Research Article

Received: 7 July 2019 Revised: 8 September 2020 Accepted article published: 2 October 2020 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.10849

Supercritical and ultrasound-assisted extracts

from Pleurotus pulmonarius mushroom:
chemical profiles, antioxidative, and enzyme-
inhibitory properties
Ivan Milovanovic,a* Gokhan Zengin,b Svetolik Maksimovicc and
Vanja Tadicd

Abstract
Background: This study provides an insight into the impact of ultrasound-assisted extraction with water as solvent (UAEW) and
extraction by supercritical carbon dioxide (SC-CO2) with 5% EtOH on antioxidant and enzyme inhibitory activity in regard to the
chemical profile of the edible and medicinal mushroom, Pleurotus pulmonarius.
Results: Extraction efficiency was between 0.36% and 63.32%, depending on the extraction technique. The main compounds in
the extracts were fatty acids. Supercritical CO2 extraction with co-solvent was the most suitable method for obtaining extracts
that were rich in ergosterol content, reaching a value of 40.1 mg g−1. The UAEW of crude mushroom powder ensured the high-
est yield, as well as the extracts with best antioxidative activity. The measurements of enzyme inhibitory activity revealed that
all types of investigated extracts exhibited only tyrosinase and amylase inhibition at a significant level.
Conclusion: Based on our results, the extraction methods significantly affected the chemical profile and bioactivity of
P. pulmonarius.
© 2020 Society of Chemical Industry

Keywords: Pleurotus pulmonarius; supercritical CO2 (SC-CO2) extraction; ultrasound-assisted extraction with water as solvent (UAEW);
chemical analysis; antioxidant; enzyme inhibition activity

INTRODUCTION Mushrooms are a highly valued nutritional food and therefore

Extraction is the first essential step that affects the chemical com-
position and bioactivity of samples.1 A supercritical carbon diox-
ide (SC-CO2) extraction technique, which uses non-toxic, cheap,
and readily available solvents, with easy achievement of liquid-
like density, high diffusivity and low viscosity in a supercritical
state, is considered to save time, resulting in high yields, and
has no toxic solvent production residues when compared to tradi-
tional methods. Applying SC-CO2 as an extraction agent can break
the cellular structure, causing the release of chemical compounds
into the solvent medium, ensuring minimal alterations in their
structure and enabling the preservation of bioactive properties
during extraction.2 Another extraction method used in the inves-
tigation of natural products is ultrasound-assisted extraction with
water as solvent (UAEW). This technique is widespread for the iso-
lation of water-soluble bioactive compounds from natural mate-
rials and has many advantages over other extraction methods,
because it is a simple and cheap process, which is non-hazardous
and possesses high process efficiency.3 These convenient tech-
niques are environment friendly, with a strong potential for
obtaining valuable substances that could find use not only in food
but also in the pharmaceutical industry.
their consumption has been increasing markedly in recent years.
The metabolic profiling of mushrooms has also become an exten-
sive research area in an attempt to discover new bioactive com-
pounds. More than 200 Pleurotus species have been described
so far, but just a few have been systematically investigated,
screened for their chemical composition, and confirmed to exhibit
beneficial effects on human health.4 Pleurotus pulmonarius (Fr.)
Quél., an edible and medicinal mushroom, has a worldwide distri-
bution, latterly known for its increasing use in the food and phar-
maceutical industries.
* Correspondence to: I Milovanovic, Faculty of Technology and Metallurgy, Uni-
versity of Belgrade, Innovation Center, Karnegijeva 4, 11000 Belgrade, Serbia.
E-mail: imilovanovic@tmf.bg.ac.rs
a Faculty of Technology and Metallurgy, University of Belgrade, Innovation Cen-
ter, Belgrade, Serbia
b Department of Biology, University of Selcuk, Faculty of Science, Konya, Turkey
c Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia
d Institute for Medicinal Plant research “Dr Josif Pančić”, Belgrade, Serbia
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 www.soci.org I Milovanovic et al.

Various bioactive compounds isolated from P. pulmonarius have
been identified. Among these, most were terpenoids, fatty acids,
phenols, proteins, lectins, proteoglucans, ergothionenine, and espe-
cially polysaccharides.4 Furthermore, this mushroom species has
been intensively studied in the areas of food science, medicine, bio-
technology and pharmacology, revealing that P. pulmonarius extracts
possess anticancer,5, 6 antinociceptive,7, 8 and antioxidant9 activity. In
recent years, many papers have been published on the applicability
of these two techniques for extraction of components of interest
from plants but with limited information on mushrooms (Table 1).
Based on the considerations mentioned above, the aim of the
present study was: (i) to explore if two green technologies UAEW
and SC-CO2 extraction as the chosen methods might provide the
improvement of the extraction process efficiency; (ii) to deter-
mine their chemical profile, and (iii) to examine the antioxidant
and enzyme inhibitory activity of P. pulmonarius fruiting body
extracts obtained employing the SC-CO2 and UAEW technologies.
MATERIALS AND METHODS
Culture collection
The basidiocarp of P. pulmonarius (Fr.) Quél. was collected from
Serbia, and identified according to the macroscopic features and
the micromorphology of the reproductive structures.16 A small
fragment of the fresh fruiting body was extracted on malt agar
medium (MA) for the isolation of pure cultures of P. pulmonarius
ICTMF111, which was then maintained in the Culture Collection
of the Innovation Centre of the Faculty of Technology and Metal-
lurgy, University of Belgrade (ICTMF).
Spawn preparation
Wheat grain was used for spawn production. The grains were
washed with water and boiled for 30 min until they softened.
Boiled grains were drained, supplemented with 2% Ca3(PO4)2
and 0.5% CaCO3 (Sigma-Aldrich, St Louis, USA), mixed manually,
placed in bottles, and sterilized in an autoclave at 121 °C for
15 min. After cooling, each bottle was inoculated with 25 mycelial
disks (Ø 0.5 cm) obtained from 7-day-old culture, and incubated at
22 ± 2 °C in the dark for 2 weeks.
Fruiting body growth
Wheat straw (small pieces) and oak dust in a ratio of 2.5:1 were
dipped in dH2O. After 12 h the wheat straw was centrifuged at
555 g force for 5 min to remove excess water. A mass of 0.7 kg
was inserted into the polypropylene bags and autoclaved at
121 °C for 2 h. The final humidity was 80%. Once the substrate
reached room temperature, inoculation with overgrown spawn
was performed. The inoculated bags were incubated at room
temperature (22 ± 2 °C) in the dark for 2 weeks. Humidity was
maintained by spraying water twice a day, until mushroom forma-
tion (about 30 days).17 Mushrooms were harvested at the third
day after mushroom formation. All harvested mushrooms were
lyophilized in a gamma 1–16 freeze-drying system (Christ, Oster-
ode am Harz, Germany) and ground to a fine powder in a Retsch
ZM 200 mill (Retsch, Haan, Germany; pore size 0.12 mm).
Supercritical CO2 extraction
Supercritical CO2 (SC-CO2) extraction from P. pulmonarius was car-
ried out in the High Pressure Extraction Adsorption (HPEA) 500 unit
(Eurotechnica GmbH) (Fig. 1). The unit can be used for the inte-
grated supercritical fluid extraction and impregnation process or
for the supercritical fluid extraction process only. Supercritical CO2
extraction was performed at 380 bar and 80 °C, with and without
the addition of co-solvent. For both experiments, 20 g of mush-
room powder was used. Mushroom powder was placed in the
280 mL stainless-steel extractor, designed to be operated at maxi-
mum pressure of 550 bar and temperature of 120 °C. In the exper-
iment with co-solvent, ethanol was subjected together with
mushroom powder in the extractor vessel in a quantity of 5 mass
percent of CO2 consumed for the SC-CO2 extraction without co-sol-
vent. Liquid CO2 supplied from a CO2 cylinder with a siphon tube
was cooled in a cryostat to prevent vaporization and pumped into
the system by a liquid metering pump (Milton Roy, France) until the
operating pressure was obtained. After reaching the operating con-
ditions, the continuous flow of supercritical fluid commenced.
Operating pressure was maintained by the back-pressure regulator
(BPR). After the SC-CO2 extraction with co-solvent, ethanol was
removed from the mixture with P. pulmonarius extract by a rotary
vacuum evaporator. The average extraction time was 2.7 h. Extrac-
tion yields were calculated after consumption of approximately
40 g CO2 g−1 mushroom material, as a result of which the mush-
room material was exhausted. The extraction yield (y) was calcu-
lated using the following equation:
me
y ð%Þ = ×100 ð1Þ
ms
where me is the mass of obtained extract, and ms is the mass of
mushroom material at beginning of the process. All the experi-
ments were performed in triplicate.

Table 1. Summary of supercritical extraction procedure and yield efficiencies from mushrooms

Species Extraction technology Yield (%) References

Lentinula edodes (Berk.) Pegler 20 MPa; 40 °С 0.55 Kitzberger et al.10

20 MPa; 40 °С; with 5%, 10% and 15% EtOH 1.10–3.81
Ganoderma lucidum (W. Curt.:Fr) P. Karst. 35 MPa; 25 °С 2.98 Fu et al.11
Agaricus blasiliensis Peck 10–30 МРа;40, 50 and 60 °С; 0.5–1.19 Mazzuti et al.12
20 МРа;50 °Сwith 2.5%, 5% and 10% EtOH as co-solvent; 1.5–4.2
Pleurotus ostreatus (Jack.:Fr.) P. Kumm. 15–25 MPa; 40–60 °С with EtOH as co-solvent — Bhattacharya and Mishra13
Hericium erinaceus (Bull.) Persoon 20 MPa; 40 °С 0.3–0.9 Parada et al.14
20 MPa; 40 °С with 5%, 10% and 15% EtOH as co-solvent 1.0–1.9
Pleurotus eryngii (DC.:Fr.) Quel. 10–30 MPa; 35–55 °С 0.05–0.45 Rodríguez-Seoane et al.15
10–30 MPa; 35–55 °С with 10% EtOH as co-solvent 0.15–0.94
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Extraction, chemical analysis, and biological potential of P. pulmonarius
Figure 1. Schematic view of HPEA, 500 unit.
Ultrasound assisted extraction with water as
solvent (UAEW)
Raw mushroom powder, and remaining mushroom powder after
the SC-CO2 extraction process, was lyophilized and used for the
UAEW. For all experiments, 30 mL of distilled water was added
to 0.6 g of mushroom powder placed in the 50 mL PP centrifuge
tube. Subsequently, the tube was immersed in the ultrasound
bath (Elmasonic P) for 1 h at 37 °C. The mixture was centrifuged
afterwards at −2490 g force for 10 min and the supernatant was
removed by pipette and filtered on Whatman paper. To obtain
crude P. pulmonarius extract, water was removed from the solu-
tion by a rotary vacuum evaporator at 60 °C. Extracts were kept
at −4 °C until the beginning of the experiment. The extraction
yield (y) was calculated using the Eqn (1). All the experiments were
performed in triplicate.
Analytical procedure
Gas chromatography/flame ionization detector (GC/FID)
A gas chromatography analysis of the extracts was carried out on
a HP-5890 Series II GC apparatus (Hewlett-Packard, Waldbronn,
Germany), equipped with split-splitless injector and an automatic
liquid sampler, attached to a HP-5 column (25 m × 0.32 mm,
0.52 μm film thickness) and fitted to flame ionization detector
(FID). The carrier gas flow rate (H2) was 1 mL min−1, split ratio
1:30, injector temperature was 250 °C, detector temperature
300 °C, and the column temperature was linearly programmed
from 40 °C to 260 °C (at rate of 4 °C min−1), and then kept isother-
mally at 260 °C for 10 min. Solutions of samples dissolved in
MeOH were consecutively injected in amounts of 1 μL. Area per-
centage reports, obtained as result of standard processing of
chromatograms, were used as base for the quantification analysis.
J Sci Food Agric 2020 © 2020 Society of Chemical Industry
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Gas chromatography/mass spectrometry (GC/MS)
The same analytical conditions as those mentioned for GC/FID
were employed for GC/MS analysis, along with an HP-5MS column
(30 m × 0.25 mm, 0.25 μm film thickness), using a HP G 1800C
Series II GCD system (Hewlett-Packard, Palo Alto CA, USA). Helium
was used as a carrier gas. The transfer line was heated to 260 °C.
Mass spectra were acquired in EI mode (70 eV); in an m/z range
of 40–450. An amount of 0.2 μL of sample solution in MeOH was
injected. The components of the oil were identified by compari-
son of their spectra to those from Wiley 275 and NIST/NBS librar-
ies, using different search engines. The experimental values for
retention indices were determined by the use of calibrated Auto-
mated Mass Spectral Deconvolution and Identification System
software (Amdis ver. 2.1), compared to those from the available
literature (Adams), and used as an additional tool to approve MS
findings.
High-performance liquid chromatography (HPLC) procedure
Methanol and water were of HPLC grade and they were pur-
chased from Merck (Darmstadt, Germany). Reference HPLC stan-
dard ergosterol was purchased from Sigma, St Louis, MO, USA
(purity was declared as >98%, based on the manufacturer's inter-
nal high-precision HPLC method). The HPLC fingerprint of the
investigated extracts and quantification of ergosterol was
achieved by HPLC, Agilent Technologies 1200. Detection was per-
formed using a diode array detector (DAD). The wavelength was
set at 282 nm for ergosterol determination. The HPLC separation
of components was achieved using a Phenomenex Syringe Hydro
RP C18 (5 μm), 150 × 4.6 mm i.d. column, at 35 °C, with a flow rate
of 1 mL min−1 and mobile phase, A [H2O], B [MeOH], elution being
the combination of gradient and isocratic mode: 5–30% A,
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Table 2. Yield results obtained after different extraction treatments of Pleurotus pulmonarius mushroom
Sample Extraction technology
Pp1 SC-CO2 extraction at 38 МРа and 80 °С
Pp2 SC-CO2 extraction at 38 МРа and 80 °С with 5% EtOH
Pp3 Ultrasound-assisted extraction with water at 37 °C (1 h)
Pp4 Ultrasound-assisted extraction with water at 37 °C (1 h)
Pp5 Ultrasound-assisted extraction with water at 37 °C (1 h)
RMP, raw mushroom powder; RMP-aET1, remaining mushroom powder after SC-CO2 extraction at 38 МРа, 80 °С; RMP-aET2 Remaining mushroom
powder after SC-CO2 extraction at 38 МРа, 80 °С with 5% EtOH.
0–20 min; 30% A, 5 min; 30–35% A, 25–30 min. The samples were
prepared, dissolving 20.00 mg of each in 1 mL MeOH, filtered
through 0.2 μm PTFE filters prior to HPLC analysis. The injected
volume was 5 μL. Standard solution for the determination of
ergosterol was prepared at a final concentration of 0.1 mg mL−1
in methanol. The identification was carried out considering reten-
tion time and spectra matching. Once spectra matching suc-
ceeded, results were confirmed by spiking with the respective
standard to achieve a complete identification by means of the
so-called peak purity test.18 Those peaks not fulfilling the requests
had not been taken into account for quantification.
Total phenolic content
Referring to our previous paper,19 total phenolic content was
determined on the basis of a standard Folin–Ciocalteu assay.
Briefly, 50 μL of an aliquot sample solution in methanol
(5 mg mL−1) was mixed with 100 μL of 1:10 Folin–Ciocalteu
reagent. The mixture was shaken well and then 75 μL of sodium
carbonate (7.5%) was added. The mixture was incubated
(120 min) in the dark. Finally, the absorbance of solution was
recorded at 765 nm. Gallic acid was used as standard and the
results were expressed as gallic acid equivalent (mg GAE g−1) for
total phenolic content.
Antioxidant properties
To evaluate the antioxidant capacity of the extracts, different
spectrophotometric experiments such as ferrous ion chelating,
phosphomolybdenum, and radical scavenging tests (ferric reduc-
ing antioxidant power (FRAP), 2,20 -azino-bis(3-ethylbenzothiazo-
line)-6-sulfonic acid (ABTS), cupric reducing antioxidant capacity
(CUPRAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)) were per-
formed as previously reported. The findings were given as stan-
dard compound equivalents of ethylenediaminetetraacetat
(EDTA) or Trolox (mg EDTAE g−1 and mg TE g−1). The concentra-
tions of the extracts were 0.5–5 mg mL−1. Concentration of the
5 mg mL−1 was selected for evaluating antioxidant and enzyme
inhibitory properties. The procedures were conducted following
assay methods given in our earlier work.19
Enzyme inhibitory activity
The in vitro enzyme inhibitory effects of extracts on ⊍-amylase,
⊍-glucosidase, cholinesterases (acetyl cholinesterase (AChE) and
butyryl cholinesterase (BChE)), and tyrosinase were evaluated, as
previously reported.19 The concentration of the extracts that were
investigated ranged from 0.5–5 mg mL−1. Concentration of the
5 mg mL−1 was selected for evaluating antioxidant and enzyme
inhibitory properties. The enzyme inhibitory potential of extracts
were assessed as equivalents of kojic acid (KAE) for tyrosinase,
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Extraction material Initial mass (g) Yield (%)
RMP 20.00 ± 0.01 0.36 ± 0.01
RMP 20.00 ± 0.01 0.93 ± 0.03
RMP 0.60 ± 0.01 63.32 ± 0.76
RMP-aET1 0.60 ± 0.01 45.14 ± 0.53
RMP-aET2 0.60 ± 0.01 46.87 ± 0.39
galantamine for acetyl cholinesterase (AChE) and butyryl cholin-
esterase (BChE), and acarbose for ⊍-amylase and ⊍-glucosidase,
respectively.
Statistical analysis
The antioxidant and enzyme inhibitory results were reported as
means ± standard deviations of three parallel experiments. A
one-way ANOVA was conducted, followed by Tukey's multiple
ranges, to investigate significant differences (P < 0.05) between
the tested samples. The statistical procedures were achieved by
GraphPad Prism 8 software.
RESULTS AND DISCUSSION
Extraction yield
The results of the mushroom extraction yield, comparing two dif-
ferent techniques, are presented in Table 2. The SC-CO2 extraction
carried out with EtOH as a co-solvent exhibited almost three times
higher yield in comparison to extraction performed using pure
SC-CO2 (0.93% and 0.36%, respectively). A difference in the extrac-
tion yield was noted when UAEW treatment of the remaining
mushroom after SC-CO2 extraction was applied (45.14% and
46.87%, for the samples previously extracted with SC-CO2 with
co-solvent and pure SC-CO2, respectively). The UAEW treatment
of crude mushroom powder also gave the highest extraction yield
(63.32%). The results are in accordance with the recently pub-
lished literature. Kitzberger et al.,10 showed that the extraction
yield of SC-CO2 from Lentinus edodes mushroom increased from
0.57% without co-solvent, to 3.81% when EtOH was added; Maz-
zuti et al.12 noted the increase in extraction yield up to 380% com-
pared with pure SC-CO2, reaching a value of 4.20% using EtOH as
co-solvent from Agaricus brasiliensis mushroom; Parada et al.14
noted two times higher extraction yield from the Hericium erina-
ceus mushroom when EtOH was used as co-solvent; the addition
of EtOH enhanced extraction yield in Pleurotus eryngii mushrooms
compared with pure SC-CO2 extraction (0.05% versus 0.94%).15
This fact might be due to an increase in the solubility of polar
components in the mixture EtOH/SC-CO2, in contrast with the sol-
ubility in pure SC-CO2, reducing the selectivity and enhancing the
yield.20 The low yield obtained using pure SC-CO2 extraction
might therefore be explained by the small quantity of non-polar
compounds present in the extracts that are investigated, whereas
the use of EtOH as co-solvent, providing better solubility of polar
compounds in the P. pulmonarius mushroom, significantly
increased the extraction yield.
The second technique, UAEW, provided a higher extraction
yield in comparison to SC-CO2 extraction. The higher results might
be a consequence of the low processing temperature used. Up to
J Sci Food Agric 2020
Extraction, chemical analysis, and biological potential of P. pulmonarius
now, UAEW was reported to be only widely used for obtaining the
extracts rich in a polysaccharide fraction.21 The data regarding the
chemical profile of UAEW extracts of crude P. pulmonaris mush-
room powder might be valuable for further research, being the
first report of that kind.
GC/MS and GC/FID analysis
The chemical compositions of the samples that were investigated
(Pp1-Pp5) are presented in Table 3. In total, 136 chemical constit-
uents were detected, while 9-oxononanoic acid, linoleic acid, oleic
acid, stearic acid, and ethyl octadecanoate were the most abun-
dant in all of the extracts that were analyzed. The major com-
pound that was identified in terms of percentage peak area was
oleic acid, a fatty acid. Although fatty acids were the most abun-
dant compounds among those identified by GC, no correlations
were detected concerning the effects of different extraction tech-
niques on composition, but it was obvious that different extrac-
tion conditions affected the difference in the chemical profile of
the same species. Furthermore, few non-polar components were
identified only in individual samples, which might be due to the
GC analysis used in this work being suited to low-polarity
substances.
The results regarding the chemical profile of the investigated
extracts revealed the complexity of the mixtures of polar and
non-polar compounds.10 The important finding was that nicotin-
amide, a vitamin from the B complex, and ergosterol, the precur-
sor of vitamin D2 (ergocalciferol) were detected in the extracts
that were investigated. The amounts of these compounds from
the SC-CO2 extract obtained with a co-solvent were significantly
higher than in the extracts obtained by applying the other extrac-
tion techniques (Table 3). Similar results were obtained by Mazzuti
et al.12 from the mushroom A. brasiliensis using SC-CO2 and low-
pressure extraction. Among the main constituents were linoleic
acid and ergosterol. Coelho et al.22 also reported a higher pres-
ence of oleic acid and palmitic acid in mushroom A. blazei extracts
obtained using the same conditions. Interestingly, although
ergosterol (1.6%) and linoleic acid (87.6%) were detected in
L. edodes mushroom extract obtained using SC-CO2 with EtOH
(which represents the highest concentrations of these com-
pounds found, based on the data published up to now), nicotin-
amide was not detected.23
The finding that fatty acids were major constituents in tested
species was consistent with results reported by Overton.23 In his
work, the presence of fatty acid esters from P. ostreatus was
responsible for flavor and odor. Hence, comprehensive chemical
analysis might contribute to further evaluation of P. pulmonarius
as a valuable species.
HPLC analysis
Our findings offer the first chemical report on the presence of
ergosterol in P. pulmonarius (Fig. 2). According to the results,
ergosterol was detected in extracts obtained using SC-CO2 extrac-
tion with EtOH as co-solvent and UAEW (40.1 ± 1.0 and 0.9
± 0.1 mg g−1, respectively); other extraction techniques were
shown to be ineffective for ergosterol extraction.
In a comprehensive study by Villares et al.,24 the ergosterol con-
tent varied significantly among nine fungal species and ranged
from 6.81 mg g−1 for Hygrophorus marzuolus to less than 1 mg g−1
for Cantharellus cibarius. Similar content was reported in
P. ostreatus and P. cystidus, and varied between 4.40 and
4.35 mg g−1; in Agaricus bisporus it ranged from 2.04–
7.80 mg g−1; in L. edodes was present in a range from 2.02–
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6.80 mg g−1.24 Supercritical carbon dioxide extraction with EtOH
as a co-solvent was shown to be an appropriate method for
obtaining the extract from mushroom P. pulmonarius, rich in
ergosterol.
Taking into account the results obtained by Mazzuti et al.,12 SC-
CO2 extraction with EtOH as co-solvent was not suitable for ergos-
terol extraction from A. brasiliensis. An investigation performed by
Kitzerberger et al.10 revealed that pure SC-CO2 extraction of
L. edodes provided extracts with 1.57% of ergosterol. Probably,
extraction conditions contributed to changes in ergosterol con-
tent or even affected its stability in mushrooms.25 Based on our
results, the SC-CO2 extraction with EtOH was confirmed to be an
efficient extraction technique for obtaining ergosterol-rich frac-
tions from P. pulmonarius mushrooms.
Antioxidative activity
The observed values for antioxidative activity (AA), including dif-
ferent assays, are shown in Table 4. Generally, the highest AA
(determined by all assays) was found in extracts obtained by pure
SC-CO2 extraction. However, the best DPPH radical scavenging
ability was noted for extracts obtained by SC-CO2 extraction with
EtOH as a co-solvent. Among the extracts obtained using UAEW, a
fraction of crude mushroom powder showed the highest
AA. Interestingly, the results for a mushroom residue after SC-
CO2 treatment were quite similar.
Mazzuti et al.12 demonstrated, using a DPPH test, that the
antioxidant capacity of A. brasiliensis mushrooms depended
directly on extraction conditions. In this study, the values for
extracts when pure SC-CO2 was applied ranged from 4.64% to
13.0%. Kitzerberger et al.10 measured the antioxidative poten-
tial of different extracts of L. edodes mushroom, revealing the
limited antioxidative activity, approximately 11% for the frac-
tion obtained using SC-CO2, without co-solvent addition. Fur-
thermore, by adding co-solvent, they noted a high positive
correlation (R2: 0.998) between AA and EtOH concentration.
Thus, AA increased with an increase in EtOH concentration up
to 72.97%. In the study by Xu et al.,6 the evaluation of the rad-
ical scavenging activity of hot water extracts from three edible
mushrooms resulted in finding that P. pulmonarius had pos-
sessed the highest antioxidant potential.
Our results indicated higher antioxidant potential for
P. pulmonarius mushroom extracts obtained using the non-polar
solvent. Nevertheless, our results regarding the UAEW extracts
showing the highest AA might direct further analysis to find a
more conclusive evaluation.
Enzyme inhibitory activity
In recent years, enzymes have represented the main players in the
prevention of global health problems in the pharmaceutical area.
The inhibition of some enzymes can alleviate the pathological
symptoms of diseases including Alzheimer's disease and diabetes
mellitus.26 For example, AChE catalyzes the hydrolysis of a neuro-
transmitter, acetylcholine, in the synaptic gap. The level of acetyl-
choline is very low in Alzheimer's patients; thus the inhibition of
AChE might increase neurotransmission and improve cognitive
function in the patients.27 Amylase and glucosidase are consid-
ered to be the main carbohydrate-hydrolyzing enzymes, responsi-
ble for the regulation of the blood glucose level. The inhibition of
these enzymes could control the postprandial glucose level in dia-
betic patients and thus symptoms could be alleviated by this.28
For these reasons, several compounds have been produced as
inhibitors (tacrine for cholinesterase, acarbose for amylase and
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Table 3. Mycochemicals identified by GC/MS analysis of of Pleurotus pulmonarius mushroom from five different extracts expressed as percentages
(based on area percentage reports, obtained by standard processing of chromatogram analysis)

No. Compound CAS number KIa Pp1 Pp2 Pp3 Pp4 Pp5

Pentanal 71-41-0 704 0.1 t 0.6

3-methyl-3-buten-1-ol 763-32-6 723 0.7
isobutyric acid 79-31-2 738 t
2,3-butanediol 513-85-9 785 t 12.5
Hexanal 66-25-1 801 0.2 0.2
methyl pyrazine 109-08-0 819 0.2
4-hydroxy-4-methyl-2-pentanone 123-42-2 831 0.1
3-methoxy-3-methyl-butanol 56 539-66-3 852 0.1
1,1-dimethoxy-pentane 26 450-58-8 868 0.1
3-methylthiopropanal 3268-49-3 901 0.2
4-hydroxybutanoic acid lactone 96-48-0 904 0.2
2,5-dimethyl pyrazine 885-65-4 908 0.2
Anisole 100-66-3 913 0.2
4-methyl-2-heptanone 6137-06-0 936 t 0.2
(2E)-heptanal 219-563-0 947 0.1 t 0.2
hexanoic acid 142-62-1 967 1.0 2.1 0.7
hexyl acetate 142-92-7 1007 0.1
p-methyl anisole 104-93-8 1015 0.2
2-methyl-N-(2-methylbutylidene)-1-butanamine 54518-97-7 1025 0.3
2-methyl hexanoic acid 4536-23-6 1033 0.1 0.7 0.3
benzene acetaldehyde 67-56-1 1036 0.1 0.1 0.1 0.8
γ-hexalactone 695-06-7 1042 0.2
4-hydroxy-2-methylene butanoic acid 24923-76-0 1085 0.1
2-ethyl hexanoic acid 1056468-55-3 1086 0.1 0.1
propyl hexanoate 626-77-7 1079 0.1
isopentylisovalerate 659-70-1 1102 0.6
nonanal 124-19-6 1102 t
phenyl ethyl alcohol 10031-71-7 1106 0.1
3-methyl-3-butenyl-3-methyl butanoate 54410-94-5 1112 0.1
Maltol 118-71-8 1122 0.1
isobutyl hexanoate 105-79-3 1149 t 0.1
2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 28564-83-2 1154 0.3 0.1 0.1 1.1
benzoic acid 65-85-0 1159 0.1 0.1
(2E)-nonenal 18829-56-6 1161 0.1
diethyl succinate 123-25-1 1166 0.1
octanoic acid 124-07-2 1167 0.2 0.2 t 0.3
benzoic acid, ethyl ester 93-89-0 1180 0.2
2-decanone 693-54-9 1190 t
hexyl butanoate 2639-63-6 1191 t
kojic acid 501-30-4 1192 0.1 0.4 0.1
(2E)-hexenylbutenoate 5339883-7 1193 0.1 0.1 0.2
3-decanol 1565-81-7 1196 t 0.3 1.0 0.6
6-oxoheptanoic acid 3128-07-2 1209
octanol acetate 112-14-1 1211 0.1 0.7
benzenacetic acid 103-82-2 1246 0.2 0.3
nonanoic acid 112-05-0 1267 0.3 0.4
(2E,4Z)-decadienal 25152-83-4 1292 0.1
2-undecanone 112-12-9 1293 0.2
30 -methoxy acetophenone 586-37-8 1298 0.1 0.1
azulene 275-51-4 1298 0.1 t
(2E,4E)-decadienal 25152-84-5 1315 0.1 t
4-hydroxybenzaldehyde 123-08-0 1323 0.1
2-dodecanone 6175-49-1 1348 1.8 0.3 0.3 0.3
γ-nonalactone 104-61-0 1358 0.4 0.3
decanoic acid 334-48-5 1369 0.1 1.4
allylnonanoate 7493-72-3 1376 0.5 0.2
6

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Table 3. Continued

No. Compound CAS number KIa Pp1 Pp2 Pp3 Pp4 Pp5

nicotinamide 98-92-0 1426 1.4 5.6 0.2

⊐-decalactone 705-86-2 1463 0.3 0.3
γ-decalactone 706-14-9 1465 0.1 0.1
dimethyl acetal, undecanal 52517-67-6 1474 0.1
9-oxononanoic acid 14436-32-9 1483 0.7 0.1 0.7 1.0 1.3
n-undecanoic acid 112-37-8 1488 0.2 0.2
5-methyl thocytosine 110-12-3 1498 0.2 0.4
1,3-dimethyl-1,5-dioxaspiro[5,5]-undecan-9-one 69225-59-8 1517 0.1 0.1
2,5-pyridinedicarboxylic acid, 2-methyl ester 5552-44-3 1524 0.1 0.1
(E)-octenylcyclopentanone 65737-52-2 1528 0.4
⊎-thujaplicinol 4356-35-8 1536 0.1 0.1
R-2(4H)-benzofuranone-5,6,7,7a-tetrahydro-4,4,7a-trimethyl 15356-74-8 1538 0.4
dodecanoic acid 143-07-7 1565 0.2 0.1
3-methyl-2-butenoic acid, nonyl ester 7786-47-2 1565 0.2 0.2
hydroxyl-6-cytosine 13484-95-2 1579 0.8
caryophyllene oxide 1139-30-6 1582 0.6
1-hexadecene 629-73-2 1588 t
Hexadecane 544-76-3 1600 t 0.1
2-acetyl naphthalene 93-08-3 1608 0.4
benzophenone 119-61-9 1626 0.5
hexyl ester, salicylic acid 118-60-5 1684 0.1
(2E)-tridecanol acetate 211-344-8 1703 0.1 0.1 0.8
14-hydroxy-4,5-dihydrocaryophyllene 508-54-3 1706 0.2
cedroxyde 71735-79-0 1714 0.3
pentadecanal 2765-11-9 1715 0.2 0.8 0.6
methyl ester tetradecanoic acid 124-10-7 1727 0.5 0.3
oplopanone 1977-78-0 1739 0.4
3-methyl-5-(2,6-dimethylheptyl)-1,5-pent-2-enolide 139007-95-7 1745 0.2
1-octadecene 112-88-9 1789 0.1
(Z)-7-hexadecanal 56797-40-1 1798 0.1
cyclopentadecanolide 106-02-5 1828 0.3 t
isoamyldodecanoate 6309-51-9 1844 t
hexahydrofarnesyl acetone 502-69-2 1845 0.6
pentadecanoic acid 1002-84-2 1867 1.7 1.4 1.1
n-hexadecanol 36653-82-4 1874 0.4
ethyl ester, pentadecanoic acid 41114-00-5 1879 0.1
nonadecane 629-92-5 1900 0.4 2.1
methyl hexadecanoate 112-39-0 1921 0.4 3.2 0.5
a) 4.6 34.1
hexadecanoic acid 57-10-3 1959 17.1 13.9 3.9 7.6
ethyl hexadecanoate 628-97-7 1992 0.5 0.4
eicosane 112-95-8 2000 1.8
n-octadecanol 112-92-5 2077 0.2 0.1 0.4
heptadecanoic acid 506-12-7 2080 0.2 0.6
methyl linoleate 112-63-0 2095 0.1 0.3 0.8
τ-palmitolactone 730-46-1 2105 0.2 0.1 0.6
(9E)-octadecenoic acid, methyl ester 39202-17-0 1209 0.1
methyl octadecenoate 112-61-8 2123 0.4 0.1
linoleic acid 60-33-3 2132 17.9 26.4 9.1 9.5 17.4
ethyl linoleate 544-35-4 2139 2.7 2.3 4.4
oleic acid 112-80-1 2141 14.9 11.3 18.5 32.2 29.3
stearic acid 57-11-4 2179 4.2 5.0 2.6 1.9 12.5
ethyl octadecenoate 111-61-5 2196 0.4 1.4 6.4 1.5 3.1
10-nonadecanoic acid 646-30-0 2256 0.1
10,13-eicosadienoic acid, methyl ester 56599-57-6 2292 0.1 2.2 0.5
7,10,13-eicosatrienoic acid, methyl ester 56554-30-4 2300 2.5
ergost-2,5,7,9(11),22-pentaene 2410 1.7 1.8 19.6 3.2
7

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Table 3. Continued

No. Compound CAS number KIa Pp1 Pp2 Pp3 Pp4 Pp5

pentacosane 629-99-2 2500 0.6 0.2

2-octadecyl propane-1,3-diol 5337-61-1 2530 0.3
3-methyl pentacosane 6902-54-1 2560 0.4 0.4 0.2
13-oxo-octadecanoic acid, methyl ester 2380-28-1 2570 0.3
3⊎,5⊍-ergost-7-en-3-ol 516-78-9 2632 0.6 0.2 2.2 4.6
ergost-4,6,8(14),22-tetraen-3-one 19254-69-4 2644
ergost-2,5,7,9(11),14,22-hexaene 2661 0.5 4.3
1-mono-linolein 2277-28-3 2697 0.8
neoergosterone 57-83-0 2708 0.5 0.4 1.2
octacosane 630-02-4 2800 1.3
dihydrosqualene 26266-08-0 2865 0.4
nonacosane 630-03-5 2900 14.5 2.5 0.8
untriacontane 630-04-6 3100 5.0
ergosterol 57-87-4 3152 4.9 0.8
2,6,10,15,19,23-hexanethyl-tetracosa-2,6,14,18,22- 153650-82-9 3183 0.1 1.7
pentaene-10,11-diol
⊎-sitosterol 83-46-5 3197 1.3
5⊍-ergosta-7,22-dien-3⊎-ol 2465-11-4 3202 0.2
tritiacontane 630-05-7 3300 0.3 0.3
ergost-5,7,22-trien-3⊎-ol 57-84-4 3400 4.1 1.9
stigmast-4-en-3-one 1058-61-3 3435 2.5 1.3
pentatriacontane 630-07-9 3500 3.3 t
3-acetoxyurs-12-en-28-oic acid 7372-30-7 3534 0.4 0.2
hexatriacontane 630-06-8 3600 0.8 t
3⊎-hydroxystigmast-5-en-7-one 127684-08-6 3610 1.9 0.3 0.4
The percentage of the total identified chemical compounds 99.4 98.9 99.5 99.2 99.5

t – trace (the percentage was less than 0.05); a) – Sample Pp3 (relative abundance: 4.6%): 48.499, m/z 396 (75), 363 (90), 341 ((100), 297 (15), 271 (25),
253 (55), 208 (50), 157 (53), 143 (48), 69 (80) (ergosterol derivative); Sample Pp4 (relative abundance: 34.1%): 48.98 m/z 396 (75), 363 (90), 341 ((100),
297 (15), 271 (25), 253 (55), 208 (50), 157 (53), 143 (48), 69 (80) (ergosterol derivative),

glycosidase, and kojic acid for tyrosinase). However, most of them
have side effects, including gastrointestinal disturbance and
toxicity.27, 29, 30 Hence, the discovery of novel inhibitors from
natural sources might be an acceptable solution for these
shortcomings.
Inhibition of several important enzymes such as acetylcholines-
terase, butyrylcholinesterase, tyrosinase, amylase, and glycosi-
dase activity was investigated (Table 5). Tyrosinase and amylase
inhibition activity was observed for all tested extracts. Inhibition
values ranged from 0.08 to 21.18 mg KAE g-1 for tyrosinase and

Figure 2. HPLC chromatograms of ergosterol standard and analyzed P. pulmonarius mushroom extracts obtained using different methods of extraction
(Pp1 – Pp5) with UV spectrum assigned to peak at 22.18 min corresponding to ergosterol standard (the wavelength was set at 282 nm).
8

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Extraction, chemical analysis, and biological potential of P. pulmonarius www.soci.org

Table 4. Antioxidant activity of P. pulmonarius mushroom obtained after different extraction treatments

Total phenolic DPPH scavenging ABTS FRAP CUPRAC Metal chelating

Samples content (mg GAE g−1) (mg TE g−1) (mg TE g−1) (mg TE g−1) (mg TE g−1) (mg EDTAE g−1)

Pp1 19.07 ± 0.16a* 11.00 ± 0.11a 26.96 ± 0.67a 27.08 ± 0.01a 61.87 ± 1.17a 14.90 ± 1.09a
Pp2 9.73 ± 0.22c 9.05 ± 0.53b 15.25 ± 2.66c 27.52 ± 0.24a 51.35 ± 0.08b 11.71 ± 1.37b
Pp3 10.24 ± 0.19b 11.43 ± 0.07a 26.93 ± 0.43a 7.72 ± 0.07d 17.46 ± 0.45c 7.31 ± 0.01c
Pp4 6.32 ± 0.11e 10.61 ± 0.23a 24.78 ± 0.11ab 9.79 ± 0.21c 14.49 ± 0.22e 1.49 ± 0.18d
Pp5 7.47 ± 0.17d 10.62 ± 0.26a 23.50 ± 0.27b 10.82 ± 0.19b 16.14 ± 0.28d 0.64 ± 0.19e
*
Values expressed represent means ± standard deviations of three parallel colorimetric measurements.
Different letters indicate significant differences in the extracts (P < 0.05).
GAE, Gallic acid equivalents; TE, Trolox equivalents; EDTAE, EDTA equivalents.

Table 5. Enzyme inhibitory activity of P. pulmonarius mushroom obtained after different extraction treatment

AChE inhibition BChE inhibition Tyrosinase inhibition Amylase inhibition Glucosidase inhibition
Samples (mg GALAE g−1) (mg GALAE g−1) (mg KAE g−1) (mmol ACAE g−1) (mmol ACAE g−1)

Pp1 Na na 21.18 ± 0.89a 0.37 ± 0.01b 0.37 ± 0.01b

Pp2 1.27 ± 0.03a* na 19.77 ± 0.93a 0.41 ± 0.02a 0.41 ± 0.01a
Pp3 0.54 ± 0.03c 0.21 ± 0.03a 3.59 ± 0.97b 0.11 ± 0.01c na
Pp4 0.58 ± 0.02c 0.03 ± 0.01b 0.98 ± 0.14c 0.04 ± 0.01d na
Pp5 0.65 ± 0.02b 0.23 ± 0.08a 2.23 ± 0.18b 0.03 ± 0.01d na
*
Values expressed are means ± standard deviations of three parallel colorimetric measurements.
GALAE, Galantamine equivalent; KAE, Kojic acid equivalent; ACAE, Acarbose equivalent; na, not active. Different letters indicate significant differences
in the extracts (P < 0.05).

0.03 to 0.41 mmol ACAE g−1 for the amylase assay. The remaining
tests, including AChE, BChE and glucosidase, did not exhibit any
activity – or at least the values obtained showed a moderate effect.
These variations between enzyme assays might be caused by
different chemical compositions of the P. pulmonarius extracts,
obtained applying different extraction treatments. To date there
have been no reports regarding anti-enzyme activity from
P. pulmonarius extracts obtained by utilizing UAEW and SC-CO2
extraction. Several investigations reported anti-tyrosinase activity
for hot water extracts from five Pleurotus species and the results of
tyrosinase inhibitions ranged from 6.52% to 60.68%.31 The same
trend was noticed for wild-growing mushrooms, exhibiting differ-
ent inhibitory actions (reported as galantamine equivalents)
against AChE (0.83–0.97 mg GALAE g−1 extract) and BChE
(0.86–1.33 mg GALAE g−1 extract).32 The tested species extracts
were good inhibitors of amylase and glucosidase, as well. Simi-
larly, Cor et al.33 showed that G. lucidum SC-CO2 extracts exhibited
AChE inhibition between 7.33% and 22.54%, while AChE inhibi-
tory activity of hot water extracts was much higher; the authors
observed 50% inhibition of AChE using the extract in concentra-
tion of 1 mg mL−1.34
Our findings could therefore be regarded as the results of a pre-
liminary investigation, necessary to investigate the use of
P. pulmonarius as a possible source of natural inhibitors.
CONCLUSION
This study provided data regarding notable differences in yield
efficiency when obtaining extracts from P. pulmonarius when
supercritical CO2 extraction, with or without EtOH as co-solvent,
was applied. Ultrasound-assisted extraction from the crude
P. pulmonarius with water as solvent ensured an even higher yield,
reaching 63.32%. Fatty acids were found to be the major com-
pounds, together with a number of less abundant components.
The P. pulmonarius extracts obtained using SC-CO2 exhibited
higher antioxidant activity than extracts obtained by UAEW.
Enzyme inhibitory potentials were quite low, with the exception
of tyrosinase and amylase inhibition activity. The research and
the results of the bioactivity investigations might open up further
possibilities for studies of correlations between extraction effi-
cacy, chemical composition, and the biological activity of the
P. pulmonarius mushroom.
ACKNOWLEDGEMENT
This research was supported by projects No. 45017 and No. 45001
of the Ministry of Education, Science and Technological Develop-
ment of the Republic of Serbia.
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