Journal of Ethnopharmacology 292 (2022) 115191

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Gastroprotective effect of hydroalcoholic extract from Agaricus blazei Murill

against ethanol-induced gastric ulcer in mice
João Francisco Câmara Neto a, Matheus da Silva Campelo a, Gilberto Santos Cerqueira b, **, João
Antônio Leal de Miranda b, Jhonyson Arruda Carvalho Guedes c, Raimundo Rafael de Almeida a,
Sandra de Aguiar Soares a, Nilce Viana Gramosa a, Guilherme Julião Zocolo c, Ícaro Gusmão
Pinto Vieira d, Nágila Maria Pontes Silva Ricardo a, ***, Maria Elenir Nobre Pinho Ribeiro a, *
a
Laboratório de Polímeros e Inovação de Materiais, Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, CE,
CEP 60440-900, Brasil
b
Núcleo de Ensino e Pesquisa em Microscopia e Processamento de Imagens, Departamento de Morfologia, Centro de Ciências da Saúde, Universidade Federal do Ceará,
Fortaleza, CE, CEP 60440-900, Brasil
c
Embrapa Agroindústria Tropical. Rua Dra. Sara Mesquita, 2270 - Pici, CEP 60020-181, Fortaleza, CE, Brasil
d
Parque de Desenvolvimento Tecnológico, Universidade Federal do Ceará, Avenida do Contorno, CEP 60455-970, Fortaleza, CE, Brasil

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: The use of mushrooms in medicine is quite old and the first report about the use
Agaricus brasiliensis of genus Agaricus in treatment of ulcers occurred in Byzantine period. This mushroom is widely consumed as
UPLC-QTOF-MSE food, tea, food supplements, as well as nutraceutical and cosmeceutical applications, being cultivated and
Nuclear Magnetic Resonance
appreciated in several countries such as Brazil, Korea, Japan and China.
Mannitol
Antioxidant
Aim of the study: This study aimed to characterize the chemical profile and the potential gastroprotective effect of
hydroalcoholic extract from Agaricus blazei Murill (HEAb).
Materials and methods: The extract was chemically characterized by elemental analysis, UPLC-QTOF-MSE, Nu­
clear Magnetic Resonance (NMR) and high-performance liquid chromatography (HPLC) techniques to elucidate
the metabolites present in the extract. The quantification of phenolic compounds and the in vitro antioxidant
activities were performed and the gastroprotective effect of this extract was evaluated against ethanol-induced
gastric ulcer model. HEAb was administered by gavage at 5, 25 and 50 mg kg− 1 and N-acetylcysteine at 300
mg kg− 1 (positive control). Furthermore, the pathways of nitric oxide (NO), Cyclic Guanylate Monophosphate
(cGMP), prostaglandins (PGs) and the involvement of ATP-sensitive K+ Channels were modulated.
Results: Mannitol, malic acid, pyroglutamic acid, L-agaritine and L-valine were putatively identified by UPLC-
QTOF-MSE in HEAb. In addition, it was possible to identify mannitol by the intense signals in the NMR spectra,
being still quantified as the main compound in the extract by HPLC. The contents of total phenols and flavonoids
corroborated with the good antioxidant activity of HEAb. This study observed that HEAb at 25 and 50 mg kg− 1
had gastroprotection effect demonstrated by the reduction of histopathological parameters and the reduction of
mastocytosis in the stomach of mice.
Conclusions: In this study was possible to conclude that HEAb has gastroprotective effect related to the
involvement of NO and PG pathways in the ethanol-induced gastric ulcer model in mice.

1. Introduction duodenum, being a disease that affects about 10% of adults at least once
in their lives (Kuna et al., 2019). It is currently known that Helicobacter
Gastric ulcers are injuries that develop in the stomach and proximal pylori infections, smoking, alcoholism, prolonged use of nonsteroidal

* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: giufarmacia@hotmail.com (G.S. Cerqueira), naricard@ufc.br (N.M.P.S. Ricardo), elenir.ribeiro@ufc.br (M.E.N.P. Ribeiro).

https://doi.org/10.1016/j.jep.2022.115191
Received 13 January 2022; Received in revised form 27 February 2022; Accepted 9 March 2022
Available online 12 March 2022
0378-8741/© 2022 Elsevier B.V. All rights reserved.
J.F. Câmara Neto et al. Journal of Ethnopharmacology 292 (2022) 115191

anti-inflammatory drugs and increased oxidative stress are the main 2.2-diphenyl-1-picrylhydrazyl (DPPH), folin-ciocalteu, sodium carbon­
factors related to the development of these injuries (Oppong et al., ate (Na2CO3), aluminum chloride (AlCl3), ferrous sulfate (FeSO4), fer­
2015). Besides that, about 4.6 million people are diagnosed with gastric rozine and D-mannitol which were used in this work are all analytical
ulcers annually, which makes it a global public health problem (Fahmy grade (>98%) and were purchased from Sigma Aldrich - St. Louis (USA).
et al., 2020a). Ethanol and methanol were purchased from the Synth company (Brazil),
Furthermore, the recurrences in hospitals after the exhaustive both of which are analytical grade (>99%).
treatments and the undesirable side effects have driven the search for
antiulcer drugs that reduce the incidence of recurrences of patients and
2.2. Mushroom sample
offer better protection against gastric ulcers (Shen et al., 2017; Wu et al.,
2018). Many studies with natural products have been stimulated to
ABM used in this work was obtained from the company specialized in
discover new compounds with gastroprotective activity (Lu et al., 2021).
mushroom planting in São Paulo - Blazei Murril® DEC Enterprises
The use of mushrooms for human treatment is quite old. The first
Comercial Ltda (SP) and the use of these species was registered in the
report of the genus Agaricus for use in the treatment of ulcers was in the
Brazilian National System of Management of Genetic Heritage and
Byzantine period (Ramoutsaki et al., 2002). Agaricus blazei Murill (ABM)
Associated Traditional Knowledge (SisGen) under registration number
is a mushroom from Brazil and trivially known as ‘Cogumelo do Sol®’
AC29F45.
and ‘Cogumelo Piedade’ in addition to being widely reported in the
literature as Agaricus blazei (Rubel et al., 2018; Soković et al., 2014).
Strains of this mushroom were taken to Japan in 1965, being produced 2.3. Preparation of hydroalcoholic extract from Agaricus blazei Murill
artificially at the Iwade Mushroom Institute (Mizuno et al., 1990).
Since that time, ABM has become widely consumed as food or tea HEAb was obtained by soaking the dried and crushed mushroom
(Kurozawa et al., 2010), food supplements (Mahmood et al., 2019) as (caps, gills and stems) (25.93 g) in 1 L of ethanol:water solution (70:30 v
well as nutraceutical and cosmeceutical applications (Taofiq et al., v− 1) under magnetic stirring (500 rpm) at room temperature for seven
2019), being cultivated and widely appreciated in countries such as days. The obtained material was subjected to vacuum filtration and
Japan, Korea and China (Kozarski et al., 2014). From an ethno­ concentrated at 60 ◦ C in a rotary evaporator under reduced pressure (40
pharmacological point of view, the Agaricus blazei Murill mushroom has rpm), then dried in a water bath at 40 ◦ C. The crude extract from ABM
been used mainly as an adjuvant agent in chemotherapy for cancer and (yield of 53.53%) was stored at 25 ◦ C until required for further use.
inflammatory diseases (Barbisan et al., 2002; Firenzuoli et al., 2008; Kim
et al., 2009; Ouedraogo et al., 2012). In addition, other mushrooms
belonging to the phylum Basidiomycetes are already traditionally used in 2.4. Elemental analysis
the treatment of gastrointestinal inflammatory diseases, such as gastric
ulcer, where Hericium erinaceus mushroom stands out (Liu et al., 2016). The carbon, oxygen, hydrogen, nitrogen and sulfur contents were
In the last decades, extracts and polysaccharides of the ABM have determined for the extract by elemental microanalysis using Perki­
become increasingly popular due to their long history as disease treat­ nElmer’s 2400 Series II CHNS equipment. Data were obtained in tripli­
ments, associated with their activities antitumoral (Sovrani et al., 2017), cate (Ribeiro et al., 2021).
immunomodulatory (Rubel et al., 2018), anti-inflammatory (Nakamura
et al., 2019), antioxidant (Zhai et al., 2015) and antiviral properties
2.5. Conditions for UPLC-QTOF-MSE analysis
(Cardozo et al., 2014), which are currently elucidated (Campelo et al.,
2021a). Extracts from Agaricus blazei prepared by the company Ando­
Chromatographic analyzes were performed using an Acquity Ultra-
San™ (ACE Co. Ltd., Gifu-ken, Japan) were used in a single-blinded
Performance Liquid Chromatography (UPLC) system containing an
placebo-controlled study and proved the quality of life in patients
autosampler and a binary pump (Waters, Milford, MA, USA). Chro­
with ulcerative colitis, due to its immunomodulatory activity (Therkel­
matographic separation of the compounds was carried out on an
sen et al., 2016). Thus, despite the lack of reports about the gastro­
analytical column Waters Acquity UPLC BEH (150 × 2.1 mm, particle
protective effect of the Agaricus blazei mushroom, the therapeutic
size 1.7 μm) at 40 ◦ C. The mobile phase used consisted of two solvents:
potential of this mushroom should not be disregarded, since it is already
water with 0.1% formic acid (solvent A) and acetonitrile with 0.1%
widely used in traditional medicine and presents pharmacological
formic acid (solvent B). The elution gradient used was 95% B (0–15
properties related to this effect.
min), 100% B (15–17 min), 2% B (17.01), 2% (17.02–19.01 min), flow
The activities of reducing cholesterol, gastric ulcers and digestive
rate 0.4 mL min− 1 and injection volume of 5 μL.
problems are also reported, being their mechanisms of action are still
Coupled to the UPLC system there is a quadrupole time of flight mass
unknown in the literature. The intense dissemination on the web and in
spectrometry (Waters, Milford, MA, USA) equipped with an electrospray
mass media for commercial purposes leads many people to use it for
ion source (ESI). The analyzes were designed in ESI− and ESI+ modes,
these types of illnesses without any scientific knowledge elucidated for
acquired in the range 110–1180 Da, the temperature of the fixed source
the mushroom (Firenzuoli et al., 2008). In addition to the various uses of
at 120 ◦ C, desolvation temperature 350 ◦ C, 500 L h− 1 desolvation gas
the ABM, Sui et al. (2010) also demonstrated that the polysaccharides of
flow. Leucine enkephalin was used as a lock mass, extraction cone of 0.5
this species have efficacy in the treatment of wounds (orally, 100 mg
V, capillary voltage of 2.6 kV in ESI− and 3.2 kV in ESI+ (Rodrigues
kg− 1 b.w.), obtaining a regeneration of 63.2% in skin burns of
et al., 2021).
Sprague-Dawley rats. Given the problem presented, its use in prophy­
lactic form, in adjuvant therapy against some diseases, and the report of
the proven healing property of the mushroom, this work aims to carry 2.6. Nuclear Magnetic Resonance (NMR)
out studies with hydroalcoholic extract from ABM in order to evaluate
its potential gastroprotective effect. NMR spectra were obtained using the Bruker Avance DRX-500
spectrometer (Fällanden, Switzerland), equipped with a 5 mm inverse
2. Materials and methods probe operating at 499.6 MHz (1H) and 125.6 MHz (13C). The extract
characterization was performed by 1H NMR, 13C NMR and DEPT 135
2.1. Reagents and standards and the chemical shifts (δ) were expressed in ppm. Sample preparation
was carried out by dissolving 20 mg of the crude extract in 0.6 mL of D2O
Formic acid, ascorbic acid, acetonitrile, dimethyl sulfoxide (DMSO), (Ribeiro et al., 2021).

2
J.F. Câmara Neto et al.
2.7. High-performance liquid chromatography (HPLC)
For mannitol quantification, an aqueous crude extract solution (1%,
w v− 1) was filtered through a 0.45 μm Millipore® membrane. The HEAb
chromatographic profile was determined on a SHIMADZU LC-10AD
chromatograph with a RID-10A refractive index detector at 40 ◦ C. The
isocratic analysis was performed using a Kromasil 100-5-NH2 column
(4.6 × 150 mm). The flow of mobile phase (deionized water/acetoni­
trile, 30/70 (v v− 1)) was 1.25 mL min− 1. The standard curve was con­
structed in a range of 1–10 mg mL− 1 of mannitol. The equation of the
line was obtained by the ordinary least squares method (y = 775732x –
27107, R2 = 0.9992). This method was used to determine the mannitol
content in the extract studied (Miki et al., 1996).
2.8. Total phenolic content and total flavonoid content
The determination of the total phenolic contents present in HEAb
was performed by the folin-ciocalteu method with some adaptations,
using the Thermo Scientific model Genesys 6 (UV/Vis) (Sousa et al.,
2007). 100 μL of HEAb methanolic solution (0.3%, w v− 1) were prepared
and added to 500 μL of folin-ciocalteu reagent, followed by stirring for
30 s in a 10 mL volumetric flask. Subsequently, 6 mL of distilled water
and 2 mL of Na2CO3 (15%, w v− 1) were added and stirred for 1 min.
Then, the volume was made up to 10 mL with distilled water and the
sample was kept in the dark. Absorbance readings were taken at 750 nm
using quartz cuvettes after 2 h of mixing the reagents. The control was
composed of methanol and all reagents except HEAb. The results were
expressed as milligram equivalent gallic acid per gram of extract (mg
GAE/g).
The determination of the flavonoid content present in HEAb was
performed using the aluminum chloride method (UV/VIS) (Funari and
Ferro, 2006). The results were expressed as milligram quercetin equiv­
alent per gram of extract (mg QE/g) All analyzes were performed in
triplicate.
2.9. Evaluation of in vitro antioxidant activity
2.9.1. DPPH radical-scavenging activity assay
The antioxidant activity was screened by DPPH assay with modifi­
cations (Brand-Williams et al., 1995). The sample (15.0 mg) was dis­
solved in 1.5 mL of methanol:DMSO (1:1). Subsequently, several
dilutions were prepared for analysis (5.000–5 μg mL− 1). Aliquots of 0.1
mL of the dilutions were added in test tubes with 3.9 mL of the a 6.5 ×
10− 5 mol L− 1 DPPH methanolic solution. After this procedure, the
sample was kept in the dark for 1 h before being read on a spectro­
photometer at 515 nm. Methanol/DMSO (1:1) was used as the control.
The sweep index (IV%) was calculated from equation (1):
IV% = (ADPPH - ASAMPLE / ADPPH) x 100 (1)
Where, ADPPH is the absorbance detected for the methanol solution with
DPPH while ASAMPLE is the absorbance obtained for the tested dilutions.
The efficient concentration (EC50, μg mL− 1) was determined through the
ratio of the new dilutions concentrations by their respective IV% for the
analysis. Ascorbic acid was used as the reference substance for the
experiment. The assay was performed in triplicate for each
concentration.
2.9.2. Ferrous ion chelating (FIC) assay
The ferrous ion chelating assay was adapted from the method
described by Campelo et al. (2021b). Aliquots of 1 mL (0.1 mmol L− 1) of
FeSO4 solution were mixed at different dilutions (0.5–4.0 mg mL− 1) of
the extract (1 mL), followed by 0.25 mmol L− 1 ferrozine (1 mL). The
resulting mixtures were kept under rest for 10 min before determining
their absorbances at 562 nm. The results were expressed as a percentage
of chelating activity according to equation (2):
3
Journal of Ethnopharmacology 292 (2022) 115191
Chelating activity (%) = [A0 − (A − Ab)/A0] × 100 (2)
Where, A0 = FeSO4 + Ferrozine without sample; A = sample + FeSO4 +
Ferrozine; and Ab = sample without FeSO4 + Ferrozine. With the results
obtained, Fe2+ chelating ability was calculated (mg mL− 1). Ascorbic
acid was used as the reference substance for the experiment. The assay
was performed in triplicate for each concentration.
2.10. In vivo gastroprotection assay
2.10.1. Animals
The animals used in this work were male Swiss mice (weight: 20–30
g; 8–12 weeks), stored in standardized cages under controlled temper­
ature (23 ± 1 ◦ C), 12 h light/12 h dark cycle and feed/water ad libitum.
Fifteen hours before the experiments, the mice were left fasting with
access only to water ad libitum. The protocol and experimental proced­
ures are under number 2071010219 approved by the Ethics Committee
on the Use of Animals at the Federal University of Ceará (CEUA-UFC).
2.10.2. Ethanol-induced gastric mucosal lesions
This ulcer model was performed according to the method of Robert
et al. (1979), with some modifications. Mice were randomly divided into
five groups (n = 8), fasting for 15 h before the experiment, but had free
access to water. Gavage was performed with HEAb (5, 25, and 50 mg
kg− 1) (doses based on literature studies, Sui et al. (2010), Xin., (2019)
and Nakajima et al. (2002)), HEAb vehicle (control) or N-acetylcysteine
(NAC) 300 mg kg− 1 (positive control), 60 min before the administration
of absolute ethanol (0.2 mL/25 g animal, p.o.), through an orogastric
metal tube. Thirty minutes after gavage with ethanol, the animals were
euthanized by anesthetic overdose (ketamine 300 mg kg− 1 and xylazine
30 mg kg− 1), followed by laparotomy to removal the stomachs. The total
and injured stomach areas (glandular portion) were measured by a
computer planimetry program (Image J®) and expressed in terms of
percentages of the ulcerated gastric area.
2.10.3. Histological assessment
For histological assessment, stomachs from all groups [control
(HEAb vehicle + ethanol), HEAb-5 (5 mg kg− 1) + ethanol, HEAb − 25
(25 mg kg− 1) + ethanol, HEAb − 50 (50 mg kg− 1) + ethanol and NAC
(300 mg kg− 1) + ethanol)] were fixed in 10% neutral-buffered formalin
solution, sectioned and embedded in paraffin. Sections (4 μm) were
deparaffinized, stained with hematoxylin and eosin, and then examined
under a light microscope. The specimens were assessed according to the
criteria of Laine and Weinstein (1988). In brief, a 1 cm length of each
histological section was assessed by scores for epithelial (0–3), edema in
the upper mucosa (0–4), hemorrhagic damage (0–4), and presence of
inflammatory cells (0–3). The sections were analyzed in a blind manner
(Cerqueira et al., 2012).
2.10.4. Evaluation of mastocytosis
To enable the identification and counting of mast cells, the paraffin
blocks with gastric segment samples, corresponding to the control,
HEAb (25 and 50 mg kg− 1) and NAC group, were selected for toluidine
blue staining, according to Michalany (1998). Digital images were
captured for the enumeration of mast cells present on the slides with the
aid of an optical microscope and an image acquisition system (LEICA,
Wetzlar, Germany). Using the software ImageJ® 1.48v (USA) it was
possible to represent the average results of 10 fields for each group
analyzed.
2.10.5. HEAb pathways of action in the ethanol-induced gastric ulcer model
2.10.5.1. Role of nitric oxide (NO) in the gastroprotective effect of HEAb.
The mice were treated with HEAb-25 (p.o.) 60 min before administra­
tion absolute ethanol (0.2 mL/animal, p.o). To assess the role of nitric
J.F. Câmara Neto et al.
oxide in the gastroprotective effect of HEAb, combinations were also
performed where L-NAME (10 mg kg− 1, s.c.) was administered 15 min
before HEAb-25 (p.o) or L-Arginine (600 mg kg− 1, i.p.) and 1 h later
injury was induced with ethanol (Gürbüz et al., 1999). After 30 min of
ethanol administration, the animals were sacrificed, the stomachs
removed, and total and injured stomach areas were measured following
the procedure described previously.
2.10.5.2. Role of Cyclic Guanylate Monophosphate (cGMP) in the gas­
troprotective effect of HEAb. The involvement of cGMP was evaluated by
administration of ODQ (10 mg kg− 1, i.p.), a specific guanylate cyclase
inhibitor, 30 min before the administration of HEAb-25 (i.p.). The
gastric lesion was induced with ethanol 30 min after the extract’s
treatment (Díaz-Triste et al., 2014). The total and injured stomach areas
were measured following the procedure described previously.
2.10.5.3. Involvement of prostaglandins (PGs). Animals were treated
with HEAb-25 (p.o.) or misoprostol (70 μg kg− 1, p.o.) 60 min before
administration of absolute ethanol (0.2 mL/animal, p.o.). Combinations
were also performed with indomethacin (10 mg kg− 1, p.o.) 120 min
before administration of HEAb-25 (p.o.) or misoprostol (70 μg kg− 1, p.
o.). Lesions were induced with absolute ethanol 60 min after extract or
misoprostol doses. After 30 min of ethanol administration, the animals
were euthanized, the stomachs were removed and then proceeded as
described above regarding the determination of the percentage of the
ulcerated area (Cerqueira et al., 2012).
2.10.5.4. Involvement of ATP-sensitive K+ channels. Mice were treated
with HEAb-25 (p.o.) (60 min) or diazoxide (3 mg kg− 1, i.p.) (30 min)
before oral administration of absolute ethanol (0.2 mL/animal). To
assess the possible participation of ATP-sensitive K+ channels in the
gastroprotective effect, combinations were performed in which gliben­
clamide (5 mg kg− 1, i.p.) was administered 15 min before treatments
with HEAb-25 (p.o.) or diazoxide (3 mg kg− 1, i.p.), and 60 min later, the
gastric injury was induced with ethanol. After 30 min of the absolute
ethanol administration, the animals were euthanized, the stomachs
removed and then proceeded as described above regarding the analysis
of the percentage ulcerated area (Cerqueira et al., 2012).
2.11. Statistical analysis
Data normality was assessed using the Shapiro-Wilk test. The
following tests were used to perform the experiments’ statistical ana­
lyses: Analysis of variance (one-way ANOVA), followed by Tukey’s
multiple comparisons post-test for normal mean values and Kruskal-
Wallis test followed by Dunn’s test for nonparametric data. The confi­
dence interval adopted was 95%, that is, results with p < 0.05 were
considered statistically significant (also considering the other levels of
significance (**p < 0.01; ***p < 0.001 and ****p < 0.0001). Statistical
analyses of the data were performed using GradPad Prism software,
version 6.0 (GraphPad Software Inc., La Jolla, CA, USA). All results were
expressed as mean ± standard error of the mean (SEM) or mean ±
standard deviation (except for the histopathological scores presented by
the median values).
3. Results
3.1. Elemental analysis
The quantification in percentage (%) of carbon, hydrogen, nitrogen,
sulfur and oxygen in HEAb was performed through elemental analysis.
The results obtained show higher levels of oxygen (47.92 ± 0.16%) and
carbon (37.72 ± 0.17%), followed by hydrogen (7.55 ± 0.06%) and
nitrogen (4.98 ± 0.22%). Low sulfur (1.83 ± 0.05%) content was
observed in this extract. In the literature, works that used an aqueous
4
Journal of Ethnopharmacology 292 (2022) 115191
extract from ABM showed carbon and nitrogen contents of 40.9 and
6.1%, respectively, corresponding to the presence of carbohydrates and
proteins (Yamanaka et al., 2012).
3.2. Ultra-performance liquid chromatography coupled with electrospray
ionization quadrupole time of flight mass spectrometry (UPLC-QTOF-MSE)
The chemical profile of HEAb was established by analyzing the
chromatograms in the negative (ESI− ) and positive (ESI+) modes. The
mass spectra were also analized. The high-intensity peaks in the samples
were considered, but also those low-intensity peaks. In Table 1, the notes
of the metabolites are displayed. Associated with the metabolites, their
deprotonated ions [M-H]- and protonated [M + H]+ ions and their
fragment ions are also presented, as well as the error in parts per million
(ppm) and its possible molecular formula. It is important to note that the
recognition of these compounds was designed based on an intense
search in the literature and the exhaustive searches considering the
chemotaxonomy (family, genus and species). The chromatograms ob­
tained for HEAb are shown in Fig. 1.
3.3. Nuclear Magnetic Resonance (NMR)
The characterization of the mannitol (Fig. 2a) in the HEAb was
performed by UPLC-QTOF-MSE and NMR. 1H NMR spectra obtained for
HEAb showed several signs between 3.0 and 5.5 ppm, wich is a region
characteristic of sugars (Ramírez-Meraz et al., 2020) (Fig. 2b).
3.4. High-performance liquid chromatography (HPLC)
The putative identification and characteristic signs of mannitol in the
extract by UPLC-QTOF-MSE and NMR required its quantification. The
presence of mannitol in the extract was confirmed by the retention time
verified in the chromatographic analysis of the HEAb sample, which was
similar to that obtained for the standard (3.77 min, Fig. 3a). It was
possible to observe a retention time of 3.75 min for HEAb (Fig. 3b).
Mannitol content of 40.81 ± 0.74% was verified in HEAb, correspond­
ing to 408.14 mg of mannitol per gram of crude extract.
3.5. Total phenolic content (TPC) and total flavonoid content (TFC)
In order to relate the results of the antioxidant activity, it was
necessary to quantify the phenolic compounds since they are reported as
one of the main classes of secondary metabolites with this property
(Otmani et al., 2021). The total phenolics and total flavonoids levels
found in HEAb were 89.23 ± 2.56 (mg GAE/g DW) and 14.33 ± 0.07
(mg QE/g DW), respectively. The study by Tsai et al. (2007), which used
aqueous and ethanolic (95%) extracts from ABM, obtained the following
results: 5.67 ± 0.09 and 5.80 ± 0.05 mg/g of crude extract, respectively.
These differences in results can be explained by the fact that the inter­
molecular interactions of these components are stronger with the
mixture of solvents when compared to extractions with just water or
ethanol, at the time of their extraction (preparation of extracts).
3.6. Determination of in vitro antioxidant activity
The antioxidant activity of HEAb was evaluated through DPPH free
radical scavenging and Fe2+ ions-chelating capacity. Ascorbic acid
showed high antioxidant activity (18.52 ± 0.75 μg mL− 1), given the low
amount of material required to inhibit 50% of DPPH radicals in the test.
For chelation activity, HEAb showed better results (0.62 ± 0.01 mg
mL− 1) when compared to the positive control (7.22 ± 0.59 mg mL− 1).
We suggest that the several compounds presents in HEAb are responsible
by its good antioxidant activity (117.04 ± 0.76 μg mL− 1), since they can
act as Lewis acids and promote the inhibition of DPPH radicals. In the
chelation of ferrous ions, the different secondary metabolites present in
HEAb promoted greater efficacy Fe2+ sequestration when compared to
J.F. Câmara Neto et al. Journal of Ethnopharmacology 292 (2022) 115191

Table 1
Components that were identified in HEAb by UPLC-QTOF-MSE.
Peak tR Metabolite Molecular Negative mode (ESI− ) Positive mode (ESI+) Reference
(min) annotation Formula -
[M-H] Fragment ions Error [M+H] +
Fragment ions Error
(MS/MS) (ppm) (MS/MS) (ppm)

1 1.32 Mannitol C6H14O6 [M+Cl]-217.0475 181.0708; − 1.8 – – – Ammar et al. (2017)

96.9655; Klewicki et al. (2017)
89.0205;
71.0111
2 1.45 Malic acid C4H6O5 133.0128 115.0013; 0.0 – – – Delgado-Povedano
89.0250; et al. (2016)
71.0147
3 2.06 Pyroglutamic C5H7NO3 128.0348 128.0357; 0.0 – – – Delgado-Povedano
acid 82.0286 et al. (2016)
4 2.10 L-Agaritine C12H17N3O4 – – – 268.1283 84.0681 − 5.2 Delgado-Povedano
et al. (2016)
5 3.37 L-Valine C5H11NO2 – – – 118.0875 72.0917 5.9 Delgado-Povedano
et al. (2016)

tR (min): Retention time in minutes; All masses were calculated using MassLynx V4.1 software.

Fig. 1. Chromatograms for the HEAb: (a) Negative ion mode of HEAb; (b) positive ion mode of HEAb.

the positive control.
3.7. In vivo gastroprotective activity
3.7.1. Investigation of the gastroprotective effect of HEAb in the ethanol-
induced gastric ulcer model
Administration of absolute ethanol produced lesions in the gastric
mucosa (Fig. 4a), which were reduced in HEAb pretreated animals (25
and 50 mg kg− 1) with 75–81% decrease. The ethanol-ulcerated area was
significantly decreased with oral administration of HEAb-25 (3.28 ±
0.72%, p < 0.01, Fig. 4d) and HEAb-50 (4.60 ± 1.51%, p < 0.05,
Fig. 4e). In the group HEAb-5 (15.39 ± 3.20%, Fig. 4c) no significant
effects were observed for treatment. N-acetylcysteine (NAC, 300 mg
kg− 1) (0.57 ± 0.11%, p < 0.0001, Fig. 4f), used as a reference drug, also
significantly reduced gastric lesions (Fig. 4f) by 95% compared with the
control (17.91 ± 2.56%, Fig. 4b).
evidencing gastroprotection when compared to the control group
(Fig. 5a), in which severe lesion characterized by hemorrhagic damage,
edema, loss of epithelial cells and presence of inflammatory cells was
observed. NAC pretreatment also prevented ethanol-induced damage at
the tested dose (Fig. 5e).
Significant differences were observed for HEAb at 25 and 50 mg kg− 1
and NAC (300 mg kg− 1), indicating thus a decrease in histopathological
scores compared to the control group. No significant difference was
found for HEAb at 5 mg kg− 1 (Fig. 5b) compared with the control group,
indicating that this dose was ineffective. Based on this results, the doses
of 25 and 50 mg kg− 1 were chosen to proceed with the modulation of the
possible pathways of action of HEAB. These observations can be seen in
Fig. 5. The pretreated groups with HEAb at 25 and 50 mg kg− 1 were able
to reduce the ulcerative lesion induced by ethanol, similar to the refer­
ence drug N-acetylcysteine. The analysis of Fig. 5 shows the mainte­
nance of cellular integrity, absence of inflammatory signs, and
preservation of mucous, submucosal, muscular and serous layers.

3.7.2. HEAb reduces histopathological parameter scores of ethanol-induced
gastric injury
The results regarding the histopathological analysis of the gastric
mucosa of the mice are shown in Table 2. The animals pretreated with
HEAb (25 and 50 mg kg− 1, p.o.) (Fig. 5c and d) showed a massive
macroscopic and microscopic difference of the injured gastric mucosa,
3.7.3. HEAb effect on mice stomach submucosa: mast cells
Administration of absolute ethanol to animals previously treated
with vehicle alone (saline, p.o.) produced a significant increase in the
number of mast cells in the gastric mucosa (connective tissue) (10.1 ±
0.74, Fig. 6a) compared to all groups tested. The number of mast cells

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J.F. Câmara Neto et al. Journal of Ethnopharmacology 292 (2022) 115191

Fig. 2. Molecular structure of mannitol (a) and Nuclear Magnetic Resonance (NMR) of: (b) 1H NMR spectrum of HEAb; (c) 13C NMR spectrum of HEAb; (d) DEPT 135
NMR spectrum of HEAb.

Fig. 3. Chromatogram for ABM extract. (a) mannitol; (b) HEAb.

was significantly decreased with previous oral administration of HEAb-

25 (4.41 ± 0.37, p < 0.0001, Fig. 6b) and HEAb-50 (4.36 ± 0.54, p <
0.0001, Fig. 6c). NAC (6.11 ± 0.45, p < 0.001, Fig. 6d), used as a
reference drug, also significantly reduced the number of mast cells in the
gastric mucosa (Fig. 6e).
3.7.4. HEAb pathways of action in the ethanol-induced gastric ulcer model
After demonstrating the gastroprotective activity of HEAb and the
dose capable of producing the maximum effect on the ethanol-induced
gastric ulcer model (25 mg kg− 1), the possible mechanisms of action
involved in HEAb gastroprotection were investigated in relation with
the involvement of nitric oxide (NO)/Cyclic Guanylate Monophosphate
(cGMP), Prostaglandins and K + ATP channels pathway. The results were
expressed in terms of percentages of mucosal lesions and can be seen in
Fig. 7.
Fig. 4. Investigation of the gastroprotective effect of HEAb in ethanol-induced
gastric ulcer model through macroscopic assessment of gastric mucosa in mice.
(a) percentages of ulcerated gastric areas for the groups; (b) Control; (c) HEAb-
5; (d) HEAb-25; (e) HEAb-50; (f) NAC 300 mg kg− 1. For statistical analysis,
Kruskal-Wallis test followed was used, followed by Dunn’s test (*p < 0.05 vs
group control; **p < 0.01 vs group control; ***p < 0.001 vs group control and
****p < 0.0001 vs group control).
4. Discussion
Cardozo et al. (2013) analyzing the polysaccharides of ABM obtained
carbon (34.42%), hydrogen (5.56%) and nitrogen (2.50%) contents
similar to the this study. In addition, when they isolated the poly­
saccharides from the mycelium, it was observed contents of 32.07% (C),
4.36% (H) and 1.63% (N). Due to the use of water as a solvent, we can
suggest the presence of polysaccharides in HEAb, considering that these

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J.F. Câmara Neto et al. Journal of Ethnopharmacology 292 (2022) 115191

Table 2
Scores of histopathological changes in the gastric mucosa of mice submitted to ethanol-induced gastric lesions, pretreated or not with HEAb and NAC.
Experimental Group Epithelial cell loss (score 0–3) Edema (score 0–4) Hemorrhage (score 0–4) Inflammatory infiltrate (score 0–3) Total (score 0–14)

Control 2.9 (2–3) 2.5 (2–3) 2 (2–4) 0 (0–1) 7.4

HEAb-5 2,5 (1–3) ns 2,5 (2–3) ns 2 (2–3) ns 0 (0 - 0) ns 7.0
HEAb-25 1.1 (0–2)*** 1 (1–2)**** 1 (0–2)**** 0 (0–0)ns 3.1
HEAb-50 1 (0–2)*** 1 (1–2)**** 1 (0–2)*** 0 (0–0)ns 3.0
NAC (300 mg kg− 1) 0 (0–0)**** 1 (1–1)*** 0.5 (0–1)**** 0 (0–0)ns 1.5

For statistical analysis, Kruskal Wallis test was used, followed by Dunn’s test (*p < 0.05 vs group control; **p < 0.01 vs group control; ***p < 0.001 vs group control;
****p < 0.0001 vs group control and not significant (ns) vs group control). Animals were treated orally with HEAb (25 and 50 mg kg− 1 p.o.), or N-acetylcysteine (NAC
300 mg kg− 1 p.o.) 60 min before of administration of ethanol (0.2 mL/25 g, p.o.). The results are presented as median, with maximum and minimum values shown in
parentheses.

Fig. 5. Effect of HEAb on the gastric mucosa of mice submitted to gastric lesions induced by ethanol. All panels were obtained on the 100 μm scale (×200). (a)
Control; (b) HEAb − 5; (c) HEAb-25; (d) HEAb-50 and (e) NAC (300 mg kg− 1). The animals treated with absolute ethanol, showed disruption of the superficial region
of the gastric gland (arrow) with epithelial cell loss (arrow green), edema (arrow blue) and intense hemorrhage (arrow red). Pretreatment with HEAb (25 and 50 mg
kg− 1, p.o.) and NAC (300 mg kg− 1) showed preservation of gastric mucosa. (For interpretation of the references to colour in this figure legend, the reader is referred
to the Web version of this article.)

macromolecules are hydrophilic.
The fragments identified in the HEAb (Table 1) for the MS spectrum
of compound 1 show a chlorine adduct [M + Cl]- in m/z 217.0475
(C6H14O6Cl), which was putatively identified as mannitol. The metab­
olite shows a loss of 36 Da [M + Cl - 36]- which could be relative to the
monosaccharide in its neutral form (C6H14O6). In addition, the MS2
spectrum shows the presence of three other peaks in m/z 96.9655
[C6H14O6 - 84]-, 89.0205 [C6H14O6 - 92]- and 71.0111 [C6H14O6 - 110]-.
Compounds 2 and 3 showed a molecular ion [M]- in m/z 133.0128
and 128.0348, putatively identified as malic acid and pyroglutamic acid
of molecular formula C4H6O5 and C5H7NO3, respectively. The fragments
of these metabolites observed in the MS2 spectrum showed losses of 18
Da [M - H2O]-, 44 Da and 62 Da for malic acid, while fragments 128 Da
and 46 Da for pyroglutamic acid were observed. Compounds 4 (L-
Agaritine) and 5 (L-Valine) showed a molecular ion [M]+ in m/z
268.1283 (C12H17N3O4) and 118.0875 (C5H11NO2), respectively. The
fragments of these metabolites observed in the MS2 spectrum showed
184 Da and 46 Da losses in the same order as described above.
For HEAb (Fig. 2b), the 1H NMR spectrum showed a signal at 3.80
ppm attributed to the H-1 of mannitol, while H-2 and H-3 showed signs
at 3.63 and 3.74 ppm, respectively. The NMR data for mannitol are
according to reported in the literature by Paula et al. (1998). The signals
referring to hydroxyl hydrogens in the extract spectra were not
observed. 13C NMR and DEPT 135 spectrum of HEAb showed three signs
at 70.82, 69.24 and 63.20 ppm, associated with the carbons C-2, C-3 and
C-1 of mannitol (Fig. 2c and d), respectively. The 13C NMR data for
mannitol in the HEAb are according to described by Rodrigues et al.
(2010) that isolated this compound from Stemodia maritime.
Some studies in the literature quantified mannitol in ABM extracts,
which resulted in 79.43 mg/g in extract, corresponding to about five
times less when compared to HEAb (Tsai et al., 2008). This difference in
results may be associated with how the extracts are obtained since, in
the methodology used, 50 mL of an ethanol solution (80%) is used to
extraction, followed by stir (45 min), washing and filtration (Tsai et al.,
2008). The methodology used in the present work, besides a longer
contact time between the raw material (macerated mushroom) and the
extracting solution there is a larger volume of solvent used for the
preparation of the extract, thus resulting in a greater intermolecular
interaction between the mannitol present in the mushroom with the
solvents used.
Lo et al. (2016) obtained high mannitol levels in relation to other
sugars that were also quantified in the ABM species (arabinitol, glucose,
myo-Inositol and trehalose). Some works show that mannitol is quite
recurrent in mushrooms of this genus and already has been reported for
the species of A. bisporus (Cardoso et al., 2019), A. meleagris (Rafighi
et al., 2019), A. campestris (Jedidi et al., 2016), A. bitorquis and

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J.F. Câmara Neto et al. Journal of Ethnopharmacology 292 (2022) 115191

Fig. 6. Effect of HEAb treatment on mast cell count for 10 fields (toluidine blue staining). Values were presented as mean ± SEM of the number of mast cells per 10
fields. All panels were obtained on the 100 μm scale (x 200). (a) Control; (b) HEAb-25; (c) HEAb-50; (d) NAC 300 mg kg− 1 and (e) mean of the number of mast cells
per 10 fields. For statistical analysis, one-way ANOVA test was used, followed by Tukey’s test (*p < 0.05 vs group control; **p < 0.01 vs group control; ***p < 0.001
vs group control and ****p < 0.0001 vs group control). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of
this article.)

Fig. 7. Modulation of HEAb gastroprotective effect pathways: (a) Role of nitric oxide (NO); (b) Role of Cyclic Guanylate Monophosphate (cGMP); (c) Role of
prostaglandins (PGs) and (d) Role of ATP-sensitive K+ Channels. For statistical analysis, one-way ANOVA or Kruskal Wallis test were used, followed by Tukey’s or
Dunn’s test (*p < 0.05 vs group control; **p < 0.01 vs group control; ***p < 0.001 vs group control; ****p < 0.0001).

A. macrosporus (Glamoclija et al., 2015).
For the phenolic compounds, Buruleanu et al. (2018) obtained in
their results higher levels of total phenols for ethanol extracts (50%)
(21.23 ± 0.01 and 69.65 ± 0.23 mg GAE/g DW) when compared to
aqueous extracts (11.30 ± 0.01 and 17.50 ± 0.01 mg GAE/g DW) for the
species of A. bisporus and A. campestris, respectively. The percentage of
total phenols obtained for the A. campestris extract (6.96%) was quite
similar to that of the present study (8.92%), but a little lower.
For the total flavonoid contents, these extracts (the same order as the
species mentioned above, respectively) showed lower results (0.83%
and 0.65% mg QE/g DW) when compared to the present work (1.43%),
which can be justified by the methodology used in the preparation of the

8
J.F. Câmara Neto et al.
HEAb (Buruleanu et al., 2018). Another study that worked with a
methanolic extract from A. blazei obtained flavonoid contents of 0.327
± 0.01 (mg EQ g− 1) (Vital et al., 2017), which suggests that the change
of extraction solvent associated with a given methodology will result in
different levels of phenolic compounds, since their intermolecular forces
will be necessarily different. The low levels of flavonoids obtained in
HEAb may justify the fact that they were not identified in the
UPLC-QTOF-MSE analysis.
Carvajal et al. (2012) carrying out studies with the fruiting bodies,
young mycelia and old mycelia of ABM obtained EC50 (μg mL− 1) (DPPH
scavenging activity) of 305 ± 14, 1413 ± 52 and 599 ± 35, respectively.
In the same study, Fe2+ chelating ability (mg mL− 1) for the same order
described above was 1.002 ± 0.07, 1.325 ± 0.09 and 0.438 ± 0.03,
respectively. The extract used in the present work proved to be more
efficient in terms of inhibiting the DPPH radical, as well as Fe2+
chelating ability (except when compared to the old mycelium). Ethyl
acetate extract from ABM showed IC50 values of 421 μg mL− 1 (DPPH
scavenging activity), which promoted an efficient response in reducing
α-glucosidase inhibition capacity in vitro (Wei et al., 2020). The HEAb
obtained in this study has about three times more antioxidant efficiency
than the previously described ethyl acetate extract.
Mannitol identified as the main compound in the present study may
have been primarily responsible for antioxidant activity since several
studies report its effectiveness in inhibiting free radicals (Ming and
WangSui, 2020; Khaleghi et al., 2019). Malic acid and pyroglutamic acid
also contributed to this effect, considering some reports in the literature
(Marques et al., 2021; Begum et al., 2018). Lu et al. (2020) studying
mushrooms of the same genus, observed in their results a direct
connection between the phenolic content of the studied mushroom
species (A. bisporus, Lentinula edodes and Boletus edulis) with the in vitro
antioxidant activities (DPPH and ORAC methods).
In addition, with the structure and conformation of α-glucans well
elucidated by Zhang et al. (2018) and the β-glucans extracted from ABM,
it can be suggested that both polysaccharides acted in synergism with
mannitol (and the phenolic compounds present in the extract),
providing satisfactory results as well as those observed by other authors
for antioxidant activity and ferrous ion chelating ability (Qi et al., 2019;
Kozarski et al., 2011, 2014). Gonzaga et al. (2020) carrying out studies
with the polysaccharides isolated from ABM obtained an efficiency of
96.5% in ferrous ion chelating activity using these biopolymers at 1.0
mg mL− 1, demonstrating the effectiveness of the polysaccharides for
such activity.
In the present study, we showed that HEAb promotes better gastro­
protective effect at 25 mg kg− 1. In this dose, a decrease in tissue damage
can be evidenced (disruption of the superficial region of the gastric
gland with epithelial cell loss, edema and intense hemorrhage, Fig. 5)
promoted by ethanol. Shaik (2018) in studies with methanolic extract
from A. bisporus at doses of 250 and 500 mg kg− 1 also obtained signif­
icant results in treating gastric ulcers induced by pylorus
ligation-induced in rats. When comparing these results with the present
study, it is noted that a smaller dose of HEAb (about ten times) is
required to obtain significant responses in the treatment of gastric
ulcers.
The same occurs in the work of Padilha et al. (2009), which studied
two extracts from ABM (aqueous and alkaline) at doses of 400 mg kg− 1
for 15 days, obtained a decrease in ulceration in 21.88% and 28.63%,
respectively, compared to the control group. Compared with the present
study, HEAb showed a greater reduction in ulceration and a decrease in
dose (about sixteen times) to demonstrate significant differences to the
control group.
Similar results were obtained in several studies investigating the
potential gastroprotective effect of extracts from mushrooms against
gastric ulcers induced by ethanol (Xin et al., 2019; Jeong et al., 2019).
However, in our study the dose effective in protecting gastric damage
induced by ethanol were significantly lower than in the other studies
reported above. Also, the chemical composition of the extract, may have
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Journal of Ethnopharmacology 292 (2022) 115191
enhanced its antiulcerogenic effect since studies show the close rela­
tionship between the increased release of reactive oxygen species and
the occurrence of gastric injury (Song et al., 2020; Fahmy et al., 2020b).
The emergence of gastric ulcers is associated with an imbalance
between aggressive and protective factors of the stomach, where
harmful factors [hydrochloric acid and pepsin secretion, stress, reactive
oxygen species (ROS), nonsteroidal anti-inflammatory drugs (NSAIDs)
and H. pylori], stand out from the defense system of the gastric mucosa
[muco-bicarbonate barrier, prostaglandins (PG), mucosal blood flow,
reserves of antioxidant systems, adequate levels of nitric oxide (NO)]
(Zakaria et al., 2016; Jeong et al., 2019; Tonchaiyaphum et al., 2021).
Another critical factor in the pathogenesis of gastric ulcers is alcohol
intake. Absolute ethanol is considered a potent aggressor agent of the
gastric mucosa, capable of interrupting the formation of gastric mucus,
which culminates in disturbance of the superficial epithelial mucosa, the
onset of oxidative stress, inhibition of prostaglandin production,
decrease in blood flow, induces mast cells degranulation, necrosis cell
and, therefore, the gastric ulcer (Brito et al., 2020; Silva et al., 2021).
Based on this, we chose to study the effect of HEAb in the ethanol-
induced gastric injury model and evaluated its gastroprotective effect
through histopathological parameters, as well as the evaluation of
possible mechanisms of action of HEAb. As previously reported, among
the compounds evidenced in the HEAb phytochemical profile, mannitol
is the major compounds in this extract. For this compound, studies
indicate its use at a dosage of 100 mg kg− 1 reduced the percentage of
ulcerated area induced by HCl, NaOH and Acetylsalicylic acid.
This drug also showed antimicrobial activity against H. pylori,
exhibiting a minimum inhibitory concentration in the range of 32–125
μg mL− 1 (Garro et al., 2015). Gharzouli et al. (2001) demonstrated that
the gastroprotective action performed by mannitol is directly propor­
tional to the increase in luminal osmolarity. This is due to the rise in
blood flow in the gastric mucosa, which can reverse the necrotic effects
generated by aggressive agents that trigger ulcers.
A clinical study with twenty-five individuals found that incorpo­
rating mannitol in alcoholic beverages can prevent damage caused by
radical species generated by excessive alcohol consumption. Besides,
this drug demonstrated a hepatoprotective effect against ethanol
administration in rats, whose biomarkers of liver damage were signifi­
cantly reduced and the endogenous antioxidant enzymes increased
(Chigurupati et al., 2016). Such results showed that mannitol can
reverse the acute and chronic deleterious effects caused by ethanol.
As the pathophysiology of ethanol-induced gastric ulcer involves
multiple factors, it is estimated that the gastric protection mechanism of
HEAb involves at least three distinct factors: reduction of oxidative
stress, reduction of the expression of inflammatory mediators and
increased luminal osmolarity. Concomitantly, there are reports in the
literature that β-glucans have also managed to reverse the ulcerative
effect induced by ethanol (Chen et al., 2019; Silva et al., 2020). In this
way, the glucans present in the extract may have acted in synergism with
the mannitol enhanced its gastroprotective effect.
Sulaieva et al. (2015) demonstrated the close relationship between
the presence of leukocytes in the ulcerated site and the risk of hemor­
rhage since the passage of these cells to the inflammation site, mainly
mast cells, is related to the worse prognosis of the gastric injury. The
mast cell degranulation promotes increased vascular permeability, the
release of inflammatory mediators and vasodilation, increasing the risk
of bleeding events. Given this, it is estimated that the decrease in the
number of mast cells is related to the gastroprotective effect.
HEAb in both dosages significantly reduced these cells in the eval­
uated tissue. Since no significant differences were observed between the
25 and 50 mg kg− 1 dosages, its effect is dose-independent. Thus, by
decreasing mast cell infiltration, HEAb possibly interferes with necrosis
and tissue damage feedback, resulting from mast cell degranulation and
release of inflammatory mediators, like histamine (Silva et al., 2018;
Brito et al., 2020).
Fig. 7a shows the evaluation results of the role of nitric oxide (NO) in
J.F. Câmara Neto et al.
the gastroprotective activity of HEAb. The administration of absolute
ethanol in animals that were previously treated only with vehicle pro­
moted a high percentage of the ulcerated gastric area (18.31 ± 3.31%).
The gastric area ulcerated by ethanol was significantly reduced with the
previous administration of HEAb-25 (p.o) (3.28 ± 0.72%, p < 0.01)
when compared to the group treated with vehicle. However, the gas­
troprotective effect of HEAb-25 was reversed by the previous adminis­
tration of L-NAME (10 mg kg− 1,i.p.), an inhibitor of NO synthesis (16.93
± 3.14%). These results were similar for treating animals with L-argi­
nine at a dose of 600 mg kg− 1, i.p., (15.89 ± 1.75%), which is known to
be an agonist of this pathway. Thus, it is estimated that the gastro­
protective effect of HEAb-25 involves molecular mechanisms related to
NO pathway.
NO improves mucosal integrity in the gastrointestinal tract. This
effect is related to its vasodilation by regulating local blood flow and
secretion of mucus. So, this action is a possible contribution to the
gastroprotective effect of HEAb (Fonseca da Silva et al., 2020). The in­
crease in NO production by the HEAb-25 dose may have influenced the
reduction of inflammation dependent on mast cells, which favored the
gastroprotective effect (Nguyen et al., 2020). From the evaluation of role
of the NO pathway in the gastroprotective effect of HEAb, we investi­
gated the role of cyclic Guanylate Monophosphate (cGMP) (Fig. 7b) and
observed that the gastroprotective effect promoted by HEAb (6.35 ±
1.20%) compared to the control group (18.31 ± 3.33%), was not
significantly inhibited by the action of ODQ, selective inhibitor of gua­
nylate cyclase (20.89 ± 4.03%). Thus, it was shown that the gastro­
protective mechanism by NO is independent of the activation of
guanylate cyclase.
To assess the involvement of prostaglandins (Fig. 7c), it was observed
that ethanol-induced ulcers were significantly reduced by prior treat­
ment with HEAb-25 (3.28 ± 0.72%, p < 0.001), as well as by miso­
prostol, a prostaglandin E1 analog (8.14 ± 1.55%, p < 0.01), compared
to control (18.31 ± 3.31%) (Fig. 7c). The indomethacin administration
significantly increased the effect of HEAb at 25 mg kg− 1 (14.23 ±
2.40%), a non-selective cyclooxygenase inhibitor. Similar results were
obtained by combining indomethacin with misoprostol (17.63 ±
2.28%). Thus, prostaglandins’ involvement in the gastroprotective effect
is performed by HEAb. In this sense, it is known that the protection of the
gastric mucosa by NO is due to the regulation of the synthesis of pros­
taglandins. The putatively identified components in HEAb, mainly
mannitol, were able to inhibit the release of gastric secretion, acting in
the preservation of epithelial cells, as well as in the protection of mice’s
gastric mucosa (Danai et al., 2021).
The investigation of the involvement of ATP-sensitive K+ channels
(Fig. 7d) in the gastroprotective effect of HEAb showed a high per­
centage of ulcerated area for the control group (18.31 ± 3.31%). The
area ulcerated by ethanol was significantly reduced with previous
administration of HEAb-25 (3.28 ± 0.72%, p < 0.01) and diazoxide (3
mg kg− 1, i.p.), (6.98 ± 1.01%, p < 0.05), when compared to the group
treated only with vehicle. The gastroprotection performed by HEAb-25
was not reversed by previous administration of glibenclamide (10 mg
kg− 1, i.p.), a drug responsible for blocking ATP-sensitive K+ channels
(8.07 ± 1.98%, p < 0,05). In contrast, the gastroprotection of diazoxide
was reversed by the action of glibenclamide (22.31 ± 2.71%). There­
fore, it can be inferred that the gastroprotective effect of HEAb is in­
dependent of the modulation of ATP-sensitive K+ channels. Thus, we can
assume that the gastroprotective effect of HEAb-25 is directly related to
its action on the nitric oxide (NO) and prostaglandin pathways (Fig. 7a
and c, respectively).
Prostaglandins and nitric oxide play a vital protective role in main­
taining cellular integrity in the mucosa through the stimulation of
mucus-bicarbonate secretion, regulation of mucosal cell renewal and
repair, and inhibition of leukocyte recruitment (Zakaria et al., 2016).
Our results converge with results evidenced in other studies with ex­
tracts rich in sugars and phenolic compounds, such as Sumbul et al.
(2011), suggesting that extract from F. vulgaris reduces induced gastric
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Journal of Ethnopharmacology 292 (2022) 115191
lesions by ethanol, due to its antioxidant properties, and also by stim­
ulating the synthesis of PGE2.
Other studies propose that cytoprotective properties of natural
compounds in the resolution of gastric ulcers are associated with
increased release of PGs and also showed that the hydroethanolic extract
from A. brachypoda (De-Faria et al., 2012), containing glycosylated
dimeric flavonoids, promotes cytoprotection and maintenance of gastric
homeostasis due to restoration of PG levels and, consequently, produc­
tion of gastric mucus (Da-Rocha et al., 2017). Abdelwahab (2013) stated
that gallic acid’s main mechanism of action as an antiulcer agent is due
to the promotion of mucosal protection by endogenous factors (NO,
PGE2 and tumor necrosis factor-α), inhibition of apoptosis induced by
oxidative stress and histamine release by mast cells.
Due to the multifaceted genesis of gastric ulcer, with several pro­
moting agents and responsible for the establishment of oxidative stress
and the inflammatory process, it becomes difficult to establish only one
mechanism of action responsible for solving this problem. Thus, we
suggest that HEAb gastroprotective effect is related to NO and PG
pathways and, therefore, decreased mastocytosis, oxidative stress and
inflammation in the injured tissue.
5. Conclusion
Putative identification by UPLC-QTOF-MSE suggested the presence
of mannitol, malic acid, pyroglutamic acid, L-agaritine and L-valine in
the HEAb. After identifying intense and characteristic signs of mannitol
in this extract by NMR, it was necessary to quantify it. HPLC analysis
confirmed mannitol as the main compound present in HEAb. The con­
tents of total phenols and flavonoids corroborated with the good in vitro
antioxidant activity of HEAb. HEAb-25 showed gastroprotective effect
demonstrated by reducing histopathological parameters and mastocy­
tosis in the mices’s stomach. In addition, it evidenced the role of HEAb-
25 in NO and PG pathways. Based on the results obtained, further
studies are needed on the possible anti-Helicobacter pylori effect of HEAb
and in vivo antioxidant assays. The investigation of the potential gas­
troprotective effect of polysaccharides from Agaricus blazei Murill it also
becomes necessary, since these biopolymers can have contributed to the
gastroprotection of HEAb.
Declaration of competing interest
Please check the following as appropriate:
● All authors have participated in (a) conception and design, or anal­
ysis and interpretation of the data; (b) drafting the article or revising
it critically for important intellectual content; and (c) approval of the
final version.
● This manuscript has not been submitted to, nor is under review at,
another journal or other publishing venue.
● The authors have no affiliation with any organization with a direct or
indirect financial interest in the subject matter discussed in the
manuscript
● The following authors have affiliations with organizations with
direct or indirect financial interest in the subject matter discussed in
the manuscript:
CRediT authorship contribution statement
João Francisco Câmara Neto: Conceptualization, Data curation,
Formal analysis, Investigation, Methodology, Writing – original draft.
Matheus da Silva Campelo: Data curation, Formal analysis, Investi­
gation, Methodology, Writing – review & editing. Gilberto Santos
Cerqueira: Funding acquisition, Investigation, Methodology, Re­
sources, Supervision, Writing – original draft, Writing – review & edit­
ing. João Antônio Leal de Miranda: Formal analysis, Methodology,
Supervision, Writing – review & editing. Jhonyson Arruda Carvalho
J.F. Câmara Neto et al.
Guedes: Data curation, Formal analysis, Methodology. Raimundo
Rafael de Almeida: Data curation, Formal analysis, Methodology.
Sandra de Aguiar Soares: Formal analysis, Methodology, Writing –
review & editing. Nilce Viana Gramosa: Formal analysis, Methodology,
Supervision, Writing – review & editing. Guilherme Julião Zocolo:
Funding acquisition, Investigation, Methodology, Resources, Supervi­
sion, Writing – original draft, Writing – review & editing. Ícaro Gusmão
Pinto Vieira: Formal analysis, Methodology, Supervision, Writing –
review & editing. Nágila Maria Pontes Silva Ricardo: Conceptualiza­
tion, Investigation, Funding acquisition, Resources, Writing – original
draft. Maria Elenir Nobre Pinho Ribeiro: Conceptualization, Funding
acquisition, Investigation, Project administration, Resources, Supervi­
sion, Writing – original draft, Writing – review & editing.
Acknowledgments
This work was funded in part by the Coordenação de Aperfeiçoa­
mento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001
(CAPES/PROEX PROCESS: 23038.000509/2020-82, Nº AUXPE: 1227/
2020), Conselho Nacional de Desenvolvimento Científico e Tecnológico
- Brazil (CNPq), (N.M.P.S. Ricardo, grant No 309795/2021-4), MCTI/
CNPq/Universal (M.E.N.P.R.-No 424179/2016-4), the Central Analítica-
UFC/CT-INFRA/MCTI-SISNANO/CAPES and The Center of Application
Northeast and Use of Nuclear Magnetic Resonance (CENAUREMN) for
the support.
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