Production and Purification of Statins from Pleurotus ostreatus
´ guila, Patricia Arancibia-Avila, Oscar Fuentes,
Enrique Zamorano-Ponce, and Margarita Herna´ndez
Facultad de Ciencias, Depto. Ciencias Ba´sicas, Universidad del Bı´o Bı´o, Chilla´n, Chile.
Fax: +56-42-20 30 46. E-mail: firstname.lastname@example.org
* Author for correspondence and reprint requests
Z. Naturforsch. 58 c
, 62Ð64 (2003); received August 9/September 18, 2002
strains were cultured in liquid medium and on wheat straw. The yields
Key words: Pleurotus ostreatus
, Statins, Lovastatin
As part of a larger study of chilean mushrooms,
we have found in the fungi Pleurotus ostreatus,
Pleuroteaceae (Basidiomycetes), an importantcholesterol reducing molecule. Here we report thepresence of statins in fruiting bodies and fermen-tation processes from Pleurotus ostreatus
collectedfrom native forest of the VIII Region of Chile.
The genus Pleurotus
is represented in Chile by two
Fig. 1. Lovastatin molecular structure.
species P. ostreatus
and P. sutherlandii
, both foundgrowing attached to the cortex of endemic Notho-fagus
trees (France et al.
, 2000). Lovastatin has
Futhermore, the fact that lovastatin is present in
been extracted from Pleurotus pulmonarus
high proportion in this edible kind of mushroom,
makes this fungi an important food supplement
1995), and Kurashige et al.
(1997) has proven anti-
for patients suffering from hipercholesterolemia.
cancerigenous properties on the same molecule.
Therefore, we report our findings obtained from
Statins are found to be an inhibitor of the en-
the screening for statins of several strains of Pleuro-
zyme hydroxymethylglutaryl coenzyme A (HMG-
in their natural environment and also
CoA) reductase that catalyzes the reduction of
grown on a variety of natural
HMG-CoA to mevalonate during synthesis of cho-
subtrates inside a greenhouse. Lovastatin and its
lesterol (Endo, 1992; Bobek et al.
, 1997). All natu-
precursors were isolated and purified from strain
ral statins have a common molecular structure, a
cultures that produce higher statins concentrations.
Material and Methods
lactone, but they differ from each other due to sidechains and a methyl group around the ring (Fig. 1).
Endo et al.
(1976) described a process for the
Fruiting bodies of Pleurotus ostreatus
production and purification of mevastatin from
lected at the forest from VIII Regio´n of Chile,
After this, lovastatin was ob-
growing on Nothofagus
sp. in autumn and spring
tained from cultures of Monascum ruber
of 2001. Mycelial cultures of the strain were de-
zoni et al.
, 1999) and in 1980 an industrial process
rived from the spore print of fruiting body. A
for commercial production was set up using Asper-
voucher specimen of the mushroom was deposited
which yielded nearly 180 mg lovas-
in the herbarium of Instituto de Investigacio´n
tatin/l (Buckland et al.
Agropecuaria, INIA-Quilamapu, Chilla´n, Chile.
” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com ·
J. Alarc´on et al.
· Statins from Pleurotus ostreatus
Fungal strain and culture conditions
The structure of lovastatin was established on
Four strains of Pleurotus ostreatus
were kept on
the basis of spectroscopic studies, GC-MS and
potato dextrose agar (PDA), and incubated for 7Ð
their spectral data was compared to data from lit-
10 days, stored at room temperature. Fermenta-
erature or from authentic sample (Table II).
tion was carried out in Hagen medium containing(g/l): CaCl
0.25, MgSO4 x 7H2O 0.15, 1.3 ml of FeCl3 1%,
The identification and quantification of statins
malt extract 3.0 and glucose 10.0; in a 3 liter fer-
were carried out on the culture filtrated and ex-
mentor with aeration and agitation (150 rpm);
tracts by HPLC, using a Merck LiChrospher 100
250 ml of well-grown culture (7 days) in the same
RP18 reverse phase column with a diode array de-
medium was used as inoculum. The fermentation
tector eluted at a flow rate 2 ml/min and elution
with gradient 90:10 water:methanol (v/v).
Results and Discusion
Mycelia cultures from the greenhouse were de-
The present study allowed to make a compari-
veloped according to the methodology of France
son between the content of lovastatin present in
(2000) on wheat straw. Fruiting bodies were
the fruiting bodies of P. ostreatus
cultivated in con-
collected after three months and were processed
servatory on straw of wheat and collected fruiting
for extraction using MeOH until exhaution. The
bodies in the forests of VIII the Region of Chile.
methanolic extract was concentrated and further
The results show that the produced fruiting bodies
extraction was done using AcOEt. Each of the ex-
in conservatories, present a lovastatin level that
tracts were analysed looking for lovastatin concen-
fluctuates between 0.40 to 2.07% measured in dry
fungus. However, the collected fruiting bodies intheir natural environment, present levels of lovas-
tatin between 0.7 to 2.8% measured in dry fungus.
Culture filtrate (3 l) obtained by filtration was
The mycelial development in liquid cultures gave
acidified to pH 3 with HCl 0.01 m and extracted
a range for lovastatin concentration between 5 and
with ethylacetate (4 ¥ 500 ml). The combined ex-
70 mg/l, depending of the seta type analyzed. It is
tracts were dried (Na2SO4) and concentrated to a
important to know that the statins are present un-
der two types of structural forms, depending on pH.
The mycelial mass was washed with 0.05 m HCl
One of them is the -hydroxyacid form being in al-
and stirred at room temperature for 1 h, then filtered
kaline solution, while the hydroxy acid and
and after acidification extracted with methylene
droxylactone form being in equilibrium in acid con-
chloride (3 ¥ 500 ml) and ethylacetate (2 ¥ 400 ml)
dition. Our GC-MS and HPLC laboratory analysis
for 1 h at 40∞ under stirring. The extract was dried
showed clearly this equilibrium, which contributes
(Na2SO4) and concentrated to a final volume of 5 ml.
to doublefold the lovastatin concentration.
J. Alarc´on et al.
· Statins from Pleurotus ostreatus
2.62 (1H, m, J
= 18,4,2, Ha)2.74 (1H, dd, J
= 18, Hb)
These preliminary results found in poor nutrient
in liquid media. The latter can be
culture conditions, suggest to us that it is possible
done modifying the nutritive quality of the media
to increase the yield of statins produced by P.
Bobek P., Ozdin L., Kuniak L., and Hromadova M.
France A., Can˜umir J. A., and Cortez M. (2000), Produc-
(1997), Regulation of cholesterol metabolism with di-
cio´n de Hongos Ostras Chilla´n, Chile. Instituto de In-
etary addition of oyster mushroom (Pleurotus ostrea-
vestigaciones Agropecuarias. Boletı´n INIA N∞ 23, 32 p.
) in rats with hypercholesterolemia. Cas Lek Cesk
Gunde-Cimerman N., and Cimerman A. (1995), Pleuro-
fruiting bodies contain the inhibitor of 3-hydroxy-
Buckland B., Gbewonyo K., Hallada T., Kaplan L., and
3-methylglutaryl-Coenzyme A reductase-lovastatin.
Masurekar P. (1989), Production of lovastatin, an in-
Exp. Mycol. 19
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Hajjaj H., Niederberger P., and Duboc Ph. (2001), Lo-
Novel Microbial Products for Medicine and Agricul-
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Kurashige S., Akusawa Y., and Endo F. (1997), Effects
of Lentinus edodes
, Grifola frondosa
Endo A. (1992), The discovery and development of
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with a carcinogen N-butyl-N-butanolnitrosamine. In-
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Manzoni, M., Bergomi, S., Rollini M., and Cavazzoni V.
zyme A reductase by ML-236A and ML-236B, fungal
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Biotechnol. Lett. 21
FEBS Lett. 72
Bladder stone discovery and treatment. Clavamox, cranberry, methionine to She pees blood for 2 days followed by blood clots for 1 day after catherization Urine cloudy , squatting frequently. inflammation/damage to bladder wall that is not completely healed Frequency noted frequent squatting that AM. Free catch examined. Riley sees struvites but no sign of bacteria. cholesterol
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