|STEROID COMPOUNDS FROM THE FUNGUS LAETIPORUS SULPHUREUS (BULL.:FR.) MURR.: RECOVERY AND QUANTITATION
Mishyn L., Gvozdkova T.
Institute of Microbiology, National Academy of Sciences of Belarus, Minsk, Belarus
It is known that the wood-rotting basidial fungus Laetiporus sulphureus is able to synthesize a number of steroid compounds, mainly, sterols and triterpenoic acids. The main sterol of this fungus is ergosterol, fungisterol was found in less quantities . Triterpenoic acids (eburicoic and sulphurenic) were shown to be present in extracts from L.sulphureus . Steroids mentioned above have a significant biological activity. Mycelium of L.sulphureus grown in submerged culture is believed to be a possible source for obtaining eburicoic acid, the amount of which can reach up to 30% of the dry mycelium weight . The common method to recover sterols and triterpenoic acids includes a number of procedures to transfer them at last to diethyl ether . This method is very labour-consuming and demands to use diethyl ether which is pretty dangerous due to its explosive properties. The main objects of this work were: 1) to make the recovery of steroids from fungal mycelium less labour-consuming; 2) to change diethyl ether to other more safe solvent; 3) to choose or to develop a convenient method for the quantitation of ergosterol and eburicoic acid and to estimate their concentrations in the extract of L.sulphureus.
Mycelium of L.sulphureus M131 was grown in 500 ml Ehrlenmeyer flasks containing 200 ml of the glucose-peptone medium on a shaker 180 r.p.m. during 144 hours at 26 - 280C. The pellets obtained by filtration were then grinded with a pestle in a mortar using the quartz sand and extracted by 96% ethanol until they got colorless. The proportion of the mycelium wet weight and the volume of ethanol was 1:100 (w/v). We have tested three methods to recover sterols and triterpenoic acids, two of which (methods 1 and 2) are modifications of the method described .
Method 1. The ethanolic extract from L.sulphureus was evaporated to dry using a rotary vacuum vaporizer, then saponified during 4 hours on a boiled water by 5% KOH solution. After the saponification, the same volume of water was added, non-saponifiable substances were extracted by hexane (2 times, 50 ml each time) in a flask at a gentle shaking. The mixture was moved to a separating funnel, the lower water layer was acidified by HCl solution up to pH 1.5 – 2.0 and extracted by hexane, the upper layer was collected for further investigations. Two fractions obtained were subjected to spectrophotometric studies and chemical analysis according to the Lieberman–Burchard method.
Method 2. In contrast to the method 1, the saponification was made by the ethanolic solution of KOH (5 %) and chloroform was used as solvent instead of hexane. Before carrying out any studies, both fractions were evaporated to dry and redissolved in 10 ml of ethanol.
Method 3 (without saponification). The mixture of the equal volumes of ethanolic extract, hexane and water was shaked gently, poured out to a separating funnel. Two layers appeared were collected and studied.
The determination of ergosterol was made according to Lieberman and Burchard (the Proskuryakov modification) . Spectrophotometric studies were made using the “Specord M 40” spectrophotometer.
Both fractions (saponifiable and non-saponifiable) obtained by the method 1 gave the absorbance spectra that are typical of ergosterol. Any spectra typical of tetracyclic triterpenoids were not found here. After acidifying the saponifiable phase up to pH 1.5 – 2.0 a precipitate was formed. The precipitate was dissolved in ethanol and gave absorbance data that are characteristic both of ergosterol and triterpenoids. So we concluded that the acidification of this phase caused to the formation of precipitate of triterpenoic acids that are insoluble in water. It is also possible that a part of ergosterol was not completely extracted by hexane and remained. For this reason, we decided to carry out the saponification in an ethanolic medium and to use for the extraction more polar solvent (chloroform) – method 2. In the non-saponifiable fraction obtained by the method 2 we have found out the below absorbance maxima (nm): 244.6 – 253.5 – 270.6 – 282.2 – 293.1 – 316.4 (fig.1).
Absorbance Absorbance Absorbance
Wavelength, nm Wavelength, nm
Fig.1. UV absorbance curve of the non-saponi- Fig.2. UV absorbance curves of the hexane (A) fiable fraction obtained by the method 2 and water-ethanolic (B) phases obtained by the
The maxima at 244.6 and 253.5 nm we connect to the presence of another sterol – fungisterol. The other absorbance maxima are typical of ergosterol.. The saponifiable fraction didn’t give any distinct peaks. The Lieberman-Burchard reaction with the non-saponifiable fraction was positive, with the saponifiable – negative. Thin-layer chromatography has revealed that non-saponifiable fraction contains a major component giving a bright spot after clearing up a chromatographic plate in iodine vapour. The Rf value of this spot corresponds to ergosterol. Non-saponifiable fraction didn’t give any clear spots. So we came to conclusion that the method 2 may be used to recover an ergosterol-rich fraction, but it doesn’t fit for the recovery of triterpenoic acids. The upper (hexane) phase obtained by the method 3 gave absorbance spectrum of ergosterol (fig.2A). In the water-ethanol phase (fig.2B) we can see a distinct peak at 244.1 nm and a bend at 252.0 nm. Such a spectrum is typical of tetracyclic triterpenoids. Taking in account that eburicoic acid is the major triterpenoid of L.sulphureus, we suppose that just the presence of this acid causes such an absorbance. The upper phase gives a color product having the absorbance maximum at 684.9 nm in the Lieberman-Burchard reaction, the lower – a color product with the maximum at 491.2 nm. On these grounds, we concluded that the method 3 can be used for the recovery both sterols and triterpenoic acids. The content of ergosterol determined in the fractions obtained by the methods 2 and 3 was approximately equal (17 –18 mg per 1 l of ethanolic extract, i.e. 0.68 – 0.72 % of the dry mycelium weight). The lack of eburicoic acid as a standard didn’t give us a possibility to determine its content in the ethanolic extract from L.sulphureus.
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