2. 1
Highlights
• Pleurotus ostreatus methanol extract (MEPO) contains many health-beneficial compounds
• MEPO showed stronger inhibition of α-glucosidase activity than α-amylase.
• MEPO showed potential for blood glucose regulation in in silico study.
3. 2
Antidiabetic and Antioxidant Potentials of Pleurotus ostreatus -derived Compounds: An in vitro and
in silico approach
1
Nnemolisa, S.C., 1
Chukwurah, C.C., 1
Edeh, S.C., 1
Aguchem, R.N., 1,2
Chibuogwu*, C.C., 1
Aham, E.C.,
3
Chukwu, M.C., 1
Obiora, M.O., 1
Anyebe, D.E. and 1
Okagu, I.U.
1 Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu,
Nigeria.
2 Institute for Drug-Herbal Medicine-Excipient Research and Development, Faculty of Pharmaceutical
Sciences, University of Nigeria, Nsukka, Enugu, Nigeria.
3 Department of Genetics and Biotechnology, Faculty of Biological Sciences, University of Nigeria, Enugu,
Nsukka.
* Correspondence: christian.chibuogwu@unn.edu.ng (ORCID: 0000-0002-9091-4310) +2347069263943
Abstract
Many health benefits have reportedly been associated with mushroom consumption. This study determined
the chemical constituents of Pleurotus ostreatus methanol extract (MEPO) and investigated its antioxidant
and antidiabetic effects using in vitro and in silico approaches. The chemical composition of MEPO was
determined using the gas chromatography-flame ionization detector (GC-FID) technique, while 2,2-
Diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods were used to
determine antioxidant activity. The antidiabetic activity was evaluated using α-amylase and α-glucosidase
inhibition assays, while molecular docking was done to give insight into the binding potentials of MEPO
constituents against α-amylase, α-glucosidase, and phosphoenolpyruvate (PEP) carboxykinase activities.
Thirteen compounds, including ephedrine, oxalate, rutin, naringin, and kaempferol, were identified in
MEPO. The extract showed moderate antioxidant activity, as observed from the DPPH (IC50 = 732.41
mg/ml) and FRAP studies. The extract also demonstrated stronger inhibition of α-glucosidase activity (IC50
= 246.58 mg/ml) than α-amylase activity (IC50 = 1074.05 mg/ml). Docking studies revealed that rutin and
naringin interacted effectively with amino acid residues crucial for α-amylase, α-glucosidase, and PEP
carboxykinase activities via hydrogen bonds. The result shows that MEPO is a rich store of beneficial
compounds which could be explored for the management of diabetes and associated complications.
Keywords: Mushroom, methanol extract, α-amylase, α-glucosidase, PEP carboxykinase.
1. Introduction
Diabetes mellitus (DM) is a group of chronic metabolic disorders characterized by an abnormal rise in
plasma glucose levels resulting from poor glucose metabolism due to abnormal insulin secretion, insulin
deficiency, and/or insulin insensitivity (Singh et al., 2022). In 2021, 536.6 million people were reportedly
living with diabetes, and a 46% increase (783.2 million) has been predicted by 2045 (Ogurtsova et al.,
2022). Most diabetics (80.6%, 432.7 million) reside in low- and middle-income nations and about half of
these individuals are more likely to experience complications because they are unaware that they have the
condition (Sun et al., 2022). These complications are the major causes of mortality in diabetic patients. One
of the factors contributing to the pathogenesis of diabetic complications from hyperglycemia is an
impairment of reactive oxygen species and antioxidant defense balance (Dehghan et al., 2016). This results
4. 3
in a state known as oxidative stress. Indeed, studies have shown that hyperglycemia-induced oxidative
stress is implicated in vascular damage of diabetes subjects (Singh et al., 2022).
Numerous consequences of diabetes may be delayed or prevented with intensive postprandial
hyperglycemic management. However, the available treatment reflects an insufficient and less effective
glycemic control for diabetic patients (Abbas et al., 2019). This has led to the exploration of more effective
therapeutic targets and approaches to glycemic control. One of the therapeutic options presently explored
for effective glycemic control is the inhibition of α-amylase and α-glucosidase activities. The α-amylase is
a calcium metalloenzyme that plays an important role in starch digestion catalyzing the hydrolysis of α-(1,
4)-D-glycosidic linkages in starch and other polymers of glucose, producing smaller fragments (Sun et al.,
2020). These products are further degraded by α-glucosidase to absorbable monosaccharides which then
enters the bloodstream. Therefore, inhibiting these enzymes would hinder the absorption of starch, expand
intestinal sugar-holding time and delay the rate of glucose absorption and consequently decrease blood
sugar level. Acarbose, voglibose and migiltol are drugs used to inhibit the activities of these enzymes in
practice. However, they cause unwanted side effects such as abdominal discomfort (Alqahtani et al., 2020)
which hinders their use in the treatment of diabetes and its complications. This has therefore led to urgent
requirement for effective substitutes which would reduce diabetes complications with fewer side effects.
Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme in gluconeogenesis that catalyzes the
carboxylation of pyruvate to form oxaloacetate in the gluconeogenic pathway (Gómez-Valadés et al., 2006).
Although this enzyme is widely implicated for its role in hepatic glucose production in diabetic conditions,
studies have shown that overexpression of PEPCK in adipose tissue is a major contributor to obesity
development due to increased glyceroneogenesis in the adipose tissue (Rines et al., 2016; Yu et al., 2021).
Therefore, it makes sense to target PEPCK activity for inhibition as a strategy in diabetes therapy.
For ages, nature has been a source of therapeutic agents and an astounding number of modern medications
have been identified from natural sources based on their usage in conventional medicine (Bhushan &
Kulshreshtha, 2018). The interest in natural substances have significantly increased in recent years, because
these substances are intended to be used as nutraceuticals to replace synthetic substances whose usage is
restricted due to their adverse effects (Omari et al., 2019). The nutraceutical potentials of mushroom have
been well documented since time immemorial; however, it is in the beginning of this century that the
nutritional and pharmacological properties of mushroom gathered much attention (Huang et al., 2022).
Mushrooms are well-known macro-fungus with a characteristic fruiting body that can either be hypogenous
or epigeous (Yadav & Negi, 2021). Mushroom cultivation has emerged as a promising agricultural business
that is land-independent with the capacity of transforming lignocellulosic waste into nutrient-rich foods, in
addition to significantly reducing environmental pollution. Nutritionally, mushroom contains a relatively
high amount of carbohydrate, protein, dietary fibers, minerals, vitamins and unsaturated fatty acids
(Nowakowski et al., 2021). Edible mushrooms are excellent sources of beneficial bioactive metabolites,
and these substances have broad-spectrum therapeutic applications, including but not limited to antiviral,
anti-inflammatory, anti-bacterial, anti-oxidant and antidiabetic effect (Mwangi et al., 2022; Suleiman et al.,
2022). Pleurotus genus is among the species with the highest production worldwide (Torres-Martínez et
al., 2022). This can be attributed to its short cultivation cycle, its ability to grow at moderate temperature
(25-30ºC) and a wide range of lignocellulosic materials (Cruz-Solorio et al., 2018). Of particular interest is
P. ostreatus which is the second most widely grown and consumed edible mushroom in the world. It is
considered a high quality food due to its high nutritional value and beneficial health effects (Lesa et al.,
5. 4
2022). In the present study, the in vitro and in silico anti-diabetic potential of methanol extract of Pleurotus
ostreatus was investigated.
2. Materials and Methods
2.1 Collection and Authentication of Material
Pleurotus ostreatus together with wood sawdust and rice bran were the materials used for this study. Wood
sawdust and rice bran were obtained from timber and rice farmers in Enugu State, Nigeria and were
pulverized using a mechanical grinder. The mushroom was cultivated at Southeast Zonal Biotechnology
Center, University of Nigeria.
2.1.2 Chemicals and Reagents
The chemicals and reagents used in this study were of analytical grade.
2.2. Mushroom Cultivation
Mushroom was cultivated following the method described by Aguchem et al., (2022). 50 kg of the substrate
was prepared by mixing the substrate with 0.40 kg of CaCO3 before adding 38 liters of water to the mixed
substrate. The substrate mixture was then packed in a limpid refractory polythene bag, each tied with rubber
bans before sterilization at 121o
C for 15 minutes at 15 psi. After sterilization, the sterile packed substrates
were allowed to cool to room temperature. The substrates were then inoculated in a germ-free manner with
the spawn and then re-tied. The inoculated bags were then taken inside a darkroom and allowed some days
to ramify with the complete ramification of the inoculated bags clearly visible by the appearance of pin
heads and fungi hyphae. Incisions were gently made on the ramified mushroom bags in a germ-free manner
before they were transferred to the growth room for the production of fruiting body. During the course of
this process, the relative humidity and temperature were maintained at 74-86% and 25o
C respectively. By
watering the bags at least once daily, wetting the floor, keeping water in tray and ice blocks close to the
mushroom bags, humid air in the growth room was achieved. The fruiting bodies of the mushroom was
then harvested and used for analysis.
2.2.1 Preparation of Mushroom Extract
Fresh whole mushroom sample (100 g) was oven-dried at 50o
C and ground into fine particles and then
macerated in 400 ml of methanol at room temperature in an air-tight container. The container was left for
72 hours with intermittent shaking to increase the efficiency of extraction of the phytochemicals. This was
followed by filtration and concentration in vacuo to obtain the crude methanol extract of P. ostreatus
(MEPO) (Figure S1 (A-F) - supplementary materials).
2.2.2 Extraction of Chemical Constituents for GC-FID analysis
Extraction of chemical constituent for gas chromatography-flame ionization detector (GC-FID)
analysis was carried out following Boisa et al., (2020) and Ojukwu et al., (2021) methods, respectively. A
test tube containing 1 g of the sample was filled with 15 ml of methanol and 10 ml of 50% w/v KOH. The
test tube was then placed in a water bath set to 60°C for 60 minutes. After that, the content was moved to a
separatory funnel. 20 ml of ethanol, 10 ml of cold water, 10 ml of hot water and 3 ml of hexane was used
successively to wash the contents of the tube which was all transferred to the funnel. This extract was then
6. 5
mixed with 10 ml of 10% v/v ethanol aqueous solution and rinsed three times. The solvent was evaporated
after the solution was dried with anhydrous sodium sulfate. The resulting sample was solubilized in 1000
µl of pyridine after which 200 µl (0.2 ml) of the solution was transferred to a vial for analysis.
2.2.3 Analysis of chemical constituent of MEPO
The chemical constituents of MEPO were analyzed on a BUCK M910 Gas chromatograph (GC) (BUCK
M910, PUB Scientist, USA) equipped with a flame ionization detector (FID). 0.1 ml of the extract was
injected into the GC machine. At an ionization voltage of 70 eV, injector temperature of 250°C and injector
mode was split with linear velocity 36.5 cm/sec and pressure was 57.5 kPa. The instrument was run in
electron impact mode. In principle, the detector uses a flame to ionize carbon-containing organic
compounds. During separation, the sample passes through a hydrogen-fueled flame in the GC column
which ionizes the carbon atoms in the sample.
2.2.4 Assay of the inhibitory effect of MEPO on α-amylase and α-glucosidase activities
The assay mixtures containing the solution of the enzyme (0.1 ml), acetate buffer (0.1 - 0.5 ml, 0.1 mol/l at
pH 5.5) and mushroom extract (32 - 160 mg/ml) were incubated for 15 minutes at room temperature. This
was followed by the addition of 0.5 ml of 2% starch and 2% sucrose to each test tube containing the
respective enzymes and incubation of the resulting mixture in a water bath at 50°C for 30 min. The reaction
was terminated with the addition of 1 ml of dinitrosalicylic acid (DNSA) reagent to each test tube and
placed in a boiling water bath for 10 minutes. To stabilize the color after heating, 1 ml of 1.4 mol/l Rochelle
salt (sodium potassium tartrate) was added to the test tubes immediately and the total volume of the solution
was adjusted to 4 ml with distilled water. The reaction mixtures were then cooled to room temperature and
absorbance was taken at 540 nm using a spectrophotometer (Spectron lab 2A, England). The test tube used
as control was prepared without mushroom extract. All tests were done in triplicate and the percentage of
inhibition of enzyme activity was calculated as Equation 1:
% inhibition =
(Abs.of control − Abs.of treatment)
(Abs.of control)
× 100 1
Where: Abs. of control = Absorbance of control at 540 nm; Abs. of treatment = Absorbance of sample
containing extract at 540 nm
2.2.5 In vitro antioxidant activity of MEPO
2.2.5.1 DPPH radical-scavenging assay
The DPPH radical-scavenging activity of MEPO was determined following the protocol of Sayah et al.,
(2017). Differently labelled test tubes received additions of MEPO at various mass concentrations (32 - 160
mg/ml) followed by the addition of MeOH to bring the volume to 250 𝜇l. Each test tube was then shaken
violently following the addition of 2 ml of a 0.18 mmol/l (0.005%) methanolic solution of DPPH after
which the mixture was allowed to stand in the dark for 30 minutes at room temperature. The same procedure
was used to prepare the control without any extract and MeOH served for baseline correction. Changes in
samples absorbances were taken at 517 nm using a spectrophotometer (Spectron lab 2A, England). DPPH
radical-scavenging potential was calculated using the method provided by Adebiyi et al., (2017).
7. 6
2.2.5.2 Ferric-reducing antioxidant power assay
The method described by Alachaher et al., (2018) was used to determine the ferric reducing antioxidant
potential of MEPO. 1 ml solution containing 32, 64, 96, 128, and 160 mg/ml each of the extracts was
combined with potassium ferricyanide (5.0 ml, 1.0%) and sodium phosphate buffer (5.0 ml, 0.2 mol/l, pH
6.6). The mixture was incubated at 50°C for 20 minutes followed by the addition of 5 mL of 10%
trichloroacetic acid and centrifuged at 1000 xg (10 min at 5°C). The upper layer of the solution (5.0 ml)
was diluted with 5.0 ml of distilled water and 0.5 ml ferric chloride (0.1% w/v) and absorbance measured
at 700 nm using a spectrophotometer (Spectron lab 2A, England). The experiment was performed thrice
and scavenging activity calculated as in Equation 2.
Scavenging activity (%) =
(Abs.of control − Abs.of treatment)
(Abs.of control)
× 100 2
Where: Abs. of control = Absorbance of control at 700 nm; Abs. of treatment = Absorbance of sample
containing extract at 700 nm
2.3 In silico molecular docking studies
2.3.1 Ligands and protein preparation
Epicatechin, naringin, catechin, kaempferol, linamarin, rutin, resveratrol, spartein, ephedrine and standard
drug (Acarbose) were the ligands used in this study. The chemical structures of the ligands were retrieved
from NCBI PubChem database (https://www.ncbi.nlm.nih.gov/pccompound) in 2D (sdf) format and were
converted to 3D (PDB) and PDBQT structures using Discovery Studio software and AutoDock Tool,
respectively.
The selected crystal structures of the proteins, α-amylase (PDB entry: 1U33), PEP-carboxykinase (PDB
entry: 1KHB) and α-glucosidase (PDB entry: 5KZW) were obtained from the Protein Data Bank
(http://www.rcsb.org/pdb). The crystallographic water as well as the existing ligands were removed. The
crystal structures were changed to PDBQT format after the addition of hydrogen atom to the geometry. The
structures were refined, and their geometries optimized with the aid of AutoDock Tool.
2.3.2 Docking
Following the protocol described by Dundas et al. (2006), identification of the proteins active sites was
done using CASTp server (Computer Atlas of Surface Topology of protein). The X, Y, Z center coordinates
and X, Y, Z grid dimensions of proteins were selected (30 x 30 x 30 Å), optimized and thereafter docked.
Docking and image preparation was performed with AutoDock Vina (Scripps Research Institute, San
Diego, CA, USA), BIOVIA Discovery Studio and PyMOL (to generate 3D structures).
2.3.4 Drug-likeness and toxicity prediction
In silico prediction of molecular descriptors and drug-like properties of the MEPO phytochemicals was
estimated using an online tool on the SwissADME server (http://http://www.swissadme.ch/index.php). MW
(molecular weight), cLogP, HBD (number of hydrogen bond donors), HBA (number of hydrogen bond
acceptors), gastro-intestinal absorption, and nViolation (number of violations of Lipinski’s rule of five)
8. 7
were calculated on the basis of Lipinski's rule of five as described by Daina et al., (2017). The ProTox
webserver was used to predict the toxicity profile of the MEPO phytochemicals.
2.4 Statistical analysis
The results obtained were analyzed using the Statistical Product and Service Solutions (SPSS) software
(version 20). Results were presented as mean ± standard deviation and tests for statistical significance done
using one way analysis of variance (ANOVA) at 95% confidence level and mean values with p≤ 0.05 was
taken to be significant. The IC50 values were estimated from the linear plot of inhibition (%) against
concentration using the equation of the line graph.
3. Results
3.1 Phytochemical constituents of methanol extract of Pleurotus ostreatus
The gas chromatography analysis of the methanol extract of Pleurotus ostreatus as presented in Table 1
showed the presence of thirteen (13) compounds. Ephedrine was found to be the highest followed by
oxalate. Rutin, naringin, kaempferol, epicatechin, phytate, resveratrol, catechin were also detected (Figure
S2 - Supplementary materials).
Table 1: phytochemical constituents of methanol extract of Pleurotus ostreatus
Compounds Concentration (𝝁g/ml)
Linamarin 8.03 ± 0.05
Catechin 4.27 ± 0.16
Sapogenin 7.97 ± 0.04
Rutin 11.19 ± 0.45
Spartein 8.18 ± 0.14
Flavan-3-ol 4.34 ± 0.15
Naringin 10.40 ± 0.15
Resveratrol 7.38 ± 0.35
Kaempferol 9.02 ± 0.13
Phytate 7.65 ± 0.29
Epicatechin 7.72 ± 0.10
Oxalate 16.68 ± 0.34
Ephedrine 33.77 ± 0.92
9. 8
3.2 In vitro antidiabetic activity of the mushroom extract
The result presented in Table 2 shows the percentage inhibition of α-amylase and α-glucosidase activity by
MEPO. The result showed a concentration-related inhibitory effect of the extract on α-amylase and α-
glucosidase activity with IC50 value of 1074.05 mg/ml and 246.58 mg/ml respectively. The highest
inhibitory effect was observed with the highest concentration of the extract (160 mg/ml) and vice versa.
Table 2: Inhibitory effect of MEPO on α-amylase and α-glucosidase activity in vitro
Sample concentration
(mg/ml)
α-Amylase activity
(%)
α-Glucosidase activity
(%)
32 4.8±0.18 33.87±0.63
64 5.1± 0.16 36.53± 0.09
96 5.14±0.11 36.69±0.6
128 8.79±0.23 41.9±0.41
160 10.03±0.46 43.43±0.16
IC50 = 1074.05 mg/ml IC50 = 246.58
mg/ml
3.3. In vitro antioxidant effects of methanolic extract of P. ostreatus
The DPPH radical-scavenging activity and ferric reducing antioxidant power of MEPO is shown in Table
3. MEPO demonstrated DPPH radical-scavenging activities in a concentration-dependent manner with an
IC50 value of 732.41 mg/ml; the highest DPPH radical-scavenging effect was observed with the highest
concentration. The same trend is also obtainable with the ferric reducing antioxidant power of the extract.
Table 3: In vitro DPPH radical-scavenging activity and ferric reducing antioxidant power of MEPO
Sample concentration (mg/ml) % Quenching of DPPH radical FRAP
32 9.73 ± 0.10a
77.00 ± 0.58a
64 9.58 ± 0.07a
84.93 ± 0.31b
96 12.21 ± 0.25b
87.18 ± 0.68c
128 14.88 ± 0.20c
89.46 ± 0.11d
160 16.48 ± 0.11d
98.13 ± 0.54e
IC50 = 732.41 mg/ml
3.4. In silico molecular docking studies
A molecular docking study was carried out to elucidate the binding interactions of MEPO phytochemicals
at the active sites of α-amylase, α-glucosidase, and PEP carboxykinase to garner an understanding into the
in vitro results. From Table 4, favorable overall binding energies were observed in ranges of -5.9 to -9.8
kcal/mol for α-amylase (1U33), -5.7 to -9.3 kcal/mol for α-glucosidase and -5.4 to -9.8 kcal/ mol for PEP
carboxykinase (1KHB). Epicatechin, naringin and rutin had the best binding energies across all three targets
when compared to Acarbose. On the other hand, kaempferol and linamarin showed better binding energies
for α-amylase and PEP carboxykinase only when compared to the Acarbose.
10. 9
Table 4: Binding energies of selected compounds and standard inhibitors against protein targets
Ligands
α-amylase
(1U33)
Proteins (Kcal/mol)
α-glucosidase
(5KZW)
PEP carboxykinase
(1KHB)
Epicatechin -9.8 -8.2 -9.3
Naringin -9.4 -9.3 -9.8
Catechin -9.3 -7.2 -7.8
Kaempferol -9.3 -6.9 -8.0
Linamarin -9.0 -7.3 -8.6
Rutin -8.7 -8.8 -9.6
Resveratrol -8.1 -6.9 -7.5
Spartein -7.7 -6.5 -6.6
Ephedrine -5.9 -5.7 -5.4
Standard (Acarbose) -7.5 -8.3 -7.5
3.5 Drug-likeness prediction of the MEPO phytochemicals
Table 5 shows the drug-likeness of MEPO phytochemicals. Apart from naringin and rutin, all the MEPO
phytochemicals tested had MW less than 500, HBA < 10 and HBD not more than 5. All the compounds
showed reasonable water solubility while only naringin and rutin violated Lipinski’s rule for orally
administered drugs similar to the standard drug.
11. 10
Table 5: Drug-likeness Prediction of the MEPO phytochemicals
Phytochemicals MW
(g/mol)
No.
HBA
No.
HBD
cLogP
value
GIA No. violation Water solubility
Epicatechin 290.27 6 5 0.85 High No Soluble
Naringin 580.53 14 8 -0.79 Low 3 Soluble
Catechin 290.27 6 5 0.85 High No Soluble
Kaempferol 286.24 6 4 1.58 High No Soluble
Linamarin 309.32 4 0 2.87 High No Moderately
soluble
Rutin 610.52 16 10 -1.29 High 3 Soluble
Resveratrol 228.24 3 3 2.48 High No Soluble
Spartein 234.38 2 0 2.36 Low No Soluble
Ephedrine 165.23 2 2 1.45 Low No Very Soluble
Acarbose 645.60 19 14 -6.09 Low 3 Highly Soluble
MW: Molecular weight (<500), HBA: Number of Hydrogen Bond Acceptors (≤10), HBD: Number of
Hydrogen Bond Donor (≤5), cLogP ≤ 5, No. Violation: Number of Violation of Lipinski’s rule.
3.6 Toxicity study of MEPO phytochemicals
Apart from ephedrine, linamarin and resveratrol, all the MEPO phytochemicals had LD50 greater than 2000
mg/kg. The toxicity prediction showed relative safety of MEPO phytochemicals except for rutin and
naringin which showed potentials for immuno-toxicity, like Acarbose. Linamarin also showed potential for
hepatotoxicity like Acarbose.
12. 11
Table 6: Toxicity Properties of the MEPO phytochemicals
Compounds Predicted
LD50 value
(mg/kg)
Prediction
accuracy
(%)
Hepato-
toxicity
Immuno
-toxicity
Cytotoxi
city
Carcino
genicity
Mutagen
icity
Epicatechin 10000 100% Inactive Inactive Inactive Inactive Inactive
Naringin 2300 70.97% Inactive Active Inactive Inactive Inactive
Catechin 10000 70.97% Inactive Inactive Inactive Inactive Inactive
Kaempferol 3919 100% Inactive Inactive Inactive Inactive Inactive
Linamarin 400 100% Active Inactive Inactive Inactive Inactive
Rutin 5000 100% Inactive Active Inactive Inactive Inactive
Resveratrol 1560 68.07% Inactive Inactive Inactive Inactive Inactive
Spartein 2850 100% Inactive Inactive Inactive Inactive Inactive
Ephedrine 404 100% Inactive Inactive Inactive Inactive Inactive
Acarbose 24000 100% Active Active Inactive Inactive Inactive
4. Discussion
Despite available pharmaceutical interventions, the current projections of the incidence of diabetes and its
associated complications have necessitated the search for alternative therapeutic approaches. In this study,
in vitro and in silico antidiabetic potential of methanol extract of P. ostreatus (MEPO) was evaluated. Our
results demonstrated that MEPO significantly inhibited α-amylase and α-glucosidase activities in a
concentration-dependent manner. α -amylase and α-glucosidase are two enzymes that play key role in
carbohydrate metabolism. While α-amylase facilitates the catabolism of starch, releasing absorbable
molecules, α-glucosidase catalyzes the end step of starch and disaccharides digestion from human diet
(Kazeem et al., 2013). The uncontrolled activity of these enzymes is one of the contributors to postprandial
hyperglycemia and has been implicated in diabetes development and complications. Hence, inhibiting their
activities has become a strategy in blood glucose control. The inhibition of these enzymes’ activities is
indicative of the potential of MEPO as a promising candidate for glycemic control in diabetes management
and this is supported by previous studies. Karim et al., (2020) and Johnny & Okon, (2013) reported anti-
hyperglycemic effects of solvent extracts of P. ostreatus, using in vivo models and from our result,
inhibition of these enzymes may be one mechanism in which P. ostreatus exerts its anti-hyperglycemic
effect.
Oxidative stress, occasioned by excessive free radical production, is implicated in both the development
and progression of many disease conditions (Yaribeygi et al., 2020); thus, antioxidant therapy remains a
viable option to mitigate the oxidative damage that is associated with many diseases. DPPH radical-
scavenging and FRAP assays are common test methods used to determine the antioxidant capacity of test
substances. Antioxidant compounds are able to stabilize/quench DPPH radicals resulting in a change of
color of reaction mixture from purple to yellow, while in the FRAP assay, antioxidant compounds prevent
13. 12
the oxidation of ferrous ion (Fe2+
) to ferric ion (Fe3+
) (Omari et al., 2019). Our result showed that MEPO
moderately scavenged DPPH radicals and reduced Fe3+
to Fe2+
in a concentration-related manner, indicating
that MEPO has phytochemicals with antioxidant properties. In agreement with our result, Mishra et al.,
(2022) and Mkhize et al., (2022) also reported good DPPH radical-scavenging activity and FRAP,
respectively, of solvent extract of P. ostreatus. The same trend was also reported by Menaga et al., (2014)
and Leong et al., (2021) for extracts of other mushroom species.
To identify the phytochemicals potentially involved in the anti-diabetic and antioxidant potentials of
MEPO, we used gas chromatography instrument coupled with a flame ionization detector (GC-FID)
ephedrine, oxalate, cyanogenic glycoside, rutin, naringin, kaempferol, epicatechin, phytate and catechin,
among others. The presence of these compounds indicates that MEPO is a rich store of health-beneficial
bioactive compounds. For example, catechin, epicatechin, rutin, and kaempferol are polyphenolic
compounds known for their antioxidant and antidiabetic properties (Ademiluyi et al., 2018; Chukwuma et
al., 2022). Thus, the presence of these phytochemicals in MEPO could have individually or in combination
contributed to the antioxidant and antidiabetic potentials observed in this study.
To clarify the contribution of each of the identified compounds to the antidiabetic properties of MEPO, we
conducted molecular docking studies. Nine MEPO phytochemicals (naringin, kaempferol, resveratrol,
linamarin, rutin, epicatechin, catechin, spartein, and ephedrine) were docked against α-amylase, α-
glucosidase and PEPCK to provide insights into the ligand-complexes orientations and interactions between
these phytochemicals and the enzymes implicated in diabetes based on the binding energies. MEPO
phytochemicals demonstrated favorable binding energies (negative binding scores) against α-amylase, α-
glucosidase and PEPCK. Negative binding scores usually indicate correctly docked compounds into target
proteins’ crystal structure and higher negative scores usually indicate stronger binding affinities toward
targets (Kumar et al., 2019).
The active site of every enzyme consists of amino acid residues with three different functions, viz, catalytic
residues (responsible for the ligand conversion to products), ligand-binding residues (responsible for the
binding of the ligand to active site), and structural residues (responsible for the maintenance of the active
site structure in its functional state). Three amino acids, Asp300, Glu233, and Asp197 are the catalytic
residues of α-amylase active site. Our study showed that naringin and epicatechin had better interactions
with α-amylase active site’s residues compared to the other MEPO phytochemicals docked. Naringin had
nine hydrogen (H)-bond interactions within the active site with His305, Asp300, Glu233, Asp197, Glu240,
Lys200, Thr163, Gln63, and Trp59. Likewise, epicatechin had five hydrogen bond interactions with
His299, Asp300, Glu233, Thr163 and Gln63. A visual representation of the 3D and 2D of the docked
ligand conformations at the active site of α-amylase (Figure 1, see appendix) indicated that interactions
occurred with the key residues at the active site, like Acarbose which had four H-bond interactions with
Asp197, Asp300, Glu233 and Asn53. Naringin and rutin showed higher binding energies for α-glucosidase
when compared to Acarbose. Naringin formed H-bonds with Arg411, Ser676, Leu678, Asp518, Met519
and Arg600. We also observed that among the six H-bond interactions observed with naringin, 2-H bonds
(Arg600 and Asp518) were also observed with Acarbose. Similarly, rutin showed H-bond interactions with
Asp616, His674, Asp404, Trp481, Asp518, and Ser679. Naringin and epicatechin showed higher binding
energies (-9.8 kcal/mol and -9.3 kcal/mol, respectively) for PEPCK compared to Acarbose (-7.5 kcal/mol).
Epicatechin formed H-bond interactions with Asn403, Ala287, Cys288, Gly289, Thr291, Val335, Lys290,
Arg436 and Asn292. Naringin also had H-bond interactions with Ala287, Gly289, Cys288, Asn292,
Lys290, Pro402 and Arg87, compared to Acarbose that formed H-bond interactions with Asn292, Phe333,
14. 13
Asp311, Lys244, Lys290, Thr291, Cys288, Gly289. Hence, because of the more negative binding energies
exhibited by the phytochemicals coupled with their interactions with higher number of active site amino
acid residue may explain why MEPO constituents are better in regulating blood glucose level than
Acarbose. This may suggest that MEPO constituent might serve as an effective alternative in diabetes
management.
According to the Lipinski's rule, a drug must have an HBD (hydrogen bond donors) count of ≤ 5 and an
HBA (hydrogen bond acceptor) of ≤ 10 in order for it to be orally active (Lipinski, 2016). All the MEPO
phytochemicals and standard drug passed the oral bioactivity test, except for rutin and naringin which had
high HBDs (10 and 8, respectively) and high HBAs (16 and 14, respectively). Similarly, rutin and naringin
displayed immunotoxicity potentials, while linamarin showed hepatotoxic effects, unlike other MEPO
phytochemicals that are predicted to be safe. Future studies should establish the safety profile of MEPO
and its phytochemicals using cellular and in vivo models.
Conclusion
Methanol extract of Pleurotus ostreatus (MEPO) is rich in health-enhancing phytochemicals and
demonstrated moderate antioxidant and antidiabetic activities as evidenced by in vitro and in silico
molecular docking studies. The presence of the identified constituents and their respective interactions with
enzymes implicated in blood glucose elevation suggest that the P. ostreatus-derived compounds may be a
promising therapeutic strategy for managing blood sugar levels, especially in diabetic conditions. Further
studies are warranted to confirm the above beneficial potentials using in vivo models.
Data Availability
The data supporting the outcome of this present study are included within the article.
Declaration of Competing Interest
None
Acknowledgement
None.
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Figure 1. An observation of the molecular interaction (left: 2D and right: 3D) between compounds with the
highest binding energies and standard against α-amylase. Epicatechin (Ai, Aii), Naringin (Bi, Bii), Catechin
(Ci, Cii), Standard (Acarbose) (Di, Dii).
Ai
Bi
Ci Cii
Bii
Aii
20. 19
Figure 2. An observation of the molecular interaction (left: 2D and right: 3D) between compounds with the
highest binding energies and standard against α-glucosidase. Epicatechin (Ai, Aii), Naringin (Bi, Bii),
Rutin (Ci, Cii), Standard (Acarbose) (Di, Dii).
Di
Ai
Bi
Ci
Dii
Aii
Bii
Cii
21. 20
Figure 3. An observation of the molecular interaction (left: 2D and right: 3D) between compounds with the
highest binding energies and standard against PEP carboxykinase. Epicatechin (Ai, Aii), Naringin (Bi,
Bii), Rutin (Ci, Cii), Standard (Acarbose) (Di, Dii).
Author contribution
Chibuogwu, C.C., Okagu, I.U. and Aham, E.C. conceived and designed the study. Nnemolisa,
S.C., Chukwurah, C.C, Aguchem, R.N., Edeh, S.C. and Obiora, M.O carried out the experiment.
Aguchem, R.O. and Anyebe, D.E. analyzed the data. Chukwu, M.C. carried out the Molecular
docking studies. Nnemolisa, S.C. and Chibuogwu, C.C. drafted the manuscript. Okagu, I.U. and
Aham, E.C. edited and revised the manuscript. All authors read and approved the final manuscript.
Declaration of Competing Interest
None declared.
Di Dii
