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Production and Purification of Statins from Pleurotus ostreatus
(Basidiomycetes) Strains
´ 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: jalarcon@pehuen.chillan.ubiobio.cl * Author for correspondence and reprint requests Z. Naturforsch. 58 c, 62Ð64 (2003); received August 9/September 18, 2002
Pleurotus ostreatus strains were cultured in liquid medium and on wheat straw. The yields Key words: Pleurotus ostreatus, Statins, Lovastatin Introduction
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 (Basi- 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- tus ostreatus in their natural environment and also CoA) reductase that catalyzes the reduction of Pleurotus ostreatus 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 were col- production and purification of mevastatin from lected at the forest from VIII Regio´n of Chile, Penicillium citrinum. After this, lovastatin was ob- growing on Nothofagus sp. in autumn and spring tained from cultures of Monascum ruber (Man- 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 gillus terreus which yielded nearly 180 mg lovas- in the herbarium of Instituto de Investigacio´n tatin/l (Buckland et al., 1989).
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 (Basidiomycetes) Strains 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 et al. (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 (Basidiomycetes) Strains 2.62 (1H, m, J = 18,4,2, Ha)2.74 (1H, dd, J = 18, Hb) These preliminary results found in poor nutrient ostreatus 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.
tus) in rats with hypercholesterolemia. Cas Lek Cesk Gunde-Cimerman N., and Cimerman A. (1995), Pleuro- 136(6), 186Ð190.
tus 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, 1Ð6.
hibitor of cholesterol accumulation in humans. In: Hajjaj H., Niederberger P., and Duboc Ph. (2001), Lo- Novel Microbial Products for Medicine and Agricul- vastatin biosynthesis by Aspergillus terreus in a chemi- ture. Society for Industrial Microbiology (A. L. De- cally defined medium. Appl. Environ. Microbiol. 67,
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ities of macrophages and lymphocytes in mice treated with a carcinogen N-butyl-N-butanolnitrosamine. In- Endo A., Kuroda M., and Tanzawa K. (1976), Competi- munopharm. Immunotox. 19, 175Ð183.
tive inhibition of 3-hydroxy-3-methylglutaryl coen- Manzoni, M., Bergomi, S., Rollini M., and Cavazzoni V.
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