Alpha-amylase inhibitors from mycelium of an oyster mushroom

<p>The α-Amylase and α-glucosidase are two main enzymes involved in carbohydrate metabolism. This study was aimed at detecting alpha-amylase inhibitory activity from edible mushroom mycelia. Oyster mushroom was collected from a natural source, from Indian Institute of Technology (Banaras Hindu University) campus and was maintained <i>in vitro</i> in mycelial form. Chloroform, acetone, methanol, and water were used separately for extraction of an active constituent from mycelial cells grown, for 7 days, in potato dextrose broth. The extracts were tested for alpha-amylase inhibitory activity. Chloroform, acetone, and methanol extracts were found to have alpha-amylase inhibitory activity, with IC₅₀ values of 1.71, 224, and 383 μg/mL, respectively. Aqueous extract had no enzyme inhibitory activity. The acetone extract inhibited α-amylase non-competitively whereas chloroform extract showed competitive inhibition. Acetone extraction yielded highest total phenolic content (TPC) of 0.524 mM of gallic acid equivalent, whereas chloroform extraction resulted in lowest TPC of 0.006 mM. The HPLC and absorbance maxima of acetone and chloroform extracts suggest that the bioactive component responsible for enzyme inhibition could be glycoproteins in chloroform extract and catechins (flavonoids) in acetone extract. Thus, the mushroom mycelia under study may be exploited for production and purification of a lead compound for the development of the α-amylase inhibitory drug.</p

Fe²⁺ chelating activity.

<p><b>(A)</b><i>T</i>. <i>cordifolia</i> stem proteins; <b>(B)</b> enzyme hydrolysates (trypsin, chymotrypsin, and pepsin) of <i>T</i>. <i>cordifolia</i> stem proteins at a concentration of 4 mg/ml for all hydrolysis time.</p

Comparison of percentage reduction of DPPH.

<p><b>(A)</b> by different concentrations of <i>T</i>. <i>cordifolia</i> stem proteins and same concentrations of Ascorbic acid; <b>(B)</b> by <i>T</i>. <i>cordifolia</i> stem proteins hydrolyzed <i>in vitro</i> for various time intervals by different gastrointestinal enzymes (Papain, Trypsin, α-Chymotrypsin, and Pepsin).</p

Analyzes on enzyme digests.

<p><b>(A)</b> Tricine SDS-PAGE (14%) of <i>T</i>. <i>cordifolia</i> stem proteins treated with papain enzyme. Lane 1, marker (GeNei low molecular weight); lane 2, protein not treated with enzyme; lanes 3 to 7, protein treated with papain enzyme for different time intervals of 30 to 120 minutes; <b>(B)</b> Tricine SDS-PAGE (14%) of <i>T</i>. <i>cordifolia</i> stem proteins treated with trypsin enzyme. Lane 1, marker (GeNei low molecular weight); lane 2, protein not treated with an enzyme; lanes 3 to 7, protein treated with trypsin enzyme for time intervals of 30 to 120 minutes; <b>(C)</b> Tricine SDS-PAGE (14%) of <i>T</i>. <i>cordifolia</i> stem proteins treated with the α-chymotrypsin enzyme. Lane 1, marker (GeNei low molecular weight); lane 2, protein not treated with an enzyme; lanes 3 to 7, protein treated with the α-chymotrypsin enzyme for different time intervals of 30 to 120 minutes; <b>(D)</b> Tricine SDS-PAGE (14%) of <i>T</i>. <i>cordifolia</i> stem proteins treated with pepsin enzyme with silver staining. Lane 1, marker (GeNei low molecular weight); lane 2, protein not treated with the enzyme; lane 3, enzyme alone; lanes 4 to 7, protein treated with pepsin enzyme for different time intervals of 60 and 120 minutes, with two different enzyme:protein ratios of 1:3.33 and 1:6.67 (wt/wt); <b>(E)</b> Tricine SDS-PAGE (14%) of <i>T</i>. <i>cordifolia</i> stem proteins treated with pepsin-pancreatin enzymes with silver staining. Lane 1, marker (GeNei low molecular weight); lane 2, protein not treated with an enzyme; lane 3, enzyme alone; lane 4, protein treated with pepsin-pancreatin enzyme for 2 and 4 hours.</p

Relative activity of trypsin and α-chymotrypsin after a pre-incubation of 90 minutes with varying amounts of <i>T</i>. <i>cordifolia</i> proteins.

<p><b>(A)</b> Increase in Trypsin enzyme inhibition with increase in amount of protein; <b>(B)</b> Increase in α-Chymotrypsin enzyme inhibition with increase in amount of protein.</p

Radical scavenging activity of fractions obtained by FPLC of papain hydrolysate.

<p><b>(A)</b> TEAC of fractions; <b>(B)</b> O^2- radical scavenging activity of fractions. The X-axis represents fraction numbers with its concentration (mg/ml) used for the assay in parenthesis.</p