh aerial plant parts an endophytic fun-gal strain was isolated. The strain was identified as B. rhodina (L.theobromae) on the basis of the ITS sequence. To monitor the pro-duction of secondary metabolites of the isolate it was cultivated infour different culture media both as stationary and as shaken cul-tures. The resulting extracts were subjected to antimicrobial activ-ity studies and cytotoxicity assays. We found that the extract froma medium 25 (M25) stationary culture exhibited the highest anti-microbial and antiproliferative activities. The ethyl acetate extractof the endophyte exhibited significant antifungal activity againstvarious test strains in agar diffusion assays (Sporobolomyces salmo-nicolor, Saccharomyces cerevisiae, Candida albicans, Penicillium nota-tum, Penicillium avellanea, and Aspergillus terreus). To identify andcharacterize the active compounds large scale fermentation (40 l)of the endophyte was carried out under the optimized culture con-dition. The crude extract was liberated from lipophilic componentsand subjected to flash chromatographic separation on silica gel,followed by open column chromatography with Sephadex LH-20.Activity-guided fractionation was performed by testing the result-ing fractions against A. terreus, which proved to be the most sensi-tive test strain towards the crude extract.

Active fractions contained botryorhodine A and B (Fig. 1), whichare chemically related as based on retention time and UV spectra,and seem to belong to a complex of four aromatic metabolites ( 14). These four compounds were isolated by open column chroma-tography 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 indicatedby HRESIMS and 13C NMR data (Table 1). The 1H NMR spectrumexhibited signals corresponding to two aromatic methyl groups(d 2.26; 2.41), three aromatic protons (d 6.47; 6.54; 6.59), one hy-droxyl proton (d 9.79) and one aldehyde proton (d 10.57). The 13CNMR spectrum revealed the presence of 16 carbons of which twowere methyl groups (d 16.7; 21.4), one aldehyde carbonyl (d191.9) one ester carbonyl group (d 164.5). From HSQC and HMBCdata (Fig. 2 and 3) the presence of two aromatic phenyl groupswas concluded. HMBC data indicated that the first ring containeda methyl group (d 2.41), which was correlated with a methine car-bon 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 car-bon C-5 and to an OH-bonded carbon (C-4, d 163.8). The secondphenyl group is substituted with two meta coupled protons at d6.54 (H-3) and d 6.47 (H-10) as well as a methyl group at d 16.7which was correlated with a methine carbon (C-10) at d 114.2and two quaternary carbons (C-6) at d 131.2 and (C-5) at d 141.2on the basis of HMBC correlations. The presence of another oxy-gen-bound carbon was deduced from the correlations observed

O

O

O

OHHO

HO

1

O

O

O

OHHO

HO

3

O

O

O

OHHO

HO4

O

O

O

OHHO

HO

2

1'

2'

3'

4'

5'

6'

1

2

34

5

6

7

8

9

a

b

N

H

O

OH5

Fig. 1. Chemical structures of compounds 15.

Table 113C NMR data for compounds 14.

Position d 13C

1 2 3 4

1 111.5, qC 114.0, qC 113.6, qC 113.7, qC2 161.8, qC 163.4, qC 162.5, qC 162.5, qC3 112.3, qC 112.1, qC 117.0, qC 117.0, qC4 163.8, qC 165.4, qC 163.0, qC 162.8, qC

5 116.9, CH 118.1, CH 116.2, CH 116.2, CH6 152.0, qC 155.6, qC 145.9, qC 146.1, qC7 164.5, qC 166.4, qC 166.0, qC 165.7, qC8 191.9, CH 194.6, CH 54.8, CH2 54.7, CH29 21.4, CH3 22.3, CH3 21.4, CH3 21.4, CH310 114.2, CH 114.3, CH 114.1, CH 115.2, CH20 155.1, qC 155.0, qC 153.9, qC 155.9, qC30 105.3, CH 116.1, qC 115.3, qC 105.9, CH40 144.0, qC 145.3, qC 144.7, qC 145.8, qC50 141.2, qC 144.0, qC 144.1, qC 143.7, qC60 131.2, qC 128.3, qC 128.6, qC 132.8, qC70 16.7, CH3 17.1, CH3 16.9, CH3 17.2, CH380 9.2, CH3 9.2, CH3

O

O

O

OH

H3C

HO

HO

1

CH3

O

O

O

OH

H3C

HO

HO

3

CH3

CH3

O

O

O

OH

H3C

HO

HO

4

CH3

O

O

O

OH

H3C

HO

HO

2

CH3

CH3H

H

H

H

H

H

H HH

H

Fig. 2. Key HMBC correlations of compounds 14.

R. Abdou et al. / Phytochemistry 71 (2010) 110116 111


3/7

for the proton at d 6.54 (H-30) with two quaternary carbons, C-50 atd 141.2 and C-40 at d 144.0. This correlation together with a strongHMBC correlation between H-10 and C-50 and a COSY correlationbetween H-10 and the methyl protons H-70 led to the completededuction of the substitution pattern of ring b. This was further-more supported by comparisons with previously reported NMRdata (Bezivin et al., 2004; Lohezic-Le Devehat et al., 2007; Pit-tayakhajonwut et al., 2006). Contrary to the downfield shift ob-served for the oxygen bonded carbons C-2, C-50 and C-40 anupfield shift was observed for C-1 (d 111.5), thus suggesting itsconnection to a carbonyl carbon. Consequently, both phenylgroups are connected by a seven-membered ring containing anether linkage and an ester bridge, revealing compound 1 to be anew depsidone, for which the name botryorhodine A wasproposed.

Compound 2 (Fig. 1) was obtained as a second product with sig-

nificant antifungal activity. From HRESIMS and 13C NMR data themolecular formula C17H14O6 was deduced, thus suggesting that 2is a homologue of 1 with an additional methyl group. The 13CNMR signal at d 194.6 and the 1H NMR signal at d 10.71 attachedto it on the basis of HSQC correlations confirmed the presence ofthe aldehyde function. Its proton was correlated with an OH-bear-ing quaternary carbon at d 165.4 and a methine carbon (C-5, d118.1) on the basis of HMBC correlations. This proton (H-5) is inturn correlated with its neighboring methyl carbon at d 22.3, aswell 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 aromaticring bears two methyl groups, a hydroxyl group and a single pro-ton, 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 oxy-gen atom (C-40, d 145.3). The protons of the second methyl groupon the other hand are correlated with the methine aromatic carbon(C-10, d 114.3), the quaternary carbon bound to the same methylgroup (C-60 d 128.3) and a carbon bound to an oxygen atom (C-50, d 144.0). This information fully established the substitution pat-tern of 2. By comparison of the deduced structure with literaturedata it was found that 2 is identical with a compound producedat first in the context of the total synthesis of the depsidonedechloropannarin, a lichen (Psoroma sp.) metabolite (Elix et al.,1982). The structure of 2 was also proposed for a minor productin an extract of Erioderma chilense solely on the basis of its chro-matographic properties (Elix et al., 1986). Here, we report the firstisolation 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 majorcompound of the extract. It showed no antifungal activity butexhibited antibacterial activity against Bacillus subtilis in agar diffu-sion assays (MIC = 1265.80lM). Its molecular formula was deter-mined as C17H16O6 on the basis of HRESIMS and

13C NMR data.The spectral data of compound 3 (Tables 1 and 2) are similar tothose of1 indicating that both might have the same basic skeleton.However, the 13C NMR spectrum lacks an aldehyde signal, butshows a methylene signal (d 54.8, DEPT). The chemical shift sug-gested that this methylene is located in the periphery of a phenyland is connected to a hydroxyl group. This was further supportedby the HMBC correlations (Fig. 2) between the methylenic protonsand the carbons C-2 (d 162.5), C-4 bearing an OH group at d 163.0and the quaternary carbon (C-3) at d 117.0. Compound 3 also dif-

fers from 1 in the additional methyl group at C-30, which is alsopresent in 2 (Fig. 1).

Compound 4 (botryorhodine D, Fig. 1), a minor product fromthefraction of compound 3, has a molecular formula of C16H14O6 asindicated by HRESIMS and 13C NMR. The 1H NMR and 13C NMRspectral data (Tables 1 and 2) showed close similarity to those ofcompound 3 suggesting that both compounds have the same basicframework. In contrast to compound 3 the 1H NMR shows only twomethyl proton signals (d 2.37, d 2.43) but three aromatic protonsignals of which one appeared at d 6.60 and two at d 6.44. Onthe basis of HMBC correlations (Fig. 2) it was confirmed that theC-80 methyl group of3 is lacking as in the b-ring of 1.

Compounds 14 belong to the group of depsidones, which aretypically found in lichens (Elix and Wardlaw, 1999; Elix et al.,

2000, 1999, 1997; Rezanka and Guschina, 1999). Only a few depsi-dones have been isolated from non-lichen sources, such as auran-ticins A and B from the mangrove fungal isolate Preussia aurantiaca(Poch andGloer, 1991), emeguisins from the ascomycete Emericellaunguis (Pittayakhajonwut et al., 2006) and the depsidones garcinis-idone BF from two species ofGarcinia plants, Garcinia neglecta andGarcinia puat (Ito et al., 2001; Xu et al., 2000). Only recently, dep-sidones from endophytic fungi have been reported: excelsionefrom an endophytic fungus from the New Zealand endemic treeKnightia excelsa (Lang et al., 2007) and phomopsidone from anendophyte of the medicinal plant Eupatorium arnottianum (Meisteret al., 2007) as well as the depsidones from the endophytic fungusBCC 8616 isolated from a leaf of the Hala-Bala evergreen forest inThailand (Pittayakhajonwut et al., 2006).

It was previously reported that lichen metabolites exert a vari-ety of biological actions including antibiotic, antimycobacterial,

O

O

O

OH

H3C

HO

HO

1

CH3

O

O

O

OH

H3CCH2

HO

HO

3

CH3

CH3

O

O

O

OH

H3CCH2

HO

HO

4

CH3

O

O

O

OH

H3C

HO

HO

2

CH3

CH3H H

H

H

H

H

H

H

H

H

Fig. 3. HSQC correlations of compounds 14.

Table 21H NMR data of compounds 14.

Position d 1H (J in Hz)

1 2 3 4

12345 6.59, s 6.73, s 6.59, s 6.60, s678 10.57, s 10.71, s 4.95, s 4.94, s9 2.41, s 2.48, s 2.38, s 2.37, s10 6.47, d (3.0) 6.46, s 6.43, s 6.44, s20

30 6.54, d (3.0) 6.44, s40

50

60

70 2.26, s 2.30, s 2.39, s 2.43, s80 2.19, s 2.12, sOH 9.79, s

112 R. Abdou et al./ Phytochemistry 71 (2010) 110116


4/7

antiviral, anti-inflammatory, and analgesic, antipyretic, antiprolif-erative and cytotoxic effects (Mller, 2001). Although variouspharmacologically relevant activities of lichen metabolites havebeen recognized, their therapeutic potential has not yet been fullyexplored. An improved access to depsidones may aid in identifyingnew leads with therapeutic potential (Mller, 2001).

One may speculate on the role of these compounds in themicrobial interaction. Depsidones have been reported to act asphotoprotectors limiting the deleterious effects of UV throughtheir antioxidant activity (Fernandez et al., 1998; Neamati et al.,1997). Furthermore, they may modulate microbial populations liv-ing within or attacking the plant. We thus evaluated the antimicro-bial efficacy of compounds 12 against test strains A. terreus and B.subtilis using nystatin as a positive control for the antifungal activ-ity, ciprofloxacin for the antibacterial activity and methanol as anegative control. Compounds 1 and 2 are active against bothmicroorganisms while 3 and 4 do not have antifungal activity (Ta-ble 4) but show weak antibacterial activity (MIC 1265.80 lM for 3and 1582.20 lM). The minimum inhibitory concentration of com-pound 1 againstA. terreus was found to be 26.03 lM, while the ref-erence nystatin has a MIC-value of 13.50 lM. Compound 2 alsoshowed antifungal activity against A. terreus (MIC 49.70 lM). Thefinding that 3 and 4, both lacking the aldehyde group of 1 and 2,showed no antifungal activity suggests the possible importanceof this functional group for antifungal activity.

To evaluate the cytotoxicity and antiproliferative effects of 1and 2 they were subjected to cytotoxicity assays against HeLa, K-562 and HUVEC cell lines. Compounds 1 and 2 exhibited potentantiproliferative activity against K-562 and HUVEC (Table 3 andFig. 4). In both assays, the two compounds showed significant anti-proliferative activity over a wide concentration range with 2 cover-ing the wider range (Fig. 4). As for the cytotoxic assay against HeLacancer cell line CC50 values of 96.97 lM and 36.41lM were ob-tained for compounds 1 and 2, respectively. Compounds 3 and 4on the contrary did not show any antiproliferative activity.

The observed antifungal activity of1 and 2 is intriguing as endo-

phytes may be producing bioactive substances that may be in-volved in a hostendophyte relationship and can have thecapacity to control plant pathogens (Deba et al., 2007). To testwhether the endophyte metabolites have the potential to protectthe host plant from fungal infections, they were also tested againstthe phytopathogen Fusarium oxysporum. Indeed, 1 and 2 were ac-tive with a MIC of 191.60 lM and 238.80 lM observed for 1 and2 respectively. Compounds 3 and 4 on the contrary were inactive(Table 4).

It seems that the plantmicrobe system presented here belongsto those where the endophyte is aiding the plant as well as itself inits survival and fitness. By producing two antifungal and two anti-

bacterial compounds the endophytic fungus thus provides broadspectrum antimicrobial activity to protect itself from competinginvaders and/or the plant from phytopathogens. This is in agree-ment with previous reports of suppressed pathogenic attack, re-moved contaminants and promoted plant growth and yield byendophytic fungi (Rosenblueth and Martinez-Romero, 2006).

In this context it may be interesting to note that we also iden-tified indole-b-carboxylic acid (5) as an endophyte metabolite inthe extract of its pure culture (by comparison with an authenticsample). Like the phytohormone indole acetic acid compound 5 be-

longs to the plant auxins, which are responsible for growth stimu-lationand elongation of plants. It has been previously reported that

Table 4

Antifungal activities of compounds 14.

Compounds 1 2 3 4

MIC (lM) against A. terreus 26.03 49.70 0 0

MIC (lM) against F. oxysporum 191.60 238.80 0 0

Table 3

Antiproliferative (GI50) and cytotoxic (CC50) activities of botryorhodines A (1) and

B ( 2).

Compounds Antiproliferative activity CytotoxicityGI50 (HUVEC) GI50 (K-562) CC50 (HeLa)

1 1.67 lM 0.84 lM 96.97 lM2 0.07 lM 0.003 lM 36.41 lM

(b) K-562

0

50

100

Concentration (g ml-1)

Proliferation(%v

s.co

ntrol)

control

botryorhodine A

botryorhodine B

(a) HUVEC

0

50

100


Proliferation(%v

s.control) control

botryorhodine A

botryorhodine B

(c) HeLa

0

50

100

0.0001 0.001 0.01 0.1 1 10 100

0.001 0.01 0.1 1 10 100

0.001 0.01 0.1 1 10 100


Cytotoxicity(%v

s.control)

controlbotryorhodine A

botryorhodine B

Fig. 4. Antiproliferative and cytotoxic activity of botryorhodines A (1) and B (2)against HUVEC, K-562 and HeLa cancer cell lines.

R. Abdou et al. / Phytochemistry 71 (2010) 110116 113


5/7

endophyte-infected plants often grow faster than non-infectedones (Cheplick et al., 1989; Gasoni and Stegman de Gurfinkel,1997). This effect is either due to the endophytes production ofphytohormones such as indole-3-acetic acid, cytokines and otherplant growth promoting substances and/or owing to the fact thatendophytes could have enhanced the hosts uptake of nutritionalelements 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 ofindole-b-carboxylic acid by its endophyte.

In conclusion, we have isolated a fungal endophyte from theimportant plant B. pilosa and identified it as a B. rhodina strain.Through bioactivity-guided fractionation we succeeded in the iso-lation and full characterization of four depsidones, botryorhodinesAD, which have not been isolated from a natural source before.Our finding that 1 and 2 are significantly active against a varietyof pathogenic fungi is not only relevant for a potential therapeuticapplication, but also suggests that endophytes might be involved inprotecting the host plant from invasion of phytopathogens. Fur-thermore, we found that the fungus produces the plant auxin in-dole-b-carboxylic acid (5), which may play another importantrole in the plantfungal interaction.

3. Experimental

3.1. General

NMR spectra were recorded on a Bruker DPX-300 and a BrukerDRX-500 at 300 MHz and 500 MHz for 1H, and 125 MHz for 13CNMR, respectively; chemical shifts are given in d values (ppm). IRspectra were recorded on a Bruker FT-IR (IFS 55) spectrometer.UV spectra were recorded on a Cary 1 Bio UVvis spectrophotom-eter (Variant). HPLC-MS measurements were recorded on an Agi-lent high performance 1100 series LC/MSD Trap module with anAPI - electrospray source, PC printer and LC/MSD chemstation soft-

ware for data aquisition and data analysis. HRESIMS were recordedon a Finnigan TSQ Quantum Ultra AM Thermo Electron. Open col-umn chromatography was performed on silica gel 60 (Merck, 0.040.063 mm, 230400 mesh ASTM) and Sephadex LH-20 (Pharma-cia). TLC: silica gel plates (silica gel 60 F 254 on aluminium foil orglass, Merck), spots were visualized by spraying with anisalde-hyde/sulfuric acid followed by heating. Analytical HPLC was con-ducted on a Shimadzu HPLC system using a Nucleosil 100-5 C18column (5 lm, 125 4.6 mm) with MeCN/0.1% TFAH2O as eluent(flow rate 1 ml min1, 15/85 to 100% MeCN in 30 min) and UVdetection at 254 nm. Preparative HPLC was performed on a Shima-dzu HPLC system using a Nucleosil 100-5 C18 column (5 lm,250 16 mm, pore diameter 100 ) using a flow rate of10 ml min1 starting elution with 25% MeCN and ending with100% MeCN in 45 min with a UV detector. All solvents used werespectral grade or distilled prior to use.

3.2. Strain isolation and taxonomic classification

The herb B. pilosa (Asteraceae) has been collected at the mu-seum of agriculture in Cairo, Egypt. To obtain the endophyte fromwithin the stem parts of the plant, surface sterilization was carriedout with diluted formaldehyde (3740% for 1 min) to kill the epi-phytic fungi from the stem parts. The surface sterilized segmentswere cut into small pieces using a sterile blade and were incubatedat room temperature for 4 weeks on agar plates containing anantibiotic to suppress bacterial growth (composition of isolationmedium: 15 g l1 malt extract, 15 g l1 agar and 0.2 g l1 chloram-

phen- icol in distilled water, pH 7.47.8, adjusted with 10% NaOHor 36.5% HCl). From the growing cultures a pure strain ofB. rhodina

was isolated by repeated reinoculation on malt agar plates. Culturepurity was determined from colony morphology.

The endophytic fungus was identified on the basis of ITS se-quence as B. rhodina at the Centraalbureau voor Schimmelculturesin the Netherlands.

3.3. Endophyte fermentation, extraction and isolation

To monitor the production of secondary metabolites the funguswas cultured in four different media, a malt extract, caseinefleshpeptone, cornsteep and dextroseyeast medium both as a shakenand stationary cultures after which the antifungal activity of eachextract was examined. Both the chemical and biological analysesshowed that the antimicrobial activity of the fungal extract andthe production of secondary metabolites were highest using a sta-tionary culture (21 days) at 23 C of M25, a cornsteep mediumcomposed of sucrose (20 g l1), soy bean starch (10 g l1), corn-steep (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 for14 days and the myceliumof each plate was cut into 12 pieces eachof which was used as an inoculum in a 1 l Erlenmeyer flask, con-taining 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 flasks) was prepared by homogenizing theculture filtrate and the mycelium and then macerating for 24 hin EtOAc, which was then collected by decantation. After evaporat-ing to dryness and defatting with n-hexane 10 g of crude extractwas obtained. The major products of the extract were compounds13. Activity-guided isolation started by subjecting the extract tofractionation on a silica gel column using (hexane: EtOAc/1:1) aseluent which resulted in nine main fractions. The fractions exhib-iting antimicrobial activity were successively purified on silicagel using CHCl3: MeOH/9:1; Sephadex LH-20 (MeOH) and finallyRP-18 Silica on preparative HPLC starting gradient elution with25% acetonitrile in H2O and ending with 100% acetonitrile after45 min, to give compounds 1 (8 mg), 2 (5 mg), 3 (2 mg) and 4

(3 mg). The plant growth promoting auxin indole carboxylic acidwas isolated using Sephadex LH-20 (MeOH) and identified by com-parison of its chromatographic, UV, IR and MS data with an authen-tic 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 (film) 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] forC16H11O6; 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 (film) 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 seeTable 1.

3.3.3. Botryorhodine C (3)

White amorphous powder; UV (MeOH) kmax (log) 209 (3.72),271 (1.16); IR (film) mmax 3335, 2340, 1670, 1450, 1199, 1146,849, 726, 666 cm1; HRESIMS m/z 315.0858 [MH] calc. m/z315.0863 [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 (film) mmax 3306, 2889, 2340, 1678,1460, 1200, 1147, 850, 727, 667 cm1; HRESIMS m/z 301.0705



6/7

[MH] calc. m/z 301.0707 [MH] for C16H13O6; NMR data seeTable 2.

3.4. Antimicrobial assay

Antifungal activities were studied qualitatively by agar diffu-sion tests according to the literature (Afonin et al., 2003; Heinischet al., 2002) and quantitatively by determination of minimal inhib-itory concentration (MIC) according to the NCCLS guidelines usingthe broth microdilution method (Heinisch et al., 2002). Fifty micro-liters of the test compound solution in methanol were serially di-luted by factor two with the culture medium (RPMI 1640 with L-glutamine, MOPS and without sodium bicarbonate, LONZA Ver-viers SPRL, Belgium). Then, the wells were inoculated with the testorganism to give a final concentration of 6 103 CFU ml1. Afterincubation of the microtiter plates at 37 C (A. terreus) for 24h,the MIC-values were read with a Nepheloscan Ascent 1.4 auto-matic plate reader (Lab systems, Vantaa, Finland) as the lowestdilution of compound allowing no visible growth. The MIC for F.oxysporum and B. subtilis was determined by the agar diffusionmethod. Fifty microliters of each of the 12 serial twofold dilutionswere filled in agar (malt extract agar from Roth, Karlsruhe, Ger-many; seeded with 0.5 ml of a pretested mycelial solution) holesof 9 mm in diameter. After incubation for 24 h the MIC was readas 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), RPMI1640 (CAMBREX 12-167F) and RPMI 1640 (CAMBREX 12-167F)respectively. All cells were grown in the appropriate cell culturemedium supplemented with 10 ml l1 ultraglutamine 1 (Cambrex17-605E/U1), 500ll l1 gentamicin sulfate (CAMBREX 17-518Z),and 10% heat inactivated fetal bovine serum (PAA A15-144) at

37 C in high density polyethylene flasks (NUNC 156340).

3.5.2. Antiproliferative assay

The assay was carried out according to previously describedmethod (Dolezal R. et al., 2009). The test substances were dissolvedin DMSO before being diluted in DMEM. The adherent cells wereharvested at the logarithmic growth phase after soft trypsinization,using 0.25% trypsin in PBS containing 0.02% EDTA (Biochrom KG L2163). For each experiment, approximately 10,000 cells wereseeded with 0.1 ml culture medium per well of the 96-well micro-plates (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 werecarried out carefully on the subconfluent monolayers of HeLa cellsafter the pre-incubation time. Cells were incubated with dilutionsof the test substances for 72 h at 37 C in a humidified atmosphereand 5% CO2.

3.5.4. Method of evaluation

For estimating the influence of chemical compounds on cellproliferation of K-562, the numbers of viable cells present in mul-tiwell plates were determined via CellTiter-Blue

assay. The indica-tor dye resazurin was used to measure the metabolic capacity ofcells as an indicator of cell viability. Viable cells of untreated con-trol retain the ability to reduce resazurin into resorufin, which ishighly fluorescent. Nonviable cells rapidly lose metabolic capacity,

do not reduce the indicator dye, and thus donot generate a fluores-cent signal. Under our experimental conditions, the signal from the

CellTiter-Blue reagent is proportional to the number of viablecells. The adherent HUVEC and HeLa cells were fixed by glutaralde-hyde and stained with a 0.05% solution of methylene blue for15 min. After gentle washing the stain was eluted with 0.2 ml of0.33 N HCl in the wells. The optical densities were measured at660 nm in SUNRISE microplate reader (TECAN). The GI50 and CC50values were defined as being where the dose response curve inter-sected the 50% line, compared to untreated control. The compari-sons of the different values were performed with softwareMagellan (TECAN).

Acknowledgements

We are grateful to Dr. Grit Walther, Centraalbureau voor Schim-melcultures for the identification of the endophytic fungus and toDr. Abdel Megid, museum of agriculture, Cairo, Egypt for the iden-tification of the plant. We are also thankful to Maria-GabrieleSchwinger for performing the cultivation and agar diffusion assays,Franziska Rhein for NMR measurements, Andrea Perner for HRE-SIMS measurements and Heike Heinecke for dereplication withref-erence 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, inthe 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 NMRstructure analysis of membrane-associated peptides. Chem. Biol. Chem. 4,11511163.

Bezivin, C., Tomasi, S., Rouaud, I., Delcros, J.G., Boustie, J., 2004. Cytotoxic activity ofcompounds from the lichen: Cladonia convoluta. Planta Med. 70, 874877.

Cheplick, G.P., Clay, K., Marks, S., 1989. Interactions between infection byendophytic fungi and nutrient limitation in the grasses Lolium perenne andFestuca arundinacea. New Phytol. 111, 8997.

Deba,F., Xuan,T.D., Yasuda, M., Tawata, S., 2007.Herbicidal and fungicidal activitiesand identification of potential phytotoxins from Bidens pilosa L. var. radiataScherff. 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 activepotential antituberculotics. Arch. Pharm. 342, 113119.

Elix, J.A., Wardlaw, J.H., 1999. Four new beta-orcinol meta depsidones fromPertusaria and Siphula lichens. Aust. J. Chem. 52, 713716.

Elix, J.A., Lajide, L., Galloway, D.J., 1982. Metabolites fromthe lichen genus Psoroma.Aust. J. Chem. 35, 23252333.

Elix, J.A., Jenie, U.A., Arvidsson, L., Jrgennsen, P.M., James, P.W., 1986. Newdepsidones from the lichen genus Erioderma. Aust. J. Chem. 39, 719722.

Elix, J.A., Wardlaw, J.H., Yashimura, I., 1997. Sublobaric acid and oxolobaric acid, twonew depsidones from the lichen Anzia hypoleucoides. Aust. J. Chem. 50, 763765.

Elix, J.A., Wardlaw, J.H., Obermeyer, W., Archer, A.W., 1999. 2-Methoxypsoromic

acid, a new lichendepsidone 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-methoxypsoralenphotobinding to protein. J. Photochem. Photobiol. B 42, 195201.

Gasoni, L., Stegman de Gurfinkel, B., 1997. The endophyte Cladorrhinumfoecundissimum 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-1,3-glucanases produced by Botryosphaeria rhodina and Trichoderma harzianumRifai. Proc. Biochem. 41, 12651271.

He, G., Matsuura, H., Yoshihara, T., 2004. Isolation of an alpha-methylene-gamma-butyrolactone derivative, a toxin from the plant pathogen Lasiodiplodiatheobromae. 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 bis-

catecholate siderophores based on secondary diamino acids and relatedcompounds. J. Med. Chem. 45, 30323040.

R. Abdou et al./ Phytochemistry 71 (2010) 110116 115
http://dx.doi.org/10.1016/j.phytochem.2009.09.024http://dx.doi.org/10.1016/j.phytochem.2009.09.024


7/7

Horiuchi, M., Seyama, Y., 2006. Antiinflammatory and antiallergic activity of Bidenspilosa 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 whatdecides? Curr. Opin. Plant Biol. 9, 358363.

Lang, G., Cole, A.L., Blunt, J.W., Robinson, W.T., Munro, M.H., 2007. Excelsione, adepsidone from an endophytic fungus isolated from the New Zealand endemictree 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 theirantioxidant activities. J. Nat. Prod. 70, 12181220.

Meister, J., Weber, D., Martino, V., Sterner, O., Anke, T., 2007. Phomopsidone, a noveldepsidone froman 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 ofHIV-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 ofantimalarial activity ofBidens pilosa roots extract correlated with polyacetyleneand flavonoids. 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 amangrove 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.

Rosenblueth, M., Martinez-Romero, E., 2006. Bacterial endophytes and theirinteractions with hosts. Mol. Plant Microbe Interact. 19, 827837.

Ryan, R.P., Germaine, K., Franks, A., Ryan, D.J., Dowling, D.N., 2008. Bacterialendophytes: recent developments and applications. FEMS Microbiol. Lett. 278,19.

Sassa, T., Ishizaki, A., Nukina, M., Ikeda, M., Sugiyama, T., 1998. Isolation andidentification of new antifungal macrophorins E, F and G as malonylmeroterpenes 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 fromendophytic

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. Threeexopolysaccharides of the beta-(1-6)-D-glucan type and a beta -(1-3; 1-6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rottingtropical 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 ofphotophosphorylation and electron transport chain in thylakoids bylasiodiplodin, a natural product from Botryosphaeria rhodina. J. Agric. FoodChem. 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.


an endophyte of the medicinal plant

Documents