Agaricus brasiliensis Sun mushroom

Journal of Nutrition and Food Science Research Agaricus brasiliensis (Sun mushroom): antioxidant capacity and HMG-CoA reductase inhibition potential of dried powder formulations in vitro and hepatic alterations in hypercholesterolemic rats Aline Mayrink de Miranda a *, Lorena Souza e Silva b , Jéssica Ariela Teixeira Costa c , Wanderson Geraldo de Lima d , Geraldo Célio Brandão e , Gustavo Hen- rique Bianco de Souza e , Marcelo Eustáquio Silva f

and Maria Lúcia Pedrosa d

a Pharmacognosy Laboratory, Department of Pharma - ceutical Sciences, University of Sã Paulo, Ribeirão Preto, Brazil

b Department of Research and Postgraduate, School of Medicinal, Dynamic Faculty of the Piranga Valley, Minas Gerais, Brazil

c Institutional Program for Scientific Initiation Scholar- ships/PROBIC/FAPEMIG/UFOP, Federal University of Ouro Preto, Minas Gerais, Brazil

d Department of Biological Sciences (DECBI), Federal Uni- versity of Ouro Preto, Minas Gerais,Brazil

e Department of Pharmaceutical Sciences, School of Pharmacy, Federal University of OuroPreto, Minas Gerais, Brazil

f Department of Foods, School of Nutrition, Federal Uni- versity of Ouro Preto, Minas Gerais,Brazil * Corresponding author Aline Mayrink de Mirandaa* Pharmacognosy Laboratory, Department of Pharmaceuti- cal Sciences,University of São Paulo, Ribeirão Preto, Brazil E-mail : [email protected] (A. M. MIRANDA) Received Date: April 19, 2022 Accepted Date: April 20, 2022 Published Date: May 19, 2022 Abstract This study investigues the chemical composition of the mushroom Agaricus brasiliens is prepared in different com- mercially available forms: dehydrated whole mushrooms, mushroom powder, and capsules. The most active form was subsequently examined in vivo to investigate its possible effects on liver tissue. For the in vitro assay, the antioxidant potential via DPPH radical and hypocholesterolemic through the activity of HMG-CoA reductase were analyzed. Regarding the in vivo test, 24 female albino Fischer rats (aged 80–95 days; average weight, 205 g) were fed either the AIN-93M modified standard diet (C) or a hypercholesterolemic dietcontaining 25 % soybean oil and 1 % cholesterol supplemented with mush- room powder (HAb) or simvastatin (HS). Animals received the diets for 8 weeks. After this period, the liver was separated for histopathological analysis. Their nutritional components were compared, as well as the antioxidant activity (DPPH free radical assay) and 2-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitory effect. All samples showed high contents of fiber and protein, plus low levels of lipid (1–2%) except for the capsule form (10.3%). They all inhibit- ed 2,2-diphenyl-1-picrylhydrazyl free radicals to a large extent (70–83%), thus demonstrating high antioxidant activity. Chem- ical characterization using HPLC revealed that tocopherol in dehydrated whole A. brasiliensis is a potent antioxidant and quenches the reactive oxygen species. The ethanolic extracts of samples were found to efficiently inhibit HMG-CoA reduc- tase expression. Significant changes were observed in the liver tissue of animals that had Agaricus brasiliensis and Simvasta- tin added to their diet. Our results, taken together, confirm the significant presence of bioactive compounds in Agaricus brasiliensis, reflecting its ability to act as a food with functional properties. These results reinforce the importance of comple- mentary studies to evaluate the conditions of cultivation and obtainment of the bioactive compounds and their possible interference in the composition and, consequently, in the bio- logical activity of Agaricus brasiliensis. Key words Agaricus brasiliensis;bioactive compounds;antioxidant activity; hypocholesterolemic activity; rats; hepatic steatosis. Abbreviations Agaricus brasiliensis, A. brasiliensis; 2-hydroxy-3-methylglu- taryl-coenzyme A (HMG-CoA) reductase, HMG-CoA reductase; 2,2-diphenyl-1-picrylhydrazyl, DPPH; (±)-6-hydroxy-2,5,7,8-te- tramethylchromane-2-carboxylic acid, Trolox; Association of Official Analytical Chemists, AOAC; Reactive oxygen species, ROS; low-density lipoprotein, LDL; neutral detergent fiber, NDF. Open Access 1www.directivepublications.org

Journal of Nutrition and Food Science Research Open Access 2www.directivepublications.org 1.Introduction Mushrooms have been used for thousands of years around the world as food and both preventative and curative med- icine. Nowadays, various mushrooms are finding new appli- cations as functional foods by combining their medicinal and culinary properties. However, data about the nutritional com- position of mushrooms mostly come from studies performed in Korea, China, and Japan, where mushrooms are widely consumed and affordable to most of the population. Howev- er, since the composition of mushrooms depends greatly on their growth environment and preparation method, there is a need to study mushrooms that are grown and used outside East Asia (Matila et al. 2000; Sales-Campos et al., 2009). The Agaricus brasiliensis mushroom is native to Brazil and considered a useful food supplement. For example, studies have shown that it displays anti-inflammatory and antioxi- dant effects (Oliveira et al., 2007; Savoie et al., 2008). It has been used in alternative medicine to treat cancer, diabetes mellitus, hyperlipidemia, atherosclerosis, chronic viral hepa- titis, and bacterial infections (Kimura et al., 2004). However, little information is available on the specific compounds that provide its antioxidant effect. Hypercholesterolemia is known to accelerate the devel- opment of cardiovascular diseases and the progression of atherosclerotic lesions. Reactive oxygen species (ROS) play a central role in the pathogenesis of hypercholesterolemia (Anandhi et al., 2013). Plant and fungi rich in phenolic com- pounds can reduce hypercholesterolemia due to their antiox- idant effect. In these organisms, the phenolic compounds are generated as secondary metabolites. Besides inhibiting free radicals, they have been shown to chelate metals and inhibit lipoxygenase (Decker, 1997). The most common, naturally oc- curring phenolic antioxidants include phenolic acids, tannins, flavonoids, and tocopherols (Angelo and Jorge, 2007). Flavo- noids are especially important due to their antioxidant, anti- platelet, anti-inflammatory, and antiallegenic effects. They may also inhibit certain enzymes related to tumorigene- sis such as lipoxygenase and cyclooxygenase. Flavonoids also have significant effects on macrophage-mediated low-density lipoprotein (LDL) oxidation. It is known that the capacity of macrophages to oxidize LDL depends on the oxidative state of the cell, which is determined in turn by the balance be- tween pro-oxidants and antioxidants. Flavonoids exert their effect by inhibiting cellular oxygenases and activating cellular antioxidants (Kim et al., 2004). The therapeutic potential of a food on cholesterol metab- olism could be evaluated by its effect on 2-hydroxy-3-meth- ylglutaryl-coenzyme A (HMG-CoA) reductase. Studies have shown that mushrooms of the genus Pleurotus can reduce blood cholesterol as they contain lovastatin, a pharmacolog- ical agent widely used to decrease serum cholesterol via in- hibiting HMG-CoA reductase (Gunde-Cimerman et al., 1995). Therefore, the effect of A. brasiliensis on cholesterol metabo- lism was examined here by evaluating its effect on HMG-CoA reductase. Mushrooms may be commercially available in raw or processed forms (canned or dried). Research has shown that the processing and storage conditions affect their com- position and therefore the functional properties (De Pauli, 2010). Initially, we tested the potential of Agaricus brasiliensis, given its characteristics, to act as a therapeutic target in the control of hyperlipedemia. However, the chemical composi- tion of the different commercial forms found of this partic- ular mushroom species, and even the way in which this composition could influence the biological activities, remain unclear and often with conflicting results. In this study, the chemical composition and therapeutic po- tential of A. brasiliensis were evaluated in three commonly available preparations (namely dehydrated whole mush - rooms, powder, and capsule), in order to evaluate the bioac- tive compounds with possible antioxidant and cholesterol-re- ducing activities. Additionally, the effects of the product that showed the best results in relation to its chemical character- ization was examined with respect to its potential as a func- tional food, specifically in relation to the effects on liver tissue. 2. Materials and Methods 2.1 In vitro test 2.1.1. Sample collection Whole dried mushrooms (Sample A) were obtained from the southern region of Minas Gerais, Brazil. According to the sup- plier, the cultivation method was adapted from that of Agari- cus bisporus (Champignon de Paris). The cultivation consisted of several phases: composting (phase I), pasteurization and conditioning of the substrate (phase II), inoculation and incu- bation (phase III), coverage of the colonized substrate with a cover layer (phase IV), and mushroom production and har- vesting (phase V). During the growing period, the average temperature ranged between 16 and 28 ºC. After harvesting, the mushrooms were cut in half lengthwise and dehydrated at a constant temperature (45 °C to 55 °C) for 6 h (Pascholati, 1998). The dehydrated mushrooms were then packaged and sold (Dias, 2010). Sample B was purchased as a powder in central Minas Gerais, and no information about the composition was provided. Sample C in capsule form was purchased from a natural food store in central Minas Gerais. According to the label, it was composed of dehydrated mushroom powder (A. brasiliensis Murril), gelatin capsule (gelatin hydrolysate 90.1% and purified water 9.9%), and silicon dioxide as antihumec- tant. The following nutritional information for one serving (1.8 g) was provided: 5 kcal/21 kJ, 1 g carbohydrates, 0 g protein, 0 g total fat, 0 g saturated fat, 0 g trans-fat, 0 g dietary fiber, and 0 g sodium. The gelatin capsule was discarded, and only the contents were used in this study.

Journal of Nutrition and Food Science Research Open Access 3www.directivepublications.org 2.1.2 Sample preparation The samples were dried in an oven at 60 °C, ground, packed in glass containers, and stored at room temperature until anal- yses. All analytical procedures were performed in triplicate.

2.1.3. Chemicals The following chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA): 2,2-diphenyl-1-picrylhydrazyl (DPPH), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and Folin-Ciocalteu reagent. All chemicals and sol- vents were of analytical grade. 2.1.4. Determination of nutritional composition of sam- ples Analyses were performed in the Laboratory of Bromatology, Department of Foods, Nutrition School, Federal University of Ouro Preto. The levels of moisture, ash, total lipids, protein, and fiber were measured in triplicate. The moisture, protein, and ash contents were determined according to the method published by the Association of Official Analytical Chemists (AOAC, 1984). Specifically, the moisture content was obtained by drying in an oven at 105 ºC for about 5 h to a constant weight, the protein content (N × 4.38) was estimated using the Kjeldahl method, and the ash content was determined in an oven at 550 °C. The lipid content was determined by ex- traction with petroleum ether in a Soxhlet apparatus, as de- scribed in the Analytical Standards of the Instituto Adolfo Lutz (Instituto Adolfo Lutz, 2008). The fiber content (cel- lulose, hemicelluloses, and lignin) was determined using neutral detergent fiber (NDF) in a fiber digestion apparatus (Tecnal TE-149, TECNAL, Piracicaba, Brazil) according to the manufacturer’s instructions. The carbohydrate content was calculated by difference. The energy content was calculated according to the equation: energy (kcal) = 4 × [mass of protein (g) + mass of carbohydrates (g)] + [9 × mass of lipids (g)]. 2.1.5. Chemical characterization of A. brasiliensis by HPLC-DAD-ESI-MS The analyses were performed using diode array high preci- sion liquid chromatography (Waters, Milford, MA, USA) with coupled diode detector (DAD), and a mass spectrometer equipped with electrospray ionization (ESI) operated under the following conditions: capillary voltage 3500 V; positive and negative ion mode; capillary temperature 320 °C; voltage source 5 kV; vaporizer temperature 320 °C; corona needle cur- rent 5 mA, and gas and nitrogen sheath of 27 psi. The full scan mode (100–2000u) was used. ESI-mass spectrometry (MS/MS) data were collected using an Acquity UPLC (Waters) system, with argon as the collision gas and a collision energy of 30 eV. Chromatographic separation was performed on Acquity BEH UPLC ™ RP-18 column (1.7 µM, 50 mm × 2 mm ID; Waters). The two eluent phases were (1) 0.1% formic acid in water (sol- vent A) and (2) 0.1% formic acid in acetonitrile (solvent B). The elution time was 0–11 min, with a linear gradient of 5% to 95% of solvent B. The flow rate was 0.3 mL/min-1, and the sam- ple injection volume was 4.0 mL. Ultraviolet (UV) absorption spectra were recorded from 190 to 450 nm. MS analysis was performed on the quadrupole instrument equipped with a negative mode electrospray source (ion spray voltage: -4 kV; orifice voltage: -60 V). 2.1.6. Sample preparation for antioxidant capacity assays Samples were extracted according to a previously reported procedure with some modification. Briefly, 80% methanol (10 mL) was added to the sample, and the mixture was stirred in a vortex apparatus for 2 min and then stood for 30 min. The supernatant was collected after centrifugation at 9279.4 × g for 8 min (Ramirez-Anguiano et al., 2007). 2.1.6.1. DPPH radical-scavenging ability The antioxidant capacity was determined using the free rad- ical DPPH according to the method described by Brand-Wil- liams, Cuvelier, and Berset (Brand-Willians, 1995). Trolox (6-hydroxy-2-5-7-8-tetrametilcromo-2-carboxylate acid; 1 mL in 80 mL of methanol) was used as a standard antioxidant. The antioxidant activity was measured in terms of light absor- bance (A) by the radical at 515 nm, and the inhibition percent- age was determined according to the equation below: % Inhi- bition of DPPH radical = (Acontrol – Asample/Acontrol) × 100

2.1.7. Determination of HMG-CoA reductase inhibition Mushroom powders (50 mg/mL) were mixed with water, eth- anol/water (1:1, v/v), ethanol, or methanol. The obtained sus- pensions were shaken in a vortexer for 1 min and centrifuged at 13362 × g for 2 min. HMG-CoA reductase activity was measured using the super- natant and commercial HMG-CoA Reductase Assay kit (Sigma, Madrid, Spain) according to the user’s manual. The assay is based on measuring the decrease in optical absorbance at 340 nm, which represents the oxidation of reduced nicotin- amide adenine dinucleotide phosphate (NADPH) by the cat- alytic subunit of HMG-CoA reductase in the presence of the substrate HMG-CoA. The mushroom supernatant (20 µL) was added to a 96-well plate, and the change in absorbance was monitored at 37 °C using a microplate reader (EL808, Elisa BioTek Instruments). Pravastatin was used as a positive con- trol. This analysis was only carried out for Sample A. 2.2.In vivo tests 2.2.1. Animals Twenty-four female albino Fischer rats were obtained (aged 80–95 days; average weight, 205 g) from the Experimental Nutrition Laboratory, Department of Food, School of Nutri- tion, Federal University of Ouro Preto. The rats were kept in individual cages in a room under controlled conditions (24 °C, 55 % humidity, 12-h light/dark cycle). During the experiment, food and water were available ad libitum. The experimental protocol for the use of animals was approved by the Ethics Committee on Animal Use of the Federal University of Ouro Preto (No. 2011/69).

Journal of Nutrition and Food Science Research Open Access 4www.directivepublications.org 2.2.2 Diet and experimental design Rats were fed either the AIN-93M modified standard diet (Reeves, Nielsen, Fahey, 1993) or a hypercholesterolemic diet containing 25 % soybean oil and 1 % cholesterol (Matos et al., 2005) supplemented with mushroom powder or simvasta- tin. The oil and cholesterol levels of the hypercholesterolemic diet were identified based on the work of Matos et al. (2005) and adapted for the AIN-93M diet. We divided the animals into four groups of 6 animals each. The control group (C) re- ceived the standard diet; the hypercholesterolemic group (H) received the hypercholesterolemic diet; the hypercholester- olemic plus simvastatin group (HS) received the hypercholes- terolemic diet plus simvastatin (in the form of macerated tab- lets); and the hypercholesterolemic plus A. brasiliensis group (HAb) received the hypercholesterolemic diet and A. brasilien- sis. The C group received the standard diet, and the H, HS, and HAb groups received the hypercholesterolemic diet in metabolic cages for 2 weeks prior to the 6-week experimental period when each group received their respective treatments. 2.2.3. Sample preparation After 6 weeks of treatment, the rats were fasted for 12 h, anesthetized with isoflurane (Cristália, Itapira, SP, Brazil), and euthanized by exsanguination. To determine the sérum component levels, blood samples were collected in 5-mL test tubes and centrifuged at 1500×g for 15 min. The livers of the animals were also removed, rinsed in saline solution, weighed, immersed in liquid nitrogen, and stored immediate- ly at −80 °C for subsequent analysis. 2.2.4. Histological analysis Liver tissue fragments were fxed in 10% bufered formalin for 72 h, dehydrated, cleared, and embedded in parafn. Tissue sections (4 µm) were cut using a microtome (Leica, Wetzlar, Germany), mounted on microscope slides, stained with Mas- son’s trichrome, and photographed at 400× magnifcation (Leica Application Suite) using a Leica DM5000 microscope coupled to a digital camera. Fibrosis and infammation quan- tifcation were assessed by evaluation of the total tissue area (1.5×106 μm2 ) using 15 images of randomly-selected felds of tissue sections, per animal, obtained using Leica QWin sof- ware. 2.3. Statistical analysis All data were analyzed using the Kolmogorov–Smirnov nor- mality test. Data that were consistent with a normal distribu- tion were evaluated by univariate analysis of variance (one- way ANOVA, followed by Dunnett’s or Bonferroni’s post-test) using GraphPad PRISM® software version 5 for Windows (San Diego, CA, USA). Differences were considered significant when p < 0.05. Data were expressed as the mean ± SD. For data that did not follow a normal distribution, the nonparametric Kru- skal–Wallis and Dunn’s post-tests were used. In this case, the data were presented as the median plus range. 3. Results 3.1. Sample composition Table 1 summarizes the wt.% composition of mushroom sam- ples. All samples showed high contents of fiber (30–50%) and protein (18–25%), while the ash and moisture contents were 6–7% and ~9%, respectively. We observed that all samples had low lipid levels (1 to 2%), except for Sample C (the capsule form, 10.3%). Table 1: Percentage composition of different formulations of A. brasiliensis. NUTRIENTS A B C Moisture and Volatile Ash Proteins† Lipids Fiber (NDF) Carbohydrates** Caloric value (kcal/100 g) 9.37 ± 0.06a 6.50 ± 0.01c 20.72 ± 1.24b 1.91 ± 0.02b 39.93 ± 1.44b 21.48 ± 1.12a 185.00±2.52c 9.03 ± 0.03b 6.32 ± 0.04c 18.80 ±0.46b 1.78 ± 0.15b 50.98 ±0.52a 13.09 ±0.70b 143.60±1.76d 9.03 ± 0.04b 7.47 ± 0.01a 20.39 ±1.20b 10.34 ±0.16a 30.19 ±1.51d 22.58 ±2.54a 264.90 ± 6.72a A: Whole dried mushroom; B: Powdered mushroom; C: Cap- sule form. * Analyses were performed in triplicate. Data are shown as the mean ± standard deviation NDF = neutral detergent fiber. † Conversion factor from nitrogen to protein: 4.38. ** Carbohydrates = 100- (% moisture + % protein + % lipids + % ash + % fiber. 3.2. Chemical characteristics Three fatty acids were identified from the mushrooms and represented by peaks 2, 3, and 5 (Table 2 and Figure 1). They are proposed to be unsaturated fatty acids. Mannitol and a tocopherol derivative were also tentatively identified (peaks 1 and 4 respectively, Table 2). Their UV absorption spectra are shown as insets in Figure 1. The mass spectra obtained were compared with data from the literature. Searches were per- formed in the available databases by the mass/charge ratio (m/z) obtained in the spectra.

Journal of Nutrition and Food Science Research Open Access 5www.directivepublications.org Table 2: Compounds identified in whole dried A. brasiliensis mushroom by HPLC-DAD-ESI-MS. Peak Compound FormulaTR (min) UV (nm) LC-MS [M-H]- (m/z) 1 2 3 4 5 Mannitol Fatty acid Fatty acid 5.7-Bis(hy- droxymethyl)-- tocopherol Fatty acid C6H13O6 C17H33O5 C18H31O3 C27H45O4 C18H31O2 0.68 4.73 6.88 7.18 8.61 - - - 222.1; 276.1 - 181.1462 329.5222 295.5479 433.4049 279.3448 * TR: retention time in minutes, UV: ultraviolet absorption peak wavelength in nanometers, LC-MS [M-H]- (m/z):liquid chromatography coupled to negative mode mass spectrom- etry, mass/charge ratio. Figure 1: HPLC-DAD-ESI-MS profile of whole dried A. brasilien- sis mushroom. Insets: UV absorption spectra (190 to 450 nm) of 1 (mannitol) and 4 (5,7-bis(hydroxymethyl)-α-tocopherol). 3.3. Antioxidant capacity All samples of A. brasiliensis were found to significantly neu- tralize the free radical DPPH, with the ability ranging from 70% in Sample C to 83% in Sample A (Figure 2). Figure 2: Antioxidant activity of the different formulations of A. brasiliensis measured by DPPH scavenging activity assay. 120

* 100 80 60 40 20 0 30’ TROLOX A B C *Data are shown as the mean ± standard deviation. Analyses were performed in triplicate using 0.1 g/mL A. brasiliensis and 1000 µM Trolox. A: Whole dried mushroom; B: Powdered mushroom; C: Capsule form; DPPH: 2,2-diphenyl-1-picrylhy- drazyl. * p<0.05 vs. Trolox. 3.4. A. brasiliensis as a source of HMG-CoA reductase in- hibitors Only the extracts of Sample A were evaluated for HMG-CoA reductase inhibition, since this sample was found to have the most promising nutritional composition and antioxidant ac- tivity. The inhibition of HMG-CoA reductase varied significant- ly (~22 to ~73%) depending on the extraction solvent (Figure 3), being the highest in the ethanol extract and lowest in the aqueous extract. Figure 3: HMG-CoA reductase inhibitory activities of A. brasil- iensis extracts obtained using whole dried mushroom in eth- anol/water (1:1, v/v), ethanol, water, and methanol.

0 20 40 60 80 100 120 Inhibition % of DPPH radical Ethanol/Water (1:1,v/v) Ethanol Water Methanol Pravastatin HMG-CoA reductase inhibitory activity (%) 1 5 3 2 4 (1) (4)

Journal of Nutrition and Food Science Research Open Access 6www.directivepublications.org * ( )Hydroalcoholic extract; ( ) Ethanolic extract; ( ) Aqueous extract; ( ) Methanolic Extract; ( ): Pravastatin. Analyses were done in triplicate. 3.5. Effects of Agaricus brasiliensis on liver tissue Histological sections of the liver stained by H&E showed that the C group presented a histological aspect compatible with the normality of the organ, as can be seen in Figure 4. The hypercholesterolemic (Fig.4) and Hypercholesterolemic + A. brasiliensis (Fig.4) groups showed moderate presence of hepatocytes presenting macrovesicular steatosis and intense presence of microvesicular steatosis. On the other hand, the hypercholesterolemic + Simvastatin group (Fig.4) showed a moderate to severe presence of microvesicular steatosis, fol- lowed by a slight presence of hepatocytes in macrovesicular steatosis. Figure 4: Photomicrographs of liver histological sections stained with Hematoxylin and Eosin. Arrowhead: Macrove- sicular steatosis; Arrow: Microvesivular steatosis. C: Control group (A); H: Hypercholesterolemic Group (B); HAb: Hyper- cholesterolemic group + 1% of Agaricus brasiliensis (C); HS: Hy- percholesterolemic group + 0.008% Simvastatin (D). Presence of steatosis: 0, none; 1, 10%; 2, 10 to 33%; 3, 33 to 66% and 4, >66%. Different superscript letters indicate a statistically sig- nificant difference between columns (p < 0.0001). Data analyzed using the chi-square test. Bar = 50 micrometers. ANOVA followed by the Bonferroni’s test. 4. Discussion Foods containing functional nutrients are in high demand, as they combine nutrition with health benefits (George et al.,2005). Edible mushrooms typically contain significant amounts of protein and carbohydrate and low levels of fat, making them nutritious especially compared to animal prod- ucts (Toor and Savage, 2006; Brito and Volp, 2010). However, as previously mentioned, there are only limited data regard- ing the composition of mushrooms that are cultivated in Bra- zil. Other researchers have examined the nutritional contents in A. brasiliensis, which are affected by numerous factors. The growing conditions, soil, climate, and the choice of substrate appear to be the most important ones according to Furlani and Godoy (2005). Additionally, there are choices in the cor- rection factor used to calculate the protein content from the organic nitrogen content. A value of 6.25 is used for most foods, i.e., assuming that proteins are fully digestible and con- tain 16% nitrogen. Nitrogen could exist in forms other than proteins, however that is usually negligible in most foods. Since mushrooms have a significant amount of chitin in their cell walls, a different correction factor should be used (Furlani and Godoy, 2005). Here, we used a value of 4.38, which as- sumes that only 70% of nitrogenous compounds in the mush- room are digestible by the human body. Using the correction actor of 6.25, Oliveira, Oliveira, Lima, and Villas Boas (1999) measured the protein percentage of dried A. brasiliensis to be 30.13%. Chemical analysis of the same mushroom by Cel- so showed similar values (28.94–35.88%), although the cor- rection factor was not specified there (Eira, 2003). Their data showed that A. brasiliensis has a higher protein content than Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). These values also varied according to the strain used and whether the basidiomata stadium was open or closed. Pedroso and Tamai (2001) found that dehy- drated A. brasiliensis basidiocarp contains 40 to 45% protein. Here, our value of 18–25% contradicts the findings of Pedroso and Tamai (2001) but is similar to that found for L. edodes (19.49%) (Eira, 2003). Edible mushrooms generally have a low lipid content (less than 5% dry weight). The values found in our study (0.9 to 1.9%) were similar to those reported by other authors for both the same species (Oliveira et al., 1999) and Pleurotus spp (Sturion and Oetterer, 1995). The exception was Sample C, which contained about 10% lipid. This value was high com- pared to those reported in a recent study for A. brasiliensis and L. edodes capsules (1.82% and 1.01%, respectively) (Car- neiro et al., 2013). Besides water, carbohydrates are the most abundant compo- nent in mushrooms, and their total content varies according to the species (typically 35–70% dry weight) (Diez and Alvarez, 2001; Longvah and Deosthale, 1998; Mau et al., 2001). In most of the literature, the carbohydrate content in mush- rooms is calculated by difference (Manzi et al., 2001; Yang Macrovesicular

Microvesicular a ab bb dc c

Journal of Nutrition and Food Science Research Open Access 7www.directivepublications.org et al., 2001). The results in our study were 13–22%, which do not agree with the results of other studies (34.78% and 38.3%) (Oliveira et al., 1999; Eira, 2003). Our values are also lower compared to those of A. bisporus (54.12%), L. edodes (69.58%), and Pleurotus spp. (65.82%) (Furlani and Godoy, 2005). Further, our phytochemical analysis using HPLC identified mannitol in A. brasiliensis. This finding corrobo- rates the literature reports that polyols such as glucose, tre- halose, mannitol, and arabinitol are the main sugars in the fruiting bodies of edible mushrooms (Barros et al., 2008; Cai et al., 2013; Martins, Reis, Ferreira, 2021). Reported fiber contents in literature vary a great deal due to the use of several different methodologies. However, many studies do not mention the method used, and so a compari- son is difficult. According to Mizuno (2002), A. brasiliensis con- tains on average of 20–28% dietary fiber that includes β-glu- cans, chitin, hemicelluloses, and pectin. Using the method of Weende (which only measured the cellulose and insoluble lig- nin fractions), Oliveira and collaborators (1999) found a fiber content of 14.57% for the same species. In comparison, this study employed the neutral detergent method that accounts for cellulose, hemicellulose, and lignin. Given the impor- tance of fiber in the diet, the choice of fiber-testing method is important for minimize possible errors. Recently, we mea- sured the amount of total dietary fiber in whole dehydrated mushrooms by the gravimetric enzymatic method, and the results revealed a fiber content of about 24% (3.64% soluble fraction and 20.4% insoluble) (De Miranda et al, 2015). These data highlight the need for a careful description of the chosen measurement method, which should be appropriate for the specific food matrix. From the above discussions, there is a great deal of variation in the reported chemical composition of A. brasiliensis, and so further work is needed to determine it more definitively. In addition, variations resulting from the processing should also be considered, as temperature and humidity can interfere di- rectly with the composition and quality of the product. Rufino and collaborators (2007) discussed numerous possible methods to measure the antioxidant capacity, such as perox- yl radical capture [oxygen radical absorbance capacity assay (ORAC) and total peroxy radical-trapping antioxidant poten- tial assay (TRAP)], metal reduction [ferric reducing ability of plasma assay (FRAP) and cupric reducing antioxidant capac- ity assay (CUPRAC)], hydroxyl radical capture (deoxyribose method), organic radical capture [2,2’- azino-bis(3-ethylben- zothiazoline-6-sulphonic acid) (ABTS) assay and DPPH assay], and quantification of the products formed during lipid perox- idation [thiobarbituric acid reactive substances (TBARS) oxi- dation of LDL, oxidation of the co-carotene]. In this study, the DPPH assay was used. In this method, the analyte is added to a methanolic solution of DPPH radicals, whose subsequent reaction can be monitored by the decrease in absorbance at 515 nm.(Brand- Willians, 1995). The results are expressed as the percentage reduction of DPPH radical concentra- tion. Our data showed that A. brasiliensis was effective in re- ducing the DPPH radicals, and that the efficacy depends on the commercial form. These results are consistent with those found by Savoie and collaborators (2008), who reported that A. brasiliensis had higher antioxidant potential than the other mushroom species tested. We found that the the more processed mushroom samples (i.e. powder and capsule) had lower concentrations of phe- nolic compounds and flavonoids than the whole ones (data not shown). The content of phenolic compounds can vary greatly due to their rapid degradation by oxidation (Carneiro et al., 2013). Therefore, a lower phenolic content would be expected in the more processed foods. Furthermore, pheno- lic compounds are known to be secondary metabolites, and their production is influenced by the fungal growth conditions (Di Pauli, 2010). This may also partly explain the variation between the different samples. Palacios and collaborators (2011) evaluated the antioxidant activity of eight species of edible mushrooms (A. bisporus, Boletus edulis, Calocybe gambosa, Cantharellus cibarius, Craterellus cornu - copioides, Hygrophorus marzuolus, Lactarius delicious, and Pleurotus ostreatus) and found their phenolic contents to vary from 1 to 6 mgGAE/g, being the highest in B. edulis. Although our work did not determine the exact structure of the phenolic compounds, studies have suggested that the an- tioxidant property of A. brasiliensis is associated with a high concentration of tocopherols (Tsai et al., 2007). A positive correlation had been reported between antioxidant activity and phenolic content in mushrooms (Tsai et al., 2007; Yen et al., 1993; Cheung and Cheung, 2005). This correlation was also observed in our study for the three kinds of mush- room samples. However, phytochemical analysis of A. brasil- iensis by HPLC performed in this study revealed a different profile from previous descriptions for this and other mush- room species. Specifically, we identified the tocopherol de- rivative (5,7-bis(hydroxymethyl)-δ-tocopherol), while Tsai and collaborators (Tsai et al., 2007) found a significant amount in tocopherol in aqueous extracts of A. brasiliensis. For these authors, the observed antioxidant activity was directly relat- ed to the phenolic content, especially the tocopherol content. Regarding the flavonoid content, no UV or MS peaks associ- ated with these compounds were observed. Flavonoids have been identified in some fungal species such as Aspergillus candidus, Phallus impudicus, and Fistulina hepatica (Ribeiro et al., 2007), however animals and fungi are not capable of synthesizing these compounds themselves (Iwashina et al., 2000). Some authors suggested that only plants have the biosynthetic pathway for producing flavonoids. For example, Barros et al. (2009) analyzed 16 edible mushroom species and found significant amounts of phenolic compounds, mostly phenolic acids, by HPLC-DAD-ESI/MS. However, the presence of flavonoids was not identified by this method (Barros et al. 2009).

Journal of Nutrition and Food Science Research Open Access 8www.directivepublications.org An increasing number of studies have demonstrated the cholesterol-lowering effects of various mushrooms (Bobek et al., 1993; Cheung, 1996; Cheung, 1998; Bobek et al., 1998; Fukushima et al., 2001; Berger et al., 2004). In general, the presence of substances such as lovastatin and eritadenina makes these fungi important in searching for treatments of cardiovascular diseases (Guillamón et al., 2010). In A. brasilien- sis, several phenolic compounds along with the β-glucans and chitin are considered responsible for the cholesterol- lower- ing effect. These phenolic compounds may reduce the risk of cardiovascular diseases by regulating lipid metabolism and preventing hypercholesterolemia. Kim and collaborators (2004) reported the antioxidant effects of lovastatin and the flavonoid naringin. According to their study, naringin was able to increase the activity of some antioxidant enzymes (super- oxide dismutase, catalase, and glutathione reductase) and at the same time reduce HMG-CoA reductase activity by about 30%. Mushrooms of the genus Pleurotus are considered po- tential producers of lovastatin—a potent inhibitor of HMG- CoA reductase. Kim and collaborators (2004) had measured the inhibitory effect of the methanol extracts. The amount of lovastatin varies depending on the age and the specific part of the mushroom. In the early growth stage, it is uniformly dis- tributed in small sporocarps, and thus there is a substantial difference in concentration between the pileus and the stipe. However, as the mushroom grows, this difference can change (Pascholati, 1998). In our study, only the HMG-CoA reductase inhibition by Sample A was measured, since the whole dried mushrooms showed the highest antioxidant effect. This en- zyme inhibition was generally between 22% (aqueous extract) to 70% (ethanol extract). Therefore, the solvent choice has a large effect on the amount of active compound extracted. This result differs from a previous study, which reported that methanol could effectively extract the compounds required for strong HMG-CoA reductase inhibition (Gil- Ramirez et al., 2011). Additionally, many studies demonstrate the therapeu- tic potential of A. brasiliensis aqueous extracts, and lovasta- tin is expected to be less soluble in water than in methanol. Therefore, it is especially surprising that our aqueous and methanol extracts were ineffective. Regarding to tests to verify its biological potential, the addition of Agaricus brasiliensis to the diet promoted significant chang- es in the liver tissue, especially with regard to macrovesicular steatosis. Nakamura et al., when evaluating the supplemen- tation of mice submitted to a diet high in fat and cholesterol for 21 weeks with Agaricus brasiliensis KA21 (AGA) found an attenuation in the development of hepatic fibrosis, which is a characteristic feature of late-stage NASH. The authors as- sociated this change with the reduction in hepatic oxidative stress induced by the administration of AGA. Yamanaka et al. (2014) found that the way the mushroom is cultivated (indoor or outdoor) directly interferes not only in its composition, but also in its ability to act in the protection of liver tissue. In this study, outdoor cultivation significantly enhanced the antiox- idative activity of A. brasiliensis fruiting bodies, being more effective at protecting against CCl4-induced liver injury than mushrooms grown in a greenhouse. The administration of A. brasiliensis hydroalcoholic extract to rats represents not only the ingestion of metabolic precursors, but also the in- gestion of substances that, even at low concentrations, can exert important signaling functions in the liver as well as in the organism as a whole. Although the study by de Oliveira et al. (2010) was carried out in part on animals on a standard diet, their research helps us to understand our results. The dose used, the form of administration of the mushroom and even the duration of the experiment are directly associated with the data found in our study. Although previous studies carried out by our group together with this group have sug- gested Agaricus brasiliensis as a possible therapeutic target for hyperlipidemias, studies are needed to establish the best way to use it so that it can exert its full biological potential. 5. Conclusions We have shown that the chemical composition of A. brasilien- sis varies greatly between differently processed samples and between different studies. In our work, the largest difference from the literature was the much higher lipid content in the capsule form. However, our results confirmed that A. brasil- iensis has significant nutritional value, being high in protein and fiber and low in fat. Furthermore, this mushroom exhibit- ed significant hypocholesterolemic and antioxidant potential, as well as proved to be an important source of phenolic com- pounds (notably tocopherol). These characteristics are de- pendent of the muchroom preparation process. Dehydrated whole mushrooms were the most effective and had a higher antioxidant content than the powdered and capsule forms. This work supports the use of A. brasiliensis as a functional food, and highlights the effects of different preparation meth- ods on the medicinal effectiveness of mushrooms. CRediT authorship contribution statement Aline Mayrink de Miranda: conceptualization; data curation; formal analysis; investigation; writing - original draft method- ology; writing - review and editing. Lorena Souza e Silva: data curation; formal analysis; investi- gation; methodology. Jéssica Ariela Teixeira Costa: helped in preparation of mush- rooms and execution of research. Wanderson Geraldo de Lima: helped in execution of histo- pathological analysis. Geraldo Célio Brandão and Gustavo Henrique Bianco de Sou- za: helped in execution of physico-chemical methods. Marcelo Eustáquio Silva: helped in the experimental design and in the supply and handling of the animals and execution of research. Maria Lúcia Pedrosa: conceptualization and supervision. Acknowledgments The authors thank the Federal University of Ouro Preto, and acknowledge the financial support provided by the follow- ing Brazilian government agencies: Fundação de Amparo à

Journal of Nutrition and Food Science Research Open Access 9www.directivepublications.org Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Na- cional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Capacitação de Pessoal de Nível Superior (CAPES/Brazil). Conflict of Interests The authors declare no conflict of interest. References 1. Anandhi R et al. Antihypercholesterolemic and antiox- idative effects of an extract of the oyster mushroom, Pleurotus ostreatus, and its major constituent, chry- sin, in Triton WR-1339-induced hypercholesterolemic rats. J Physiol Biochem 2013; 69:00:00 313–323. https.: //doi:10.1007/s13105-012-0215-6. 2. Angelo PM, Jorge N. Phenolic compounds in foods – A brief review. Revista do Instituto Adolfo Lutz 2007; 66: 1–9. 10. 3. Association of Official Analytical Chemists (AOAC). Of- ficial methods of analysis of the Association of Official Analytical Chemists. 14th ed. Arlington: AOAC, 1984. 4. Barros, L et al. Antioxidante activity of Agaricus sp. Mushrooms by chemical, biochemical and electro- chemical assays. Food Chemistry 2008; 111(1): 61-66. 5. Barros, L et al. Phenolic acids determination by HPLC–DAD–ESI/MS in sixteen diferente Portuguese wild mushrooms species. Food and Chemical Tox- icology 2009; 47: 1076-1079. https:// doi: 10.1016/j. fct.2009.01.039. 6. Berger A et al. Cholesterol-lowering properties of Ganoderma lucidum in vitro, ex vivo, and in hamsters and minipigs. Lipids Health Dis 2004; 3: 2. https:// doi: 10.1186/1476-511X-3-2. 7. Bobek P et al. Dose-and time-dependent hypocho- lesterolemic effect of oyster mushroom (Pleurotus ostreatus) in rats. Nutrition 1998; 14(3): 282–286. https:// doi: 10.1016/s0899- 9007(97)00471-1. 8. Bobek P et al. Oyster mushroom (Pleurotus ostreatus) accelerates the plasma clearance of low- density and high-density lipoproteins in rats. Nutr Res 1993; 13: 885–890. 9. Brand-Willians W et al. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci Technol 1995; 28: 25–30. 10. Brito JC, Volp ACP. Cisteína como composto de rele- vância nutricional. Nutrição em Pauta 2010; 18(105): 20–24. 11. Cai, H et al. Isolation, purification and identification of nine chemical compounds from Flammulina veluti- pes fruiting bodies. Food Chemistry 2013; 141: 2873- 2879. https:// doi: 10.1016/j.foodchem.2013.05.124. 12. Carneiro AA et al. Chemical composition and an- tioxidant activity of dried powder formulation of A. blazei and Lentinus edodes. Food Chem 2013; 138: 2168–2173. https:/ doi: 10.1016/j.food- chem.2012.12.036. 13. Cheung LM, Cheung PCK. Mushrooms extracts with antioxidant activity against lipid peroxidation. Food Chem 2005; 89: 403–409. 14. Cheung PCK. Plasma and hepatic cholesterol levels and fecal neutral sterol excretion are altered in ham- sters fed straw mushroom diets. J Nutr 1998; 128: 1512–1516. https:// doi: 10.1093/jn/128.9.1512. 15. Cheung PCK. The hypocholesterolemic effect of two edible mushrooms: Auricularia auricula (Tree-ear) and Tremella fuciformis (White jelly-leaf) in hypercholes- terolemic rats. Nutr Res 1996; 16(10): 1721–1725. 16. De Miranda AM, Rossoni Júnior JV, Souza E Silva L, Dos Santos RC, Silva ME, Pedrosa ML. Agaricus brasil- iensis (sun mushroom) affects the expression of genes related to cholesterol homeostasis. Eur J Nutr. 2017;56(4):1707-1717. https:// doi:10.1007/s00394- 016-1217-x 17. De Pauli PA. Avaliação da composição química, com- postos boativos e atividade antioxidante em cogume- los comestíveis. Dissertação. (Mestrado em Ciências dos Alimentos) - Faculdade de Ciências Farmacêuti- cas, Universidade Estadual Paulista “Júlio de Mes- quita Filho”, Araraquara, 2010 (dissertation). 18. Decker EA. Phenolics: prooxidants or antioxidants? Nutr Rev 1997; 55: 396–398. https:// doi: 10.1111/ j.1753-4887.1997.tb01580.x. 19. De Oliveira AL, Eler GJ, Bracht A, Peralta RM. Puriner- gic effects of a hydroalcoholic Agaricus brasiliensis (A. blazei) extract on liver functions. J Agric Food Chem. 2010 Jun 23;58(12):7202-10. https://doi: 10.1021/jf100804k. PMID: 20507067. 20. Dias ES. Mushroom cultivation in Brazil: Challenges and potential for growth: Revision. Ciênca e Agrotec- nolgia 2010; 34: 795–803. 21. Díez VA, Alvarez A. Compositional and nutritional studies on two wild edible mushrooms from north-

Journal of Nutrition and Food Science Research Open Access 10www.directivepublications.org west Spain. Food Chem. 2001; 75(4): 417–422. 22. Eira AF. Importância dos cogumelos. Cultivo do Cogu- melo Medicinal. Campina Grande: Aprenda Fácil, 2003. 23. Fukushima M et al. Cholesterol-lowering effects of Maitake (Grifola frondosa) fiber, Shiitake (Lentinus edodes) fiber, and Enokitake (Flammulina velutipes) fiber in rats. Exp Biol Med 2001; 226(8): 758–765. https:// doi: 10.1177/153537020222600808. 24. Furlani RPZ, Godoy HT. Nutritional value of edible mushrooms: a revision. Revista do Instituto Adolfo Lutz 2005; 64: 149–154. 25. George S et al. Rapid determination of polyphenols and vitamin C in plants-derived products. J Agric Food Chem 2005; 53: 1370–1373. https:// doi: 10.1021/ jf048396b. 26. Gil-Ramirez A et al. Edible mushrooms as potential sources of new hypocholesterolemic compounds. Proceedings of the 7th International Conference on Mushroom Biology and Mushroom Products 2011; 110–119. 27. Guillamón E et al. Edible mushrooms: Role in the pre- vention of cardiovascular diseases. Fitoterapia 2010; 81: 715–723. https:// doi: 10.1016/j.fitote.2010.06.005. 28. Instituto Adolfo Lutz. Normas analíticas do Instituto Adolfo Lutz: métodos químicos e físicos para anális- es de alimentos, 4th ed. Brasília: Instituto Adolfo Lutz, 2008. 29. Iwashina, T. The structure and distribution of the fla- vonoids in plants. Journal of Plant Research 2000; 13 (3): 287-299. 30. Kim SH et al. Anti-inflammatory and related phar- macological activities of the n-BuOH subfraction of mushroom Phellinus linteus. J Ethnopharm 2004; 93(1): 141–146. https:// doi: 10.1016/j.jep.2004.03.048. 31. Kimura Y et al. Isolation of an anti-angiogenic sub- stance from A.blazei Murill: Its antitumor and anti- metastitic actions. Cancer Sci 2004; 95: 758–764. https:// doi: 10.1111/j.1349- 7006.2004.tb03258.x. 32. Kubo K, Nanba H. Anti-hyperliposis effect of maitake fruit body (Grifola frondosa). Biol Pharm Bull 1997; 20: 781–785. https:// doi: 10.1248/bpb.20.781. 33. Longvah T, Deosthale YG. Compositional and nutri- tional studies on edible wild mushroom from north- east India. Food Chem 1998; 63(3): 331–334. 34. Manzi P et al. Nutritional value of mushrooms widely consumed in Italy. Food Chem 2001; 73(3): 321–325. 35. Mattila P et al. Functional properties of edible mush- rooms. Nutrition 2000; 16: 694–696. https:// doi: 10.1016/s0899-9007(00)00341-5. 36. Mau JL et al. Non-volatile taste components of several specialty mushrooms. Food Chem 2001; 73(4): 461– 466. 37. Mizuno T. Medicinal properties and clinical effects of culinary-medicinal mushroom A. blazei Murrill (Agar- icomycetideae). Int J Med Mushrooms 2002; 4: 299– 312. 38. Oliveira ECM et al. Composição centesimal do Cogu- melo do Sol (A. blazei). R Uni Alfenas 1999; 5: 169–172. 39. Oliveira OMMF et al. Antioxidant activity of A. blazei. Fitoterapia 2007; 78: 263–264. https:// doi: 10.1016/j. fitote.2007.01.002. 40. Palacios I et al. Antioxidant properties of phenolic compounds occurring in edible mushrooms. Food Chem 2011; 128: 674–678. 41. Pascholati S. Cogumelos: cultivo e comercialização (Shiitake e Cogumelo do Sol). Mato Grosso, SEBRAE, 1998. 42. Pedroso A, Tamai F. Análise e composição química do Agaricus Blazei Murril. Apostila de estudo da USP – Faculdade de Ciências Farmacêuticas, 2001. 43. Ribeiro, B et al. Phenolic compounds, organic acids profiles and antioxidative properties of beefsteak fun- gus (Fistulina hepatica). Food and Chemical Toxicolo- gy 2007; 45(10): 1805- 1813. https:// doi: 10.1016/j. fct.2007.03.015. 44. Savoie J-M et al. Radical-scavenging properties of ex- tracts from the white button mushroom. Agaricus bis- porus. J Sci Food Agric 2008; 88: 970–975. 45. Toor RK, Savage, GP. Effect of semi-drying on the an- tioxidant components of tomatoes. Food Chem 2006; 94: 90–97. 46. Tsai S-Y et al. Antioxidant properties of A. blazei, Agro- cybe cylindracea, and Boletus edulis. LWT – Food Sci Technol 2007; 40: 1392–1402. 47. Yang J-H et al. Non-volatile taste components of sev-

Journal of Nutrition and Food Science Research Open Access 11www.directivepublications.org eral commercial mushrooms. Food Chem 2001; 72(4): 465–471. 48. Yen C-C, Duh P-D. The relationship between antioxi- dant activity and maturity of peanut hulls. J Agric Food Chem 1993; 41: 67–70.

This is an automatically generated text version. For the formatted version of record, download the PDF →