142 Journal of Basic Microbiology 2009, 49, 142–151

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Full Paper

Endophytic fungi found in association with Smallanthus sonchifolius (Asteraceae) as resourceful producers of cytotoxic bioactive natural products

Margareth B. C. Gallo1, Fernanda O. Chagas1, Marília O. Almeida1, Cláudia C. Macedo1, Bruno C. Cavalcanti2, Francisco W. A. Barros2, Manoel O. de Moraes2, Letícia V. Costa-Lotufo2, Cláudia Pessoa2, Jairo K. Bastos1 and Mônica T. Pupo1

1 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

2 Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil

Smallanthus sonchifolius is a traditional Andean plant which has been cultured mainly in Brazil,

Japan and New Zealand due to its medicinal properties. A study of the endophytic fungi

associated to the plant was carried out in order to characterize new cytotoxic agents. Thirty

two fungal strains were isolated and submitted to cultivation and extraction producing

186 extracts. Of these, 12% displayed moderate to high cytotoxic activities and were considered

promising anticancer compound sources. The ethyl acetate fractions of Nigrospora sphaerica and

Phoma betae liquid fermentations contained the synergistic compounds 8-hydroxy-6-methoxy-3-

methylisocoumarin and (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one which are potential com-

pounds for drug discovery. Another isolated compound, pimara-7,15-dien-3-β-ol diterpene is

being characterized for the first time through a detailed spectroscopic analysis including

GC/MS, homo- and hetero-nuclear correlated NMR experiments (HMQC, HMBC, COSY and

NOEdiff) along with its optical rotation.

Keywords: Nigrospora sphaerica / Phoma betae / Yacon / Aphidicolin / Isocoumarin

Received: March 19, 2008; accepted: June 30, 2008

DOI 10.1002/jobm.200800093

Introduction*

The Asteraceae Smallanthus sonchifolius (Poepp. and Endl.)

H. Robinson, a commonly employed Andean-originated

medicinal plant, has antidiabetic and anti-inflamma-

tory effects. Some unique substances like the anti-

microbial melampolide-type sesquiterpene lactones

[1–3] are present in the plant. Its tuberous roots, rich

in enzymes and fructose derivatives (FOS, GF2-9), but

deficient in starch, are indicated and consumed as

healthy diet foods and prebiotics, specially in South

America, New Zealand and Japan [4–7]. The plant is

also a special and unusual biological niche for fungal

colonization.

Correspondence: Dr. Mônica T. Pupo, Faculdade de Ciências Farma-cêuticas de Ribeirão Preto, USP, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil E-mail: mtpupo@fcfrp.usp.br Phone: +55 16 36024710 Fax: +55 16 36024178

Interactions involving plants and fungi are quite

complex. They may have qualitatively different rela-

tionships with a single host plant, from parasitism to

mutualism, and/or interact with other fungi inside the

same plant. As mutualists, endophytic fungi are con-

sidered innocuous to the host plant. They live in a

reciprocally balanced stable state that disadvantages

none of the partners, including the absence of external

manifestation of diseases in the host [8]. Associated

fungi may produce substances with specific functions

inside and outside the host plant in which they nor-

mally reside. Thus, several bioactive natural products

have been isolated from endophytic fungi showing a

range of effects, for instance antimicrobial and anti-

cancer [9].

As part of the bioprospecting program in endophytic

fungi associated to therapeutic Asteraceae [10–13], it

was considered that S. sonchifolius, commonly named

Yacon, could host endophytic fungi able to produce

Journal of Basic Microbiology 2009, 49, 142–151 Production of new cytotoxic agents 143

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

bioactive secondary metabolites for pharmaceutical

and biotechnological purposes. Using this approach,

32 endophytes have been isolated and characterized

yielding several cytotoxic extracts and compounds.

Some of the compounds isolated in this research are

being reported for the first time as secondary metabo-

lites produced by the studied fungi. The relevance of

the cytotoxic outcome in the fungi and host plant asso-

ciations is discussed.

Materials and methods

Isolation, identification and preservation of fungal strains Yacon leaves, roots and stems were collected in Febru-

ary 2006, in the city of Ribeirão Preto (S 21°11.933′

NO 47°46.699′), State of São Paulo, Brazil. A voucher

specimen was deposited at the herbarium of the Fac-

ulty of Philosophy, Sciences and Humanities of Ribeirão

Preto campus, University of São Paulo (FFCLRP-USP)

Ribeirão Preto, Brazil.

The collected plant material was washed with run-

ning tap water and dried with absorbent paper. Surface

sterilization procedures for epiphytic microbes were

carried out by sequentially submerging the plant mate-

rial in 70% aqueous ethanol (2.5 min), 2.5% aqueous

sodium hypochlorite (30, 60 or 90 min), 70% aqueous

ethanol (1 min) and sterile distilled water (1 min). The

borders of the sterilized segments were discarded and

four tiny pieces each about 5 mm in length were ex-

cised with a sterilized scalpel from the remaining plant

tissue. The excised pieces from each tissue type were

plated on triplicate Petri dishes containing PDA me-

dium supplemented with pentabiotic (100 μg/ml) and

incubated initially at 30 °C for 48 h, followed by 72 h at

23 °C. The effectiveness of the sterilization procedure

was verified by plating aliquots of the final water rinse

on the same medium used for the endophyte cultures.

Each grown fungus from plant tissues was repeatedly

subcultured on PDA antibiotic-free medium until pure

cultures were obtained.

Conidia producing strains were identified at the De-

partment of Mycology, Center of Biological Sciences

of the Federal University of Pernambuco, PE, Brazil.

Sporulating fungi were stored at 10 °C in tubes contain-

ing silica gel (6 to 12 mesh, grade 40, desiccant

activated). The isolates, deposited at the Laboratory of

Industrial Enzymology, Faculty of Pharmaceutical

Sciences of Ribeirão Preto, University of São Paulo

(FCFRP-USP), have been maintained by serial transfers

onto PDA slants at 4 °C.

Cultivation and extraction procedures Two culture media, Czapek and rice, were selected for

cultivation and screening of biological activities of the

isolated fungi, respectively, in liquid and solid cultures.

Liquid cultures were conducted in two steps (Fig. 1).

First, the silica or slant stored fungi were inoculated

onto PDA-containing medium in Petri dishes and incu-

bated at 30 °C for 7 d. After this period, aqueous sus-

pensions obtained either with fungal conidia or mycelia

were transferred to 1 l Erlenmeyer flasks containing

200 ml of seed medium [14] and incubated at 30 °C for

48 h in a rotatory shaker (120 rpm). As a second step,

this resulting pre-culture was filtered on a Büchner

filter through gauze or filter paper and the obtained

mycelia was transferred to a 2 l Erlenmeyer flask con-

taining 400 ml of Czapek medium [15] and incubated at

the same pre-culture conditions for 20 d. This proce-

dure was carried out for a small scale extract produc-

tion for bioprospection. Fifteen Erlenmeyer flasks were

used for a large scale extract production, aiming to

isolate secondary metabolites. Fungi in the 20 day cul-

tures were killed by addition of 50 ml of methanol

(MeOH), the suspension was vacuum-filtered and the

filtrate fractionated by liquid-liquid partition with

ethyl acetate (EtOAc). The resulting organic layer was

evaporated under reduced pressure to produce the

ethyl acetate fraction of liquid cultures (EaL) and a

residue (RL), which was lyophilized. The mycelial mass

collected from the filter was soaked overnight in EtOH,

followed by filtration and solvent evaporation to pro-

Figure 1. Schematic outline of endophytic fungi cultivation and extraction procedures (experimental details in text “Cultivation and extraction procedures”).

144 M. B. C. Gallo et al. Journal of Basic Microbiology 2009, 49, 142–151

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

duce the mycelial ethanolic extract (EtM). An Erlen-

meyer flask containing seed medium without inoculum

was submitted to the same preculture conditions. The

resulting solid filtrate was transferred to Czapek me-

dium and treated in the same way for control purposes.

For the solid culture (Fig. 1) 500 ml Erlenmeyer flasks

containing 90 g of parboiled rice in 90 ml distilled wa-

ter per flask, previously twice autoclaved at 120 °C for

40 min, were employed. For a small scale preparation,

one flask sufficed, but thirty flasks were needed for

large scale extract preparations. Agar plugs (about

2 × 2 cm) cut from the 7 day-old original cultures on

PDA agar were used for inoculation. One flask, without

inoculum, was kept for control use. After 20 days incu-

bation in a BOD (Biological Oxygen Demand) device at

30 °C, EtOH was added to each flask and the contents

were allowed to stand overnight at room temperature.

The EtOH was filtered and evaporated under reduced

pressure yielding the ethanol extract (EtS), which was

submitted to the previously described liquid-liquid

partition affording the ethyl acetate fraction (EaS) and

the residual fraction (RS).

Isolation of compounds EaS fraction (20.2 g) of Nigrospora sphaerica was fraction-

ated by chromatography over a XAD-4 column

(22.0 × 4.5 cm) using water, MeOH and EtOAc as eluents

Figure 2. Schematic outline of isolation of compounds from Nigrospora sphaerica. A: XAD column and elution with water, MeOH, EtOAc; B, C, F: silica gel column, gradient elution from hexane :EtOAc 95 :5 to MeOH 100%; D, E: silica gel column, gradient elution from hexane :EtOAc 95 :5 to EtOH 100% (ex-perimental details in text “Isolation of compounds”).

(step A, Fig. 2). Fraction M2EaS (9.9 g) was fractionated

over silica gel column (CC, 230–80 mesh, 26.0 × 4.5 cm)

using a gradient from with hexane:EtOAc (95:5) to

MeOH (100%; step B) to provide 22 fractions. Fraction

13 (30.1 mg) was identified as compound 1. Fraction

M1EaS (3.1 g) was crystallized from a heated solution of

MeOH/H2O 9:1 yielding compound 3 (101.0 mg; Fig. 2).

Fraction EEaS (1.64 g) chromatographed over silica gel

(400–200 mesh, 25.5 × 5.0 cm) and eluted with the

same gradient as above mentioned (step C) produced 9

fractions. Fraction 5 (246.6 mg) was rechromatographed

over silica gel (CC, 230–80 mesh, 28.0 × 3.0 cm) and

using a gradient from hexane: EtOAc (95:5) to EtOH

(100%; step D) to yield 9 fractions. Fraction 6 (46.3 mg)

was rechromatographed again on silica gel (CC, 230–

80 mesh, 20.0 × 1.3 cm) using the same gradient elution

(step E) to yield compound 2 (16.9 mg).

N. sphaerica fraction EaL (474.3 mg) was fractionated

over silica gel (CC, 400–200 mesh, 24.0 × 3.5 cm) by

elution with gradient hexane:EtOAc (95:5) to EtOH

(100%) (step F) yielding 7 fractions. Fraction 7 crystal-

lized spontaneously to afford compound 3 (98.0 mg).

Fraction EaS (16.5 g) originated from 20 day cultures

of Phoma betae (19 flasks) was chromatographed over

silica gel (400–200 mesh, 11.0 × 12.0 cm) under gra-

dient elution from hexane:EtOAc (9:1) to MeOH (100%;

step G; Fig. 3) to afford 9 fractions. Fraction 5

(686.7 mg) was rechromatographed over silica gel (CC,

230–80 mesh, 25.0 × 4.0 cm) by using gradient elution

from hexane:EtOAc (8:2) to MeOH (100%; step H) to

Figure 3. Schematic outline of isolation of compounds from Phoma betae. G, J: silica gel column, hexane :EtOAc gradient elution from 9 :1 to MeOH 100%; H: silica gel column, gradient elution from hexane :EtOAc 8 :2 to MeOH 100%; I: precipitation with CHCl3; K: normal phase HPLC, hexane :EtOAc 9 :1 elution; L: reversed phase C-18 HPLC, MeOH elution; M: sephadex LH-20 column, EtOH elution; N: crystallization (experimental details in text “Isolation of compounds”).

Journal of Basic Microbiology 2009, 49, 142–151 Production of new cytotoxic agents 145

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afford 12 fractions. Compound 2 (14.7 mg) was precipi-

tated from fraction 5 (99.7 mg) using CHCl3 (step I).

Fraction EaS (888.2 mg) originated from 32 day

cultures of P. betae (1 flask, fungus sporulation time)

was chromatographed over silica gel (400–200 mesh,

27.0 × 3.0 cm) using gradient elution with hexane:

EtOAc (9:1) to MeOH (100%; step J, Fig. 3) to provide

13 fractions (Pb-1 to 13). Fraction 4 (Pb-4, 21.9 mg) was

submitted to HPLC using an isocratic solvent system

containing hexane:EtOAc (9:1) delivered to the a semi-

prep Nucleosil 100–7 column (250 × 10 mm I.D.) at a

flow rate of 1.5 ml min–1, and monitored at 254 nm

detection wavelength at 25 °C (step K). Compound 4

(12.1 mg) was collected at 41 min. The residual fraction

(5.0 mg) collected at 45 min was resubmitted to HPLC

employing a prep C18 Shimadzu column (250 × 20 mm

I.D.) at a flow rate of 2.5 ml min–1, using MeOH as elu-

ent, and monitored at 254 nm (step L). Compound 5

(0.6 mg) was collected at 28 min. Fraction 13 (Pb-13,

426.8 mg) was chromatographed over sephadex LH-20

column (60.0 × 2.5 cm) using EtOH as eluent (step M) to

afford 7 fractions. Fraction 4 crystallized spontaneously

to yield compound 6 (18.8 mg; step N).

Spectroscopic data of compound 1 Pimara-7,15-dien-3β-ol (1): white powder; molecular

formula: C20H32O; [α]D

26 -3.7° (c 2.15, CHCl3); EI/MS m/z (rel.

int.): 288 (30), 273 (60), 255 (100), 187 (38), 145 (35), 119

(55), 107 (70), 91 (55), 80 (48), 55 (42); 1H NMR (500 MHz,

CDCl3): δ 5.69 (1H, dd, J = 10.8 and 18.0 Hz, H-15), 5.10

(1H, m, H-7), 4.82 (1H, dd, J = 1.5 and 18.0 Hz, H-16a),

4.79 (1H, dd, J = 1.5 and 10.4 Hz, H-16b), 3.13 (1H, dd,

J = 4.4 and 11.6 Hz, H-3), 0.90 (3H, s, Me-18), 0.89 (3H, s,

Me-17), 0.88 (3H, s, Me-19), 0.73 (3H, s, Me-20); 13C NMR

(125 MHz, CDCl3): δ 39.2 (C-1), 29.1 (C-2), 79.0 (C-3), 37.1

(C-4), 52.8 (C-5 and 9), 35.8 (C-6), 113.2 (C-7), 38.9 (C-10

and 12), 20.9 (C-11), 35.2 (C-13), 44.1 (C-14), 145.4 (C-15),

110.8 (C-16), 28.1 (C-17), 35.6 (C-18), 21.6 (C-19), 15.4

(C-20), C-8 was not observed.

Spectroscopic and chromatographic equipment 1D and 2D nuclear magnetic resonance (NMR) spectra

were recorded in deuterated solvents (CDCl3, acetone-d6

or MeOH-d4) on Bruker spectrometers (DRX-400 or DRX-

500), working at 400 or 500 MHz for 1H, and at 100 or

125 MHz for 13C. Tetramethylsilane (TMS) was used as

internal reference. Mass spectra were performed in

a high-resolution eletrospray Micromass Quattro LC

spectrometer (HR/ESI/MS) or in a Shimadzu GC/EI/MS

OP2010 employing an AOC-20i auto-injector and a DBS-

MS (30 m × 0.25 mm × 0.25 μm) column. The high per-

formance liquid chromatography (HPLC) system con-

sisted of a Shimadzu LC-6AD pump, and a SPD-20A UV-

vis detector with a CBM-20A interface. Data acquisition

was carried out on LC Solution software version 1.1.

Tumor cell cultures and cytotoxic bioassay Human tumor cell lines MDA-MB435 (breast), HCT-8

(colon), SF295 (brain) and HL-60 (promyelocytic leuke-

mia) were obtained from National Cancer Institute

(Bethesda, MD, USA). All cell lines were cultured in

RPMI1640 medium, supplemented with 10% fetal bo-

vine serum and 1% antibiotics (penicillin and strepto-

mycin). Cultures were maintained in a humidified in-

cubator at 37 °C and 5% CO2 atmosphere. The cytotoxic

effect was assessed using the MTT-dye reduction assay

for cell viability described by Mosmann [16] according

to an established protocol [17]. Briefly, tumor cells were

placed in 96-well plates at the following densities:

0.7 × 105 (HCT-8), 0.6 × 105 (SF295), 0.3 × 106 (HL-60) and

0.1 × 106 (MDA-MB435) cells/ml. Extracts and fractions

were tested in a single concentration (100 μg/ml) and

pure substances in a range between 0.048 and 25 μg/ml

during 72 h. Control groups received the same amount

of vehicle and doxorubicin (Doxolem®, Zodiac Produtos

Farmacêuticos S/A, Brazil), which was used as a positive

control. Growth inhibition rates were quantified as the

percentage of control absorbance by the reduced dye at

550 nm in accordance with the following equation:

Inhibition rate = [(OD control well – OD treated well)

× (OD control well)–1] × 100 .

Each sample was tested in two independent experi-

ments performed in triplicates. The results consisted of

the average value for each experimental unit. The IC50

values and their 95% confidence intervals were ob-

tained by non linear regression using the GraphPad

Prism program (Intuitive Software for Science, San

Diego, CA). An activity scale was utilized to appraise the

cytotoxic potential of the tested samples: inactive sam-

ples (I), samples with low activity (LA, cell growth inhi-

bition between 1–50%), moderated activity (MA, cell

growth inhibition between 50–75%), and high activity

(HA, cell growth inhibition between 75–100%).

Results and discussion

Isolation and identification of fungal strains Altogether 32 species of fungi were isolated and labeled

using the plant name initial letters and a number, for

example, SS29 (Table 1). The results indicated an un-

even distribution of endophytic fungi within S. sonchi-

146 M. B. C. Gallo et al. Journal of Basic Microbiology 2009, 49, 142–151

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Table 1. Endophytic fungi occurrence in Smallanthus sonchifolius tissues.

Tissue ocurrence Fungal strain Taxonomic identification

Leaves Stems Roots

SS13 Papulaspora immersa Hotson P A P SS16 Preussia minima (Auersw.) von Arx. P A A SS20 NI P A A SS21 NI P P P SS22 Mycelia sterile P A A SS26 NI P A A SS29 Aspergillus aculeatus Lizuka P A A SS32 Glomerella cingulata (Stoneman) Spaulding et Schrenk P A A SS33 Drechslera ravenelli (Curt.) Subram. et Jain. P A A SS36 Glomerella sp. P P A SS40 Colletotrichum sp. A P A SS42 Curvularia lunata (Wakker) Boedijn var. aerea

(Bat., Lima et Vasconcelos) M.B. Ellis A P P

SS43 NI A P A SS44 NI A P A SS46 Fusarium oxysporum A A P SS50 Fusarium oxysporum A A P SS55 NI A A P SS62 NI A A P SS65 NG A P P SS67 Nigrospora sphaerica (Sacc.) E. W. Mason A P P SS68 NI A A P SS70 Glomerella sp. A A P SS73 NI A P A SS74 Mycelia sterile A P A SS75 NG A P A SS76 Aspergillus fumigatus Fresenius A P A SS77 Alternaria tenuissima (Kunze) Wiltshire A P A SS78 Mycelia sterile P A A SS79 NI P A A SS82 Aspergillus niger van Tieghen A P A SS83 NI A P A SS84 Phoma betae Frank A P A

NI = not identified. NG = the fungus did not grow. A = absent. P = present.

folius tissues possibly demonstrating tissue specificity.

Hence, it was possible to isolate eleven, six and nine

species from stems, roots and leaves, respectively. A

single species, SS21, displayed a systemic colonization

and five other species occurred simultaneously in two

tissues. Species SS13 was recovered from leaves and

roots, SS36 grew from leaves and stems, while SS42,

SS65 and SS67 were isolated from both stems and roots.

Some strains did not sporulate or grow in the culture

conditions employed. They remain unidentified and

were named sterile mycelia. Isolates that sporulated

were identified by traditional taxonomical methods

based on morphological features as established in

Table 1.

The isolated genera Drechslera, Fusarium, Phoma, Alter-

naria, Curvularia, Nigrospora, Glomerella and Aspergilus

have been previously reported as plant pathogens in a

range of hosts [18–20]. They also have proven to be

endophytes in several plant species [21–26] strengthen-

ing the argument that disease is an exception in the

plant-microbe interaction, which could be more prop-

erly regarded as an unbalanced status in a symbiosis

[27]. Genus Papulaspora is a well-known competitive soil

mold in the culture of mushrooms [28, 29] and it has

just been recently reported as an endophyte in a few

species as Vitex negundo [24], Manilkara bidentata [30],

Taxus chinensis [31], Amomum siamense [32] and Solanum

tuberosum [25]. Preussia, however, is recognized either as

a soil or a coprophilous genus [33, 34] but has never

been mentioned before as an endophyte.

Cytotoxic activity of extracts From the 32 fungal isolates, 186 extracts were obtained

and tested against three tumor cell lines. EaL fractions

had the most pronounced cytotoxic effects together

with EaS fractions (Table 2), whereas fractions EtM, RL

and RS were practically inactive (data not shown). The

exceptions were RLSS33, RLSS75 and RLSS84, which

Journal of Basic Microbiology 2009, 49, 142–151 Production of new cytotoxic agents 147

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Table 2. Cytotoxic activity of the ethyl acetate fractions from liquid (EaL) and solid (EaS) cultures of the endophytic fungi isolated from Smallanthus sonchifolius.

Cell growth inhibition (%) Fungus

HCT-8 MDA-MB435 SF295

EaL fraction EaS fraction EaL fraction EaS fraction EaL fraction EaS fraction

SS13 100.0 (HA) 54.5 (MA) 95.2 (HA) 81.3 (HA) 98.1 (HA) 34.2 SS16 32.0 1.6 31.3 25.7 35.7 4.1 SS20 NC 10.3 NC 11.0 NC 4.1 SS21 27.2 I 37.0 I 23.3 10.9 SS22 100.0 (HA) I 100.0 (HA) 11.8 100.0 (HA) I SS26 31.9 19.9 17.1 15.9 41.2 24.9 SS29 40.1 68.0 (MA) 60.6 (MA) 27.9 33.1 62.9 (MA) SS32 56.6 (MA) 33.7 20.0 33.0 47.7 15.1 SS33 86.7 (HA) 3.6 71.2 (MA) I 84.5 (HA) I SS36 52.4 (MA) 35.3 38.7 44.7 49.0 19.7 SS40 3.6 I 10.7 I 12.0 I SS42 30.9 41.0 36.3 52.0 (MA) 21.2 27.1 SS43 33.9 54.2 (MA) 49.2 59.4 (MA) 25.0 54.5 (MA) SS44 62.7 (MA) 23.3 50.7 (MA) 12.1 48.1 3.2 SS46 85.6 (HA) 13.0 83.2 (HA) 30.3 85.8 (HA) 9.7 SS50 30.2 I 57.9 (MA) I 12.3 I SS55 55.3 (MA) I I I 14.2 I SS62 99.8 (HA) 27.2 51.4 (MA) 32.2 67.5 (MA) 31.8 SS65 NC 1.4 NC 10.8 NC 14.2 SS67 79.2 (HA) 86.2 (HA) 48.2 41.9 73.0 (MA) 82.2 (HA) SS68 39.2 55.2 (MA) 36.4 23.3 26.9 54.4 (MA) SS70 22.1 32.8 32.1 41.7 18.5 18.9 SS73 28.4 I 6.6 I 9.1 I SS74 24.6 83.9 (HA) I 81.0 (HA) 8.4 71.3 (MA) SS75 19.5 22.2 41.0 4.9 7.4 28.1 SS76 100.0 (HA) 48.9 42.6 30.4 52.7 (MA) 40.3 SS77 94.7 (HA) 72.9 (MA) 82.0 (HA) 53.0 (MA) 78.3 (HA) 69.2 (MA) SS78 4.9 I 19.3 3.3 6.4 I SS79 26.2 5.1 I 12.4 8.4 I SS82 87.6 (HA) 9.1 45.7 1.4 68.2 (MA) I SS83 49.3 8.8 27.7 32.5 51.0 (MA) 16.5 SS84 100.0 (HA) 12.4 92.4 (HA) 10.1 95.6 (HA) 15.0 control 25.9 2.2 34.6 20.1 25.1 4.7

displayed moderate to high activities against colon and

breast tumor cells; RSSS65 and RSSS78 inhibited about

50% of colon cells proliferation and EtMSS13, EtMSS67

and EtMSS76 were efficient inhibitors of HCT-8 tumor

cells growth. It is noteworthy that only ethyl acetate

fractions from both culture types of P. immersa (SS13),

N. sphaerica (SS67) and A. tenuissima (SS77) produced good

results on all tested cells indicating that production

of bioactive molecules by these fungi does not depend

on the culture medium employed. These observations

emphasize the significance of these species in future

biotechnology studies.

Cytotoxic effects and ecological importance of isolated compounds N. sphaerica (SS67) and P. betae (SS84) were chosen for

large scale cultures and isolation of bioactive com-

pounds due to the cytotoxicity of their extracts.

Three substances were isolated from the N. sphaerica

EaS fraction (SS67, Fig. 4): compound 1 was identified as

pimara-7,15-dien-3β-ol, compound 2 as ergosterol per-

oxide and compound 3 as aphidicolin. Spectral data of

compound 2 were in agreement with literature data

[35], while compound 1, recently detected in a plant

essential oil, was not characterized by the authors [36].

This is the first report of compound 1 obtained from a

fungal source and having its structure completely elu-

cidated by 1H and 13C NMR, HMQC, HMBC, MS and

NOEdiff spectral data. Both compounds 1 and 2 showed

weak cytotoxic activity against the tumor cell lines

tested (Table 3).

Compound 3, isolated from the EaL fraction of

N. sphaerica (SS67) had its structure established as the

diterpene aphidicolin (Fig. 4) on the basis of its NMR

data that are in agreement with reported data [37].

Aphidicolin showed a remarkable cytotoxic activity

148 M. B. C. Gallo et al. Journal of Basic Microbiology 2009, 49, 142–151

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Figure 4. Structures of compounds isolated from Nigrospora sphaerica and Phoma betae. Pimara-7,15-dien-3β-ol (1); ergosterol peroxide (2); aphidicolin (3); (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (4); 8-hydroxy-6-methoxy-3-methylisocoumarin (5); dimethyl terephthalate (6).

against all cell lines (Table 3), but its direct in vivo ad-

ministration is difficult due to its poor water solubility

and fast metabolization. Aphidicolin also may damage a

fragile site of the mammalian genome [38, 39]. How-

ever, it is a valuable tool to study eukaryotic DNA syn-

thesis and cell cycles.

P. betae (SS84) EaL fraction actually showed maximal

efficacy against all the three cancer cell lines and was

Table 3. Inhibitory effect on cultured cell growth of compounds isolated from Nigrospora sphaerica (NS) and Phoma betae (PB), endophytes in Smallanthus sonchifolius.

IC50 (µg/ml) [CI95]a Compound (Fungus)

HCT-8 MDA-MB435 SF295 HL-60

1 (NS) >25 >25 >25 22.60 [18,57–27,64] 2 (NS and PB) >25 >25 >25 >25 3 (NS) 0.05 [0.037–0.079] 0.20 [0.116–0.367] 0.16 [0.081–0.338] 0.09 [0.063–0.174] 4 (PB) 6.24 [4.96–7.85] 14.11 [11.87–16.78] 17.03 [11.40–25.43] 5.29 [4.31–6.50] 5 (PB) >25 >25 >25 >25 6 (PB) >25 >25 >25 >25 Doxorubicin 0.01 0.48 0.24 0.02

a 95% confidence interval.

Journal of Basic Microbiology 2009, 49, 142–151 Production of new cytotoxic agents 149

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

considered highly active (Table 2). In liquid potato-

sucrose medium, P. betae is reported to produce an as-

sortment of bioactive diterpenes [40, 41], including ap-

hidicolin (3), an specific inhibitor of DNA polymerase.

Hence, the low cytotoxic activity of SS84 EaS fraction

was intriguing (Table 2) and led us to investigate its

chemical composition. For this purpose the fungus was

cultivated for two different time periods: for 20 days

according to the general protocol and for 32 days leading

to sporulation. EaS fraction from 20 day cultures was

subjected to chromatographic procedures and the resul-

tant subfractions were monitored by GC/MS and 1H NMR

techniques. Several fatty acids were detected but only

compound 2 was isolated corroborating the initial low

activity. However, the EaS fraction resulting from 32 day

cultures yielded compound 6, which was identified as

dimethyl terephthalate (Fig. 4) [42], and a subfraction

that inhibited 100% the proliferation of tumor cell lines

tested. The subfraction 1H NMR spectrum revealed a

mixture of two substances in a 1:1 ratio, later separated

and identified as (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-

one (compound 4) and 8-hydroxy-6-methoxy-3-methyl-

isocoumarin (compound 5) in accordance with literature

data (Fig. 4) [43, 44]. When compounds 4 and 5 were

tested alone they showed moderate and weak activities,

respectively (Table 3). The low cytotoxicity of compound

4 against other tumor cell lines had been previously

described [45, 46] but the results in this report suggest

that isocoumarin (compound 5) and ergosteroid (com-

pound 4) are acting synergistically. In fact, it has been

reported that an isocoumarin propionic acid derivative

has recently entered clinical trials as an anticancer and

anti-angiogenic agent with the property of potentiating

or improving either the therapeutic effects of some

chemotherapeutic drugs [47, 48] or radiotherapy [49].

Our results suggest that compounds 4 and 5 might be

considered as hits for drug development of a novel anti-

cancer combination therapy.

The amounts of pesticides employed in S. sonchifolius

cultivation are relatively small leading some authors to

suggest that sesquiterpene lactones [50] and kaurene

diterpenoids [51] occuring in plant leaves may act as

resistance factors. In fact, the endophytes P. betae and

N. sphaerica and their host have similar biosynthetic path-

ways [52, 53], and the fungal cytotoxic compounds may

provide extra protection for the plant (Table 1). N. spha-

erica is also known to cause both reduction of spore ger-

mination [54] and growth inhibition of some pathogenic

fungi, i.e, Fusarium oxysporum [55, 56], one of the isolated

endophytic species in Yacon roots (Table 1). Therefore,

maintenance of N. sphaerica and P. betae as endophytes

seems to be an ecological benefit to S. sonchifolius.

Acknowledgements

This study was funded by grants from the Fundação de

Amparo à Pesquisa do Estado de São Paulo (FAPESP)

within the Biota-Bioprospecta-FAPESP – The Biodiver-

sity Virtual Institute Program (www.biota.org.br), grant

n° 04/07935-6, and the Brazilian Government’s National

Council for Scientific and Technological Development

(CNPq). The authors thank Silvana França dos Santos

for technical assistance. Margareth B. C. Gallo acknowl-

edges FAPESP for the postdoctoral fellowship (grant n°

05/56259-6).

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((Funded by:

• Fundação de Amparo à Pesquisa do Estado de São

Paulo (FAPESP) within the Biota-Bioprospecta-FAPESP;

grant number: 05/56259-6

• The Biodiversity Virtual Institute Program

(www.biota.org.br); grant number: 04/07935-6

• Brazilian Government’s National Council for Scien-

tific and Technological Development (CNPq) ))