Pleurotus sp. known for its medicinal values and considered as king of all edible mushrooms. Three species of oyster mushrooms (Pleurotus spp.) that are cultivated mostly throughout the year in the plains of India were studied for their antioxidant properties with few modifications like ultra-sonication and standardizing various factors like solvent, buffer, pH, time, incubation time and enzyme concentration. Using buffer extraction/homogenization, there was remarkable increase (increase of two-fold) in enzyme activity from 13.35 to 26.70U/mg in Pleurotus djamor var. roseus, 8.47 to 19.05 U/mg in P. ostreatus and 11.74 to 22.12U/mg in P. florida after ultrasonication which has not been reported earlier while using different solvents i.e. Methanol, ethanol, and water. Maximum inhibition was observed in natural extract (96.38%), followed by buffer (91.84%) and ethanolic extract (91.74%). Comparative efficiency of homogenization of mushroom samples in buffer system with or without ultrasonication was evaluated, that has not been reported earlier for study of antioxidative stress enzymes in Pleurotus species. Maximum SOD (26.70U/mg) and POX (0.51U/ml) was recorded at pH 6.0 (potassium phosphate buffer, 1M) with P. djamor var. roseus and (22.12U/mg) and (0.39U/ml) for P. florida (phosphate citrate buffer, pH 6.5). Optimum SOD (29.95U/ml) and POX (0.63 U/ml) activity for P. djamor var. roseus was reported at 40 °C and 37 °C respectively at different time of incubation. Though the anti-oxidative stress enzymes could make a significant contribution to the antioxidant activity in these edible mushroom extracts, yet the chemical characteristics of these components are required to be investigated.
Pleurotus (edible mushroom) widely consumed globally has been reported to possess good antioxidant activity . Pleurotus are reported to be a good source of cysteine, methionine and aspartic in comparison to Agaricus bisporus (brown, white) and Lentinus edode  and bioactive compounds (Phenolic, terpenes, polyketides) . In addition, Pleurotus species reported to possess excellent free radical scavenging and therapeutic potential (Levostatin) for treating hypercholesterolemia . Pleurotus extracts could be used in the treatment of infections commonly associated with the micro -organisms and in treatment of skin diseases. Among various species of Pleurotus, Pleurotus djamor var. roseus has remained less studied till now, definite its use as potent mushroom. Efforts made in past to study the anti-oxidative stress enzymes from microbial as well as plant sources. The present investigation was carried out to test the anti-oxidative potential of medicinally important species of Pleurotus growing in the region.
Different Pleurotus sp viz Pleurotus ostreatus, Pleurotus djamor var. Roseus and Pleurotus florida and Pleurotus djamor var. roseus were procured from vikas benal mushroom farm, thakur mushroom farm and Directorate of mushroom research (DMR, Solan) respectively located in Himachal Pradesh, India. Pleurotussamples were morphologically analysedfor differences in colour, presence of scaling on their surface and characterized for anti-oxidative stress enzymes; SuperOxide Dismutase (SOD) and Peroxidase (POX).
Briefly, the collected Pleurotus samples were washed under tap water, surface sterilized (70% ethanol) and stored at -20 °C for one week. The freeze-dried samples were homogenized in liquid nitrogen using mortar and pestle in three different freshly prepared buffer solutions (Phosphate citrate, Potassium phosphate, Tris-HCl; 0.1 M) having pH 5.0 to 9.0 respectively in
the ratio of 1:10 (w/v). Ultra -sonication of the homogenised
samples was done (PUL 59, AMP1 32%, Temp. 8 °C) to obtain
the maximum disruption of cells. Post-ultracentrifugation, the
colloidal solution was centrifuged (15,400rpm for 30 min. at 4
°C) to remove cell debris and the supernatant was used as crude
enzyme solution for carrying out the enzyme assay.
All the chemicals were of high quality analytical grade and
procured from Sigma (USA), Merck (Germany) and Hi-Media
Pleurotus samples were dried at 27 °C for 24 hrs. and
grounded to powder using mortar and pestle. The extraction
was done using different mushroom sample with three different
solvent (methanol, ethanol and water) under the shaking
conditions (150 rpm) in the ratio of 1:10 (w/v) for 24 to 48 hrs.
at 30 °C. The mixture was filtered and the assay of anti-oxidative
stress enzymes i.e. superoxide dismutase (SOD) and (POX) was
performed in solvent extracts, supernatant and pellet.
Super Oxide Dismutase (SOD) assay based on the generation
of the superoxide radicals in PMS-NADH systems by oxidation
of NADH . Briefly, one-unit SODenzyme activity (U) is that
amount of enzyme required for 50% inhibition of NBT reduction
in one minute at 560 nm under assay conditions. Similarly,
one-unit peroxidase (POX) activity isthe amount of peroxidase
required for the conversion of substrate (1μM) per minute under
assay conditions  and involved O-PhenyleneDiamine (OPD)
(chromogen) and hydrogen peroxide (H2O2) as substrate.
The most suitable buffer and pH was determined by utilizing
three different buffer systems namely phosphate citrate (1M;
pH 5.0-8.0), potassium phosphate (1M; pH 6.0-8.0) and Tris-HCl
(1M; pH 8.0-9.0) for homogenization of sample and measuring
enzyme activity in each case.
The effect of incubation temperature and time on the enzyme
activity was evaluated by varying the range of temperature
(20, 25, 30, 35, 40 and 45 °C) for SOD, (25-45 °C) for POX and
incubation time (80-105 second) for SOD, incubation time (5-30
min) and enzyme was assayed in both cases. The influence of
reaction volume on enzyme activity was observed by varying the
volume of crude enzyme (100μl-500μl) and enzyme assay done
in each case.
Three different species of Pleurotus i.e. Pleurotus ostreatus,
Pleurotus florida and Pleurotus djamor var. roseus showed
distinct morphological characteristics of whitish color, white grey
color and pink color respectively. Samples were processed using
different solvent extracts and screened for two intracellular antioxidative
enzymes superoxide dismutase (SOD) and peroxidase
The different samples subjected to various solvent extracts
showed maximum SOD activity and POX activity. In ethanolic
extract, Pleurotus djamor var. Roseus showed maximum
(23.71U/mg) activity, followed by Pleurotus florida and (22.09
U/mg) in methanolic extract. Ramkumar et al.  reported that
the methanol extraction of Pleurotus strains had the highest
antioxidant activity. Free radical scavenging potential of ethanolic
extract recorded for Pleurotus djamor var. roseus (90.74%) was
found to be comparable to Pleurotus pulmonarius extract (90%)
as reported earlier . Similarly, ethanolic extract of Pleurotus
djamorvar. Roseus and methanolic extract of Pleurotus florida
showed (0.47U/ml) and (0.35U/ml) POX activity respectively.
However, aqueous extract emerged as poorest SOD and POX
activity for Pleurotus djamorvar. roseus and Pleurotus florida.
Methanolic extraction of Pleurotus florida exhibited (84.97%)
potent superoxide radical scavenging activities which is similar
to that reported earlier by Jose and Janardhanan . Antioxidant
mechanisms of the Pleurotus species extract; Pleurtus ostreatus
(84.52%), Pleurotus djamor var. roseus (90.74%) and Pleurotus
florida (84.97%) attributed by hydrogen-donating and
superoxide, free radicals scavenging ability .
Maximum enzyme activity was observed in Pleurotus djamor
var. roseus and Pleurotus florida whereas Pleurotus ostreatus
showed minimum activity. Therefore, these were selected
for the optimization of reaction parameters for superoxide
dismutase (SOD) and peroxidase (POX) enzymes.A comparison
of the results of the previous and the present investigations on
Pleurotus species showed that the intracellular antioxidative
stress enzymes are variably secreted among the different
Among the three-buffer system (1M), namely potassium
phosphate, phosphate citrate and Tris-HCl with varied pH [5-9],
maximum SOD activity was recorded at pH 6.0 with potassium
phosphate buffer in case of P. djamor var. Roseus (26.70 U/mg)
while for optimum SOD activity for P. florida, pH 6.5 (phosphate
citrate buffer) resulted best with 22.12 activity units. Ramkumar
et al.  has also recorded 20.29U/mg of SOD and 4U/mg of POX
in dried samples of P. djamor. Increasing pH beyond 6.5 for all
three buffers, showed a sharp decline in SOD activity.
For POX activity in three different buffer systems with pH
ranging from 5-10, Pleurotus djamor var. Roseus reported
maximum POX activity (0.51U/ml) with potassium phosphate
buffer (1M, pH 6.0), followed by phosphate citrate0.39U/ml at
pH 6.5. The activity of peroxidase enzyme in case of P. florida was
recorded highest (0.39U/ml) at pH 6.5 with phosphate citrate
buffer. Tris-HCl showed least activity SOD and POX activity
(0.22U/ml) in the three buffer systems with both samples of
Pleurotus spp. was subjected to varying temperature ranged
from 20-45 °C to estimate the SOD activity. Temperature range
from 30-45 °C was found to be suitable for optimum SOD enzyme
activity, with maximum at 40 °C for P.djamor var. roseus and P.
florida i.e. 29.95U/ml and 22.74U/ml respectively. However,
any variation in temperature (below and above 40 °C) resulted
in decline in enzyme activity. Pleurotus djamor var. Roseus and
Pleurotus floridaexhibit maximum POX activity of 0.63U/ml and
0.46U/ml respectively at 37 °C when subjected to temperature
range of (25-45 °C). A sharp decline in POX activity was recorded
beyond 37 °C. Different time intervals for SOD (80-105 sec) and
POX (5-30 min) were tested to determine the time dependent
activity of SOD and POX. The maximum SOD activity for Pleurotus
djamor var. roseus (27.24U/mg) and Pleurotus florida (23.29U/
mg) was recorded at 90 seconds and 100 seconds respectively.
However, Pleurotus djamor var. Roseus showed maximum
POX activity (0.59U/ml) at 15 min and Pleurotus florida gave
maximum activity of 0.42U/ml after 20 minutes of incubation
. Further decrease in SOD and POX activity was
reported with increase in incubation time.
The concentration of enzyme used in the reaction mixture
has its own significance in converting the substrate into the
product and therefore influence the activity of SOD and POX.
The crude enzyme extract from Pleurotusdjamorvar.roseus
showed 27.59U/mg SOD activity at concentration of 300 μl
whereasPleurotusflorida showed 24.09U/mg activity with
400μl crude enzyme extract. Further purification/processing
may attribute to higher enzyme activity. The crude enzyme
from Pleurotusdjamor var. roseus showed 0.72U/ml POX
activity at 70μl concentration followed by 0.59 activity units in
Pleurotusfloridaat 90μl. A further increase in volume of crude
mushroom extract resulted in abrupt decline in the POX activity.
The high inhibition value of Pleurotus may be due to the high
phenolic residues in the extracts .
In this work, variable results were observed in Pleurotus
species (Pleurotuso streatus, Pleurotus djamor var. roseus
and Pleurotus florida) for the extraction of intracellular antioxidative
enzymes (Superoxide dismutase and Peroxidase) using
different parameters. Solvent extracts, homogenization with
buffer systems of Pleurotus spp. with or without ultrasonication
showed different levels of SOD activity and inhibition patterns of
NBT reduction causing variable inhibition. Anti-oxidative stress
enzymes (SOD and POX) revealed maximum activity in ethanolic
extract of Pleurotus djamorvar. roseus with potassium phosphate
buffer followed by methanolic extract of Pleurotus florida with
phosphate citrate buffer. Maximum SOD (26.70U/mg) and POX
(0.51U/ml) was recorded at pH 6.0 (potassium phosphate buffer,
1M) with P. djamor var. roseus and (22.12U/mg) and (0.39U/
ml) for P. florida (phosphate citrate buffer, pH 6.5). The levels of
anti-oxidative enzymes as reported in the present work are quite
encouraging and indicate their usefulness for such enzymes, but
more efforts and extensive studies are needed to derive and to
reach a definite conclusion.