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IJNP _______________

____N2___2013

Isolation of the terpestacin from the
endophytic fungus Drechslera ravenelii
Eliane O. Silva, Willian J. Andrioli, Adriana A. Lopes, Mônica T. Pupo
and Jairo K. Bastos*
Faculty of Pharmaceutical Sciences of Ribeirão Preto. University of São Paulo. Ribeirão Preto – SP,
Brazil.

In our studies about biprospection in endophytic fungi isolated from Smalanthus
sonchifolius, the fermentation in solid medium rice-oat by 720 hours of the
fungus Drechslera ravenelii (SS33) led to the isolation of sesterpene
terpestacin. The molecule terpestacin has attracted the scientific community
attention due to inhibitory activity in the syncytium formation caused by the HIV
virus and also by presenting unusual chemical structure. After fermentation in
the rice-oat medium, the hydroalcoholic extract was submitted to the liquidliquid partition with n-hexane, dichloromethane, ethyl acetate and butanol. The
dichlorometanic fraction was subjected to chromatographic processes to obtain
19.0 mg of terpestacin. The structure of this compound was were elucidated by
detailed spectroscopic analyses. This study reported the isolation of terpestacin
from the endophytic fungus Drechslera ravenelii for the first time.
Keywords: Drechslera ravenelli, endophytic fungus, secondary metabolites,
terpestacin
Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson, Asteraceae, is a
medicinal plant popularly known as Yacon and cultivated primarily in Brazil,
Japan and New Zealand. Its roots are rich in sugars not absorbable by the human
organism, being labeled as food and dietary prebiotic (Pedreschi et al., 2003).
Endophytes are microorganisms (fungi or bacteria) able to colonize and
live in healthy tissues of its host without causing symptoms for at least one
period of its life cycle. However, it may cause disease after an incubation period
or latency (Petrini, 1991). The association between endophyte and its host can
be symbiotic. Therefore, the host plant (macrophyte) protects and feeds the
endophyte, which “in return” produces bioactive metabolites to enhance the

*

Correspondence: Dr. Jairo K. Bastos
Faculdade de Ciências Farmacêuticas de Ribeirão Preto. Universidade de São Paulo. Ribeirão PretoSP, Brasil. Avenida do Café, s/n, 14040-903, Ribeirão Preto – SP, Brasil. Phone: +55 16 3602 4181.
Fax: +55 16 3633 1092. E-mail: jkbastos@fcfrp.usp.br

2

IJNP _______________

____N2___2013

growth and competitiveness of the host and protect it from herbivores and plant
pathogens (Dreyfuss and Chapela, 1994).
The endophytic fungi can produce substances typically produced by the
host plant. In this context, there is the Taxol (paclitaxel) production by the
endophytic fungus Taxomyces andreanae, which host plant is the Taxus
brevifolia, original source of Taxol (Borges et al., 2009).
There are few reports in the literature on studies involving the endophytic
fungus Drechslera ravenelii. It is known that it is able to produce pigments
yellow-orange belonging to anthraquinone class of compounds (pigments called
chrysophanol and helminthosporin) or derivatives of xanthone group (pigments
called ravenelin) (Eijk and Roeymans, 1981).
The aim of the present work was to investigate the
production of secondary
metabolites
by
the endophytic
fungus D. ravenelii under different culture conditions, as well as to identify the
secondary metabolites.
Results and discussion
Regarding the study to investigate the production of secondary
metabolites from D. ravenelii, the chromatographic procedures undertaken for
the cultures furnished 12 fractions with yieldings ranging from 76.5 mg for
CH2Cl2 (PDB/216 h) to 4.636 mg for CH2Cl2 (rice-oat/720 h). The CH2Cl2
fraction from rice-oat medium was submitted to different chromatographic
procedures to afford terpestacin (Figure 1). Spectral data obtained of the
compound are in agreement with those reported by Oka and co-workers (1993).
It is important to point out that this is the first report of terpestacin in the
endophytic fungus D. ravenelii.
20 CH

3

O
4
3

HO

17

1
24
16

HO

5

18

15

2

6

7

CH3
23

8

23

CH3

14

13

10

25

12

H3C

22

CH3

21

9

11

OH

Figure 1. Structure of terpestacin isolated from endophytic fungus Drechslera ravenelii

During the search for drugs to cure AIDS a research group of BristolMyers Squibb's Co isolated terpestacin from a culture broth of Arthrinium sp.,
and it displayed a promising activity as a novel syncytium formation inhibitor,
3

IJNP _______________

____N2___2013

which is expected to be an anti-HIV drug. They have also determined the
absolute configuration of terpestacin mainly by NMR studies and X-ray singlecrystal analysis to be a bicyclo 5,15-fused sesterpene. Terpestacin has also been
reported to inhibit angiogenesis on the basis of assays in bovine aortic
endothelial cells and in chorioallantoic membrane from chick embryos (Jung et
al., 2003; Oka et al., 1993). Furthermore, terpestacin has been reported to have
only modest antimicrobial activity, suggesting that it is not an indiscriminate
cytotoxin and may therefore be a useful lead compound for the development of
anticancer as well as anti-AIDS chemotherapeutics (Myers, Siu and Ren, 2002).
In 2001, Schlegel and co-workers isolated terpestacin from an
Ulocladium fungus, and the interest in this compound was due to both the novel
carbon skeleton and the pharmacological activity (Schlegel et al., 2001)
As a consequence of the novelty of the structure as well as the promising
activity, Berger and Tius (2007) reported the total synthesis of (±)-terpestacin
after 15 step synthesis with 6.4% yield. In our work we report the isolation of
the (-)-terpestacin with 0.5 % (w/w) yield.
Experimental
Isolation and culture of the endophytic fungus
Drechslera ravenelli was isolated from Yacon (Smallanthus sonchifolius
(Poepp.) H. Rob.; Asteraceae) leaves, and it was collected in February 2006, in
Ribeirão Preto (S 21°11.933′ NO 47°46.699′), SP, Brazil, and it was identified
by the staff of the Department of Mycology, Center of Biological Sciences of
the Federal University of Pernambuco, PE, Brazil. The microorganism has been
maintained in PDA slants (Potato Dextrose Agar) and at 4 °C.
The endophytic fungus was submitted to pre-fermentation according to
the methodology described by Jackson and co-workers (1998). The culture
media used in the fermentation were: I) Czapek (Atlas, 1995); II) PDB (Potato
Dextrose Broth) and III) rice-oat (40.0 g of rice, 20.0 g of oat and 42.0 mL of
solution aqueous containing 0.9% of peptone and 9.0% of MnCl2).
The cultures were conducted in two steps: a) pre-fermentative, in which
the fungi was inoculated into Falcon tubes containing 10 mL of seed medium
Jackson, followed by incubation at 30 °C for 48 hours in a rotary shaker (120
rpm); b) fermentative, in which the mycelium biomass of the pre-culture
medium was transferred to Erlenmeyer flasks containing 100 mL of either
Czapek or PDB medium, and incubated at the same pre-fermentative conditions
for 216 and 480 hours, respectively. Pre-cultures were also transferred to
Erlenmeyer flasks containing rice-oat medium and incubated at 30 ºC for 720
hours.
Extraction and isolation of secondary metabolite
Ethanol was added to each culture and macerated overnight. Then, the
suspension was separated from mycelium by filtration under vacuum. The
4

IJNP _______________

____N2___2013

filtrate was extracted with n-hexane, dichloromethane, ethyl acetate and nbutanol, in sequence, and the organic solvents were concentrated under vacuum
to furnish the crude fractions.
Table 1. 1H- , 13C-NMR, HMBC and COSY data of terpestacin (500 MHz and 125 MHz,
respectively, CD3OD, δH and δC in ppm, J in Hz).
Position

δ 1H (multiplicidade, J em Hz)

Δ

HMBC

COSY

13C/HMQC

1

-

50.23

-

-

2

1.78 (m); 2.37 (m)

40.54

C-15

H-3

3

5.31 (m)

123.34

-

-

4

-

138.78

-

-

5

2.03 (m); 2.32 (m)

41.50

-

H-6

6

2.35 (m)

25.01

C-4; C-7

-

7

5.18 (m)

125.60

-

H-6

8

-

134.06

-

-

9

1.78 (m); 2.06 (m)

36.05

-

H-10

10

1.64 (s); 1.79 (m)

31.23

-

H-9; H-11

11

3.99 (dd, 9.50, 4.10 )

77.34

C-13

-

12

-

137.57

-

-

13

5.40 (m)

130.42

C-11; C-22

H-14

14

1.92 (m); 2.45 (d, 17.05)

30.28

-

-

15

2.75 (dd, 1.87, 11.37)

51.07

C-1; C-13; C-14; C16 ; C-17

-

16

-

152.46

-

-

17

-

149.24

-

-

18

-

210.43

-

-

19

0.96 (s)

17.17

C-1; C-2; C-15; C-18

-

20

1.65 (s)

15.83

C-3; C-4; C-5;

H-3

21

1.65 (s)

15.66

C-7; C-8; C-9;

H-7

22

1.56 (s)

10.58

C-11; C-12; C-13

H-13

23

2.64 (sextuplete)

38.95

C-16; C-17; C-24

H-25

24

3.70 (dd, 6.69, 10.71); 3.83 (dd,
7.14, 10.71)

66.18

C-16; C-25

H-23

25

1.27 (d, 7.36)

14.88

C-16; C-23; C-24

-

The concentration of CH2Cl2 fraction from rice-oat medium under
vacuum gave a brown residue (4.636 g), which was subjected to vacuum liquid
chromatography (silica 60 H) eluted with a hexane-ethyl acetate gradient to
obtain 13 fractions. The 7th fraction (419.4 mg) was submitted to silica gel
column (230–60 mesh, 34.0 × 2.5 cm) eluted with hexane-ethyl acetate
gradient, affording 17 fractions. The 5th fraction (120 mg) was purified on
5

IJNP _______________

____N2___2013

preparative HPLC (C18 reversed-phase Shimadzu column (250 × 10 mm), using
the mobile phase CH3OH:H2O (60:40 to 100%), at a flow rate of 6.0 mL min–1
and detection at 262 nm, to give terpestacin (19 mg).
Terpestacin: white solid, Rf (HPLC): 27.0 min; [α]D25 - 27.5 (c 0.2, MeOH);
HEREIMS: m/z 425.3203 [M+Na]+ (calcd. for C25H38O4: 425.2668); 1H- and
13
C- NMR, HMBC and COSY: see Table 1.
Analytical Methods
All NMR data were collected on a Bruker DRX500 spectrometer with 1H
and 13C observed at 500 and 125 MHz, respectively, and the chemical shifts
were expressed in δ (ppm) relative to SiMe4 (the internal standard) with
coupling constants J in Hz. EI–MS were performed in a high-resolution
eletrospray Ultro-TOF - Bruker-daltonics spectrometer (HREIMS). Optical
rotations were measured in MeOH using a Jasco Digital Polarimeter – DIP 370
at 25 ºC. The high performance liquid chromatography (HPLC) system
consisted of a Shimadzu with UV-Vis detector.
Conclusions
Our results provide a new source for the production of terpestacin, which
displays interesting biological activities and an unusual chemical structure.
Furthermore, the obtention of terpestacin from D. ravenelii is advantageous
because it is enantioselecive and shows good yield in comparison for terpestacin
generated by synthesis strategy.
Acknowledgements
We are grateful to “Fundação de Amparo a Pesquisa do Estado de São
Paulo (FAPESP)” Brazil for financial support
References
Atlas, R.M. (1995). Handbook of microbiological media, CRC Press: Boca
Raton
Berger, G.O., Tius, M.A. (2005) Terpestacin Core Structure. Control of
Stereochemistry. Organic Letters, 7:5011-5013.
Borges, W.S., Borges, K.B., Bonato, P.S., Said, S., Pupo, M. T. (2009).
Endophytic Fungi: Natural Products, Enzymes and Biotransformation
Reactions. Current Organic Chemistry, 13:1137-1163.
Dreyfuss, M.M., Chapela, I.H. (1994). In The Discovery of Natural Products
with Therapeutic Potential, Butterworth-Heinemann: Boston.

6

IJNP _______________

Eijk,

____N2___2013

G.W., Roeymans, H.J. (1981). Revenelin, chrysophanol, and
helminthosporin, pigments from Drechslera holmii and Drechslera
ravenelii. Experimental Mycology, 5:373-375.

Jackson, M., Karwoswski, J.P., Humphrey, P.E., Kohl, W.L., Barlow, G.J.,
Tanaka, S.K. (1993). Calbistrins, novel antifungal agents produced by
Penicillium restrictum. The journal of Antibiotics, 46:34-38.
Jung, H.J., Lee, H.B., Kim, C.J., Rho, J., Shin, J., Know, H.J. (2003). Antiangiogenic activity of Terpestacin, a Bicyclo Sesterterpene from
Embellisia chlamydospora. The Journal of Antibiotics, 56:492-496.
Myers, A. G., Siu, M., Ren, F. (2002). Enantioselective synthesis of (-)terpestacin and (-)-fusaproliferin: clarification of optical rotation
measurements and absolute configurational assignments establishes a
homochiral satructural series. Journal of American Chemical
Society,124:4230-4232.
Oka, M., Limura, S., Tenmyo, O., Sawada, Y., Sugawara, M., Ohkusa, N.,
Yamamoto, H., Kawano, K., Hu, S., Fukagawa, Y., Oki, T. (1993).
Terpestacin, a new syncytium formation inhibitor from Arthrinium sp.
The Journal of Antibiotics, 46:367-374.
Petrini, O. (1991). Fungal endophytes of tree leaves; Andrews, J. H. and Hirano,
S.S., Eds. Microbial Ecology of Leaves. Spring-Verlag, New York, p. 179197.
Pedreschi R., Campos D, Noratto G, Chirinos R, Cisneros-Zevallos L. (2003).
Andean Yacon Root (Smallanthus sonchifolius Poepp. Endl)
Fructooligosaccharides as a Potential Novel Source of Prebiotics.
Journal Agriutural and Food Chemistry, 51:5278.
Schlegel, B., Schmidtke, M., Dörfelt, H., Kleinwächter, P., Gräfe, U. (2001). ()-Terpestacin and L-tenuazonic acid, inducers of pigment and aerial
mycelium formation by Fusarium culmorum JP 15. Journal of Basic
Microbiology, 41:179-183.

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