SPECTRAL FEATURES OF ETHANOLIC EXTRACT FROM THE BASIDIAL FUNGUS LAETIPORUS SULPHUREUS (BULL: FR.) MURR.
Mishyn L., Gvozdkova T.
Institute of Microbiology, National Academy of Sciences of Belarus, Minsk, Belarus
Spectral features have a great significance for the estimation of the molecular structure of organic substances. Absorbance spectra over the ultraviolet and visual ranges reflect transitions of coupled and uncoupled electrons in moleculars. Since the wavelength of the absorbed light corresponds to a definite transition, peaks recorded by any appropriate device show the presence of known molecular structures. Thus spectrophotometric data help to identify both color and colorless compounds.
As has been described earlier [1], an ethanolic extract from the fungus Laetiporus sulphureus have a pronounced antioxidative activity. So it was of interest to us to carry out spectrophotometric studies in attempt to find some molecular structures or substances that may be responsible for such an activity.
Mycelium of L.sulphureus M131 was grown in 500 ml Ehrlenmeyer flasks containing 200 ml of the glucose-peptone medium on a rotary shaker 180 r.p.m. during 144 hours at 26 - 280C. The extract was made extracting 1 g of washed and pressed out wet mycelium by 100 ml of ethanol after pounding mycelium with a pestle in the presence of the quartz sand. Spectral investigations were made by the “Specord M 40” scanning spectrophotometer. The ethanolic extract was diluted 6-fold by 96% ethanol before spectral investigations. The recovery of lipophilic compounds from the ethanolic extract was made as follows. Hexane was added to the extract to the proportion 1:1 (v/v), the mixture was mixed gently and then moved to a separating funnel. For the better separation of layers distilled water was added there to make the final hexane:ethanol:water proportion 1:1:1. Two phases obtained (upper – hexane, lower – water-ethanol) were collected and studied. Qualitative reactions to discover steroids were made according to the Lieberman-Burchard method [2]: 0.1 or 0.2 ml of the ethanolic extract was added to 2.1 ml of the Lieberman-Burchard reagent. The mixture was shaked and keeped at room temperature. All changes in color were observed and noted.
The absorbance spectrum of the ethanolic extract of L.sulphureus over the wavelength range 200 – 600 nm is given in the fig.1. A very high absorbance was fixed at the range 200 – 220 nm. Two peak groups can be distinguished over the wavelength range 230 – 300 nm.: 1) having absorbance maxima at 244 and 252 nm; 2) having maxima at 271 and 282 nm. One big peak at 450 nm is observed over the visual range (total carotenoids). To make two peak groups more legible we have changed the settings of the spectrophotometer over the range 230 – 300 nm and have got a more distinct picture (fig.2).
Absorbance Absorbance
Wavelength, nm Wavelength, nm
Fig.1. Absorbance curve of the ethanolic extract Fig.2. UV absorbance curve of the ethanolic of L.sulphureus over the UV-Vis ranges extract of L.sulphureus at the changed tu-
ning of the spectrophotometer
Taking in account that ethanolic extract of the fungus may contain non-lipid substances that could have similar absorbance maxima (i.e., aromatic aminoacids, proteins etc.), we treated the extract by hexane to recover a fraction of lipophilic substances.
Absorbance Absorbance Absorbance
Wavelength, nm Wavelength, nm
A B
Fig.3. Absorbance curves of the hexane phase of the Fig.4. Absorbance curve of the water-ethanolic ethanolic extract of L.sulphureus over the visual (A) phase of the ethanolic extract of L.sulphureus
and UV (B) ranges over the wavelength range 335 – 600 nm
Spectrophotometric studies of the hexane phase at the visual spectrum has revealed a three-peaked absorbance curve that is typical of carotenoids (fig.3a). The most marked UV absorbance is observed over the wavelength range 230 – 300 nm (fig.3b). We believe that this group of maxima corresponds at least to two compounds. During the investigation of the water-ethanolic layer a very high absorbance at the wavelength 200 – 220 nm was registered that is characteristic of the ethanolic extract itself. Several peaks are observed over the range 335 – 600 nm: a typical of carotenoids curve with the maximum at 429 nm and two smoothed shoulders and two pretty small peaks with the maxima at 354 and 383 nm (fig.4).
As a result, we have concluded that hexane recovers one of the minor carotenoids of L.sulphureus (the least polar) and compounds that absorb over the range 230 – 300 nm. According to the literature data [3] compounds that are isolated from wood-rotting Basidiomycetes and have the absorbance maxima at 244 and 252 nm are classified as tetracyclic triterpenoids with the skeleton of lanostan. Since L.sulphureus is able to synthesize large quantities of eburicoic acid [4] that have a structure of tetracyclic triterpenoid, we have supposed that just the presence of it causes the absorbance maxima at 244 and 253 nm. Alternatively, such an absorbance may be caused by sterols. The second group of peaks with the absorbance maxima at 261.2 – 270.9 – 281.9 – 293.8 nm corresponds to ergosterol which has the specific absorbance spectrum due to the presence of coupled double bonds. The appearence of green color during the Lieberman-Burchard reaction in 2 –3 min after adding a sample confirms that the ethanolic extract contains steroids..
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