1.
Extraction of mushroom (oyster mushroom & button mushroom)
Water extraction:
The fruiting body was air-dried under shade for five days, and then prepared the
powder by using a grinder.
The magnetic-stirrer-based extraction of [Link] was performed. For the
extraction, we took 100 gm powder of [Link] and mixed with 1000 mL milli q
water and kept at 60°C temperature for 72 hours.
During this time continuous stirring by magnetic bead was allowed.
The extract was filtered twice by Whatman no.1 filterpaper and dried by using water-
bath at 60°C. The total percentage yield of extract was 25% w/w.
2. Ethanolic extract:
Freshly-harvested whole mushrooms were shadedried and then finely powdered.
Five grams of the powder was extracted with 100 ml of 95% ethanol using a
Soxhlet apparatus.
The solvent was evaporated under reduced pressure at 50 °C and dried in
vacuum.
The dried extract (5.86% yield) thus obtained was used directly for the
determination of anti-diabetic activities and analysis of the anti-diabetic
components.
3. Extraction and isolation of polysaccharides:
Four grams of powdered mushrooms were suspended in 200 mL of 80% ethanol and
extracted in a rotary shaker at 80 degreeC for 60 min.
The ethanolic extract containing low molecular weight compounds was removed by
centrifugation (3755x g, 20 min) and the solid residue was washed twice with 80%
ethanol and centrifuged.
The alcohol insoluble fraction was then re-suspended in deionised water (ratio 1:50
w/v) and autoclaved at 115 degree C for 180 min.
The obtained slurry was cooled and centrifuged (3755x g, 20 min) and the supernatant
was concentrated with a rotary evaporator and precipitated with three volumes of 2-
propanol (24 h, at 5 degree C).
The precipitate was then centrifuged, washed twice with 80% methanol, re-dissolved
in hot deionised water, lyophilised and weighed. The extraction process was done in
triplicate.
4. Phytochemical analysis of mushrooms (oyster mushroom & button mushroom):
Alkaloids:
Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms.
Procedure: 10 ml of the plant extract was dissolved in 1.5% HCL and was filtered 0.2 ml of
filtrate was added with 1 ml of Mayer’s reagent (Mercuric chloride- 1.36 gm, Potassium
iodide- 5 gm in 100 ml distilled water), formation of white precipitate indicated the presence
of alkaloids.
Flavonoids:
Flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized,
according to chemical structure into flavonols, flavones, flavanones, isoflavones, catechins,
anthocyanides and chalcones.
Alkaline reagent test: Crude extract was mixed with 2 ml of 2% solution of NAOH, intense
yellow colour which turns into colourless solution on few drops of dilute HCL indicated the
presence of flavonoids.
Saponins:
Saponins are amphipathic glycosides grouped phenomenologically by the soap like foaming
when shaken in aqueous solutions. Structurally, they possess one or more hydrophilic
glycoside moieties combined with a lipophilic triterpene derivative.
Foam test: Foam test is a usual way to test presence of saponins. One gram of plant extract
was taken in a test tube and small amount of water and sodium bicarbonate was added to it
and shaken vigorously for 5 min. Formation of foam indicated the presence of saponins.
Tannins:
Tannins are polyphenolic compounds, widely spread in nature and are present nearly in all
plant parts. Tannins are of two types hydrolysable tannin and condensed tannin. Hydrolysable
tannin contains a polyhydric alcohol. Condensed tannins are mostly flavonols and cannot be
hydrolysed to simple compounds.
Procedure: To the crude extract 2ml of 2% ferric chloride solution was added. Formation of
blue green coloration indicated the presence of tannins.
Test for terpenoids (Salkowski test):
Five ml of each extract was mixed in 2 ml of chloroform, and concentrated H2S04 (3 ml)
was carefully added to form a layer. A reddish brown colouration of the inter face was formed
to show positive results for the presence of terpenoids.
Steroids:
Sterols are special forms of steroids, with a hydroxyl group at position-3 and a skeleton
derived from cholestane. Procedure: Crude extract was mixed with 2ml chloroform, 2ml
conc. H2SO4 and 2ml acetic acid. Formation of green colour indicated the presence of
steroid.
Test for cardiac glycosides (Keller-killani test):
Cardiac glycosides, which are highly toxic and found in a number of plants, are usually
phytochemicals consisting of an aglycone (structurally related to steroid hormones) linked to
one or more sugar molecules. The aglycones of cardiac glycosides can be divided into two
chemical groups, the cardenolides and bufadienolides, with the former group being the most
implicated in cardioactivity. Procedure: A mixture of Acetic acid glacial (2 ml) with 2 drops
of 2% FeCl3 solution was added to the plant extract and conc. H2SO4 . A brown ring
produced between the layers indicated the entity of cardiac steroidal glycosides.
Test for carbohydrates Benedict’s test:
To 0.5 ml of the filtrate, 0.5 ml of Benedict’s reagent was added. The mixture was heated on
boiling water bath for 2 min. A characteristic red colored precipitate indicates the presence of
sugar.
Test for phenolic compounds Ferric chloride test:
The extract was diluted to 5 ml with distilled water. To this a few drops of neutral 5% ferric
chloride solution was added. A dark green color indicates the presence of phenolic
compounds.
