Supercritical fluid extraction of Agaricus brasiliensis: Antioxidant and antimicrobial activities

J. of Supercritical Fluids 70 (2012) 48–56

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Supercritical fluid extraction of Agaricus brasiliensis: Antioxidant and antimicrobial activities Simone Mazzutti a , Sandra R.S. Ferreira a , Carlos A.S. Riehl b , Artur Smania Jr. c , Fabio A. Smania c , Julian Martínez d,∗ a

Chemical and Food Engineering Department, Federal University of Santa Catarina, EQA/UFSC – C.P. 476, CEP 88040-900, Florianópolis, SC, Brazil Chemistry Institute, Federal University of Rio de Janeiro, IQ/UFRJ – C.P. 68535, CEP 21941-972, Rio de Janeiro, RJ, Brazil c Microbiology and Parasitology Department, Federal University of Santa Catarina, CCB/UFSC, Brazil d School of Food Engineering, University of Campinas, FEA/UNICAMP, CEP 13083-862, Campinas, SP, Brazil b

a r t i c l e

i n f o

Article history: Received 13 March 2012 Received in revised form 18 June 2012 Accepted 19 June 2012 Keywords: Supercritical fluid extraction Antioxidant activity Antimicrobial activity Agaricus brasiliensis

a b s t r a c t The objective of this study was to obtain extracts from the mushroom Agaricus brasiliensis using supercritical fluid extraction (SFE) with pure CO2 and with CO2 plus 2.5%, 5.0% and 10.0% (w/w) of ethanol as co-solvent. In order to evaluate the high-pressure method in terms of process yield, extract composition and biological activity, low-pressure methods, such as maceration with ethanol (Mac), Soxhlet (Sox) with different organic solvents, and hydrodistillation (HD), were also applied to obtain extracts. The SFE conditions were temperatures of 313.15 K, 323.15 K and 333.15 K and pressures from 10 to 30 MPa. The SFE kinetics was investigated through the overall extraction curve (OEC). The extracts obtained by Sox with water and ethanol showed the best results for the global extraction yield. The best conditions in the studied range to obtain high yields using pure CO2 resulted to be 30.0 MPa and 323.15 K. The antioxidant potential of the extracts was evaluated by the DPPH method and by the Folin–Ciocalteau method. Maceration extract presented low yield, but good results of antioxidant activity by DPPH assays and total phenolic. The antimicrobial activity of the extracts was also studied. The main identified compounds in the extracts were linoleic and palmitic acids. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Mushrooms are high value food products, with low energetic levels and large amounts of minerals, essential amino acids, vitamins and fibers. Agaricus brasiliensis is mushroom that reached the top ranking of the best medicinal and culinary mushrooms [1]. It was formerly known in the literature as Agaricus blazei Murril (sensus Heinemann), but after the clarification of its botanical name, many publications refer to A. brasiliensis as the cultivated mushroom originated from Brazil [2,3]. This mushroom has been widely studied in the areas of food science, medicine, biotechnology and pharmacology [3]. Concerning medical aspects, many works have reported that A. brasiliensis presents antibacterial [4], antioxidant [5–7], antidiabetic [8], antiangiogenic [9], and anticancer [10–13] activities. Extracts from vegetable sources and mushrooms have important roles on the compositions of high added value food products and supplements, as well as in cosmetics and pharmaceuticals. In the recent years consumers have increased their concerns about

∗ Corresponding author. Tel.: +55 1935214046; fax: +55 1935214027. E-mail address: [email protected] (J. Martínez). 0896-8446/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2012.06.010

health, searching for natural products of high quality and free from organic solvent traces. Furthermore, the legislation restrictions have been increased, since accidents involving toxic substances represent risks to environment and to human health. This context has created a demand for processes that should be able to minimize environmental impacts, by reducing or completely eliminate the discard of toxic residues. Supercritical fluid extraction (SFE), which uses high-pressure solvents, is considered a clean technology, since the extracts obtained with this process have high purity. SFE with carbon dioxide (CO2 ) has many advantages when compared to other separation techniques, such as: (1) the solvent can be easily removed through pressure reduction or temperature elevation; (2) thermally sensible compounds can be separated at low temperatures; (3) the heat demand is much lower than in a distillation process; (4) the extraction is fast due to the solvent’s low viscosity, high diffusivity and solvation power [14]. Concerning processes that use supercritical fluids, carbon dioxide is often the preferred solvent. Carbon dioxide offers many advantages over other solvents, since it is cheap, nonflammable, chemically inert, and without risk of explosion, being able to produce natural extracts with high purity [15]. Supercritical carbon dioxide extraction represents an alternative to classic organic solvent extraction of plants material and

S. Mazzutti et al. / J. of Supercritical Fluids 70 (2012) 48–56

mushrooms. Abdullah et al. [16] studied the supercritical fluid extraction of carboxylic and fatty acids from Agaricus spp. mushrooms. SFE resulted in the characterization of acids not previously reported from theses sources. Coelho et al. [17] studied the supercritical CO2 extraction of lipids, from A. blazei, and the influence of several parameters, namely pressure, temperature and flow rate. The best extraction conditions of secondary metabolites, whereby the degree of solubilization is the highest, were obtained with pure CO2 at 40 MPa, 70 ◦ C and a flow rate of 5.7 g CO2 /min. In order to make the industrial application of SFE possible, it is necessary to determine the process’ efficiency, which can be evaluated by the combination of the product’s quality, cost and process yield. In the present work, process yield and product’s quality were the evaluated issues. The extract yield, chemical composition, antioxidant and antimicrobial activities of the extracts from A. brasiliensis obtained by SFE with pure CO2 and CO2 with ethanol as modifier were determined. To evaluate yield and quality in the SFE extracts, low-pressure techniques such as hydrodistillation, maceration with ethanol, and Soxhlet extraction with different solvents were also applied to the raw materials. 2. Materials and methods 2.1. Raw material and sample preparation The dried A. brasiliensis mushrooms were provided by Mushroom Research Center of São Paulo State University (UNESP), São Paulo, SP, Brazil. The mushrooms, with moisture content of 6.6%, were ground in a knife mill (De Leo, Porto Alegre/RS, Brazil). The mean particle diameter of the samples was determined through the micrographs from the Scanning Electron Microscopy (SEM), performed in microscope (JEOL JSM-6390LV, USA), by means of the software Size Meter, version 1.1 [18]. The samples were stored at 255.15 K in a domestic refrigerator until the extractions were performed. 2.2. Supercritical fluid extraction (SFE) The SFE of mushroom A. brasiliensis was performed in a dynamic extraction unit previously described by Zetzl et al. [19]. A cosolvent (CS) pump (Constametric 3200, Thermo Separation Process, EUA) was connected to the extraction line in order to supply the modifier (organic solvent at high-pressure) at pre-established flow rate, to be mixed with CO2 flow before entering the extraction vessel. The extraction procedure was described by Michielin et al. [20] and performed up to 3.5 h, according to the kinetics assays. Briefly, the extraction procedure consisted of placing 15 g of dried and milled material inside a stainless steel column (329 mm length × 20.42 mm inner diameter, and internal volume of 100 mL) to form the fixed particle bed, followed by the control of the process variables (temperature, pressure and solvent flow rate). The extraction was performed at the established conditions and the extract was collected in amber flasks. The kinetic assays were performed to obtain the overall extraction curve (OEC) for the SFE, where the extract samples were collected at pre-established time intervals. These assays were carried on using supercritical CO2 at 20 MPa, 313.15 K and solvent flow rate of 12 ± 2 g CO2 /min. Taking the OEC into account, SFE time for the following steps was chosen in order to recover all the extractable material. The subsequent SFE assays were divided in two groups, and performed in duplicate: (a) global yield (X0 ) assays with pure CO2 as solvent at temperatures of 313.15 K, 323.15 K and 333.15 K, pressures from 10 to 30 MPa and constant solvent flow rate of 12 ± 2 g/min, during 3.5 h of extraction; and (b) co-solvent (CS)

49

assays, where the organic solvent added to supercritical CO2 was ethanol in fractions of 2.5%, 5.0% and 10.0% (w/w) to produce the solvent mixture. These extractions were performed at 323.15 K and 20 MPa at constant solvent flow rate of 12 ± 2 g/min, during 3.5 h. The co-solvent was separated from the solute by low-pressure methods (see Section 2.4).

2.3. Low-pressure extractions (LPE) Soxhlet extraction (Sox) was performed according to the 920.39C method of A.O.A.C. [21]. The procedure consisted of 150 mL of solvent recycling over 5 g of dried sample, in a Soxhlet apparatus for 6 h at the boiling temperature of the solvent used. The extraction was performed at least in duplicate, with the following solvents: hexane (Hx), dichloromethane (DCM), ethyl acetate (EtAc) ethanol (EtOH) and water, with polarities of 0, 3.1, 4.4, 5.2 and 9, respectively [22]. The choice of solvent was based on previous studies performed in the laboratory and have demonstrated good results [23]. The maceration (Mac) method consisted of a cold maceration of the mushroom A. brasiliensis particles in ethanol, to avoid thermal degradation. The extraction was performed with 50 g of dried grounded sample placed in 200 mL of ethanol for 7 days at room temperature and one daily manual agitation. The resulting extract was evaporated to 10% of the initial volume to obtain the crude extract (CE), the ethanolic fraction (Mac-EtOH), as presented in Section 2.4. Then, the CE was partitioned with 40 mL of each solvent Hx, DCM, EtAc and water, as described by Mezzomo et al. [24]. The organic solvents were 99% pure and used in sequence according to their ascending polarity values of 0.0, 3.1, 4.4 and 9.0, respectively [22]. The hydrodistillation (HD) method consisted in placing 50 g of grounded mushroom A. brasiliensis inside a 2 L flask of a Clevenger type apparatus with 700 mL of distilled water for HD, according to Mezzomo et al. [24], and carried out at least in duplicate during 6 h. Two milliliters of hexane were introduced in the decantation part of the Clevenger apparatus to dissolve the volatile oil.

2.4. Solvent–solute separation The resulting mixtures from each technique with different solvents were separated under reduced pressure, by evaporating the solvents in a rotary evaporator, in order to obtain the extracts. The global yield (X0 ) for each technique was obtained by the mean value from the duplicate experiments, and calculated by the ratio between mass of extracted oil and mass of raw material used.

2.5. Antioxidant potential 2.5.1. Free radical scavenging activity (DPPH) The free radical scavenging of the A. brasiliensis extracts was evaluated using the 1,1-diphenyl-2-picrylhydrazil (DPPH) method, as described by Mensor et al. [25] and Benelli et al. [26]. Briefly, each extract was mixed with a 0.3 mM DPPH ethanol solution, to give final concentrations of 5, 10, 25, 50, 125, 250 and 500 ␮g extract mL−1 DPPH solutions. After 30 min at room temperature, the absorbance values were measured at 517 nm in spectrophotometer and converted into percentage of antioxidant activity (AA%). This activity was also presented as the effective concentration at 50% (EC50 ), i.e., the concentration of the test solution required to give 50% decrease in the absorbance of the test compared to that of a blank solution, and expressed in ␮g of extract mL−1 DPPH. The EC50 values were calculated from the linear regression of the AA% curves obtained for all extract concentrations. The

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S. Mazzutti et al. / J. of Supercritical Fluids 70 (2012) 48–56

AA% and EC50 for all extracts were obtained considering the mean value of triplicate assays.

2.5.2. Total phenol content (TPC) The TPC was determined according to the Folin–Ciocalteau method [27]. Briefly, the reaction mixture was composed by 0.1 mL of extract (concentration of 1667 mg L−1 ), 7.9 mL of distilled water, 0.5 mL of Folin–Ciocalteau reagent (a mixture of phosphomolybdate and phosphotungstate) and 1.5 mL of 20% sodium carbonate, placed in opaque flasks. The flasks were agitated, held for 2 h, and their absorbance was measured at 765 nm. The TPC was calculated according to a standard curve, prepared previously with chlorogenic acid as standard. The results (mean value of the triplicate assays) were expressed as milligrams of chlorogenic acid equivalent (CAE) per gram of the extract (mg CAE g−1 ).

2.6. Antimicrobial activity 2.6.1. Minimum inhibition concentration (MIC) The extracts obtained from A. brasiliensis by different methods were submitted to evaluation of their antimicrobial activity against Gram-positive bacteria Staphylococcus aureus and Bacillus cereus and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Müeller–Hinton agar and culture broth were used for growing the bacteria. An overnight culture of each bacterial species grown in Müeller–Hinton broth was diluted in fresh medium to achieve a final concentration of approximately 108 CFU/mL. All bacterial cultures were incubated in aerobic conditions [28]. The antimicrobial activities of different extracts were evaluated through the determination of the minimum inhibition concentration (MIC) by the microdilution method in culture broth [29]. The extracts (5 mg) were dissolved in 250 ␮L of dimethyl sulfoxide (DMSO) 25% because DMSO does not offer inhibition to the microorganism growth. Further, serial dilutions of the extracts (final concentration ranging between 2000 and 1.95 ␮g/mL) were prepared [29] and distributed (10 ␮L) in microdilution plates with 96 wells. In each well, 85 ␮L of Müeller–Hinton broth was added. One sample (10 ␮L) of each diluted solution and the samples for both growth and sterility controls (containing sterile culture medium and DMSO, and no antimicrobial agents, respectively) were distributed in microdilution plates with 96 wells. For each test and for the growth control well, a 5 ␮L inoculum of the bacterial suspension of E. coli, B. cereus, S. aureus or P. aeruginosa (107 CFU/mL) was added. All experiments were performed in duplicate and the plates incubated for 24 h at 36 ◦ C. Bacterial growth was first detected by optical density and afterwards by addition of 20 ␮L of an alcoholic solution (0.5 mg/mL) of 2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride. The MIC was considered as the lowest concentration of the substance that inhibited the bacterial growth, after the incubation period. The results were expressed in ␮g/mL [30]. 2.6.2. Gel diffusion assay The extracts obtained from A. brasiliensis by different methods were submitted to evaluation of antimicrobial activity against three species of Candida: C. albicans, C. parapsilosis and C. krusei. The inoculums consisted of a cell suspension with adjusted turbidity to a tube equivalent to a 0.5 McFarland standard. Müller–Hinton Agar supplemented with 2% glucose was used as culture medium. The cell suspensions were inoculated on the agar surface with a sterile swab. Further, the agar surface was perforated with 7 mm diameter holes, aseptically cut and filled with 50 ␮L of a suspension of each extract prepared at 20 mg/mL in 10% DMSO. The plates were incubated at 308.15 K for 48 h and, then, examined to verify the

inhibition. Diameter inhibition zone was measured in millimeters. The tests were performed in duplicate [31]. 2.7. Chemical profile The identification and relative quantification of the compounds present in the extracts were achieved by gas chromatography coupled to mass spectrometry analysis (GC–MS). All the SFE and LPE A. brasiliensis extracts were evaluated, except those obtained by Sox-water, Sox-EtOH and Mac, because the solubilization of these extracts in dichloromethane was not possible. The analyses were performed in a gas chromatograph coupled with a mass detector (GC-Varian 3800, MS/MS-Varian 1200L; Varian, Inc., CA, USA) and VF5-MS capillary column (30 m × 0.25 mm, 0.25 ␮m; Varian, Inc., CA, USA). The samples were dissolved in dichloromethane and injected at a 1:10 rate for analysis following the conditions: initial column temperature of 323.15 K and final temperature of 593.15 K, with heating rate of 283.15 K/min, during 20 min and helium as carrier gas at 1 mL/min flow rate. The major extract components were evaluated by recognition using the database for natural products Standard Reference Data Series of the National Institute of Standard and Technology (NIST Library – Mass-Spectral Library with Windows Search Program – Version 2) [32], where the mass spectrometer results were compared. 2.8. Statistical analysis The yield (X0 ) and antioxidant activity results were statistically evaluated by a one-way analysis of variance (ANOVA), using the Software Statistica for Windows 7.0 (Statsoft Inc., USA) in order to detect significant differences between values in function of SFE temperature and pressure, and among the percentage of antioxidant activity. The significant differences (p < 0.05) were analyzed by Tukey test. The antimicrobial activity was not evaluated by ANOVA because the results obtained by the experiments (mean value of the duplicate assays) were coincident and showed no standard deviation. 3. Results and discussion 3.1. Global yield (X0 ) of LPE and SFE The global yields obtained by the different extraction methods and solvents (Sox, Mac, HD and SFE) are presented in Table 1, together with the polarity index of the organic solvents used and CO2 density at each temperature/pressure applied. Among Sox extractions, those carried out with water and EtOH provided the highest yields. The conditions of temperature, solvent recycle and solvent/solutes interactions, in Sox method, contribute to the highest solubilization of components from raw material (maximum yields). Also, on Sox extraction, performed at the solvent boiling temperature, the surface tension and viscosity are low compared to those at lower temperatures. Therefore, the solvent reaches the active sites inside the solid matrix far more easily, promoting solubilization [33]. Beyond the contributing factors, the highest yields obtained with EtOH and water indicate that the mushroom A. brasiliensis contains many intermediate to high polarity compounds, due to the polarity of the solvents (5.2 and 9, respectively). The Mac extraction provided extraction yields lower than Sox with water and EtOH, and presented total yields without significant difference to those achieved by HD. Probably, these low yields occurred due to the low processing temperature, in contrast with Sox extraction. Thus, the surface tension and viscosity remain relatively high, making difficult the access to soluble compounds on the matrix and reducing the extraction yield, even for equal solvents.

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a

LPE

b

Solvent

Polarity Index

Maceration Hydrodistillation

Hx DCM EtAc EtOH Water EtOH Water

0.0 3.1 4.4 5.2 9.0 5.2 9.0

SFE

Solventa

CO2 (kg/m3 )c

Soxhlet

d

X0 (%)

1.9c 2.21c 3.2c 37b 57a 2.4c 0.004c

± ± ± ± ± ± ±

0.1 0.01 0.2 1 4 0.5 0

1.4

Extraction yield (%)

Table 1 Global yield (X0 ) of mushroom Agaricus brasiliensis extracts obtained by lowpressure extraction (LPE) and supercritical fluid extraction (SFE).

1.2 1.0 0.8 0.6

313.15 K/10 MPa 313.15 K/30 MPa 323.15 K/10 MPa 323.15 K/30 MPa 333.15 K/10 MPa 333.15 K/30 MPa

CO2 CO2 CO2 CO2 CO2 CO2

629 911 385 871 295 830

0.85b 0.98ab 0.6bc 1.19a 0.5c 1.11ab

± ± ± ± ± ±

0.05 0.01 0.1 0.01 0.1 0.01

a Hx, hexane; DCM, dichloromethane; EtOH, ethanol; EtAc, ethyl acetate; CO2 , carbon dioxide. b Solvent polarity index [22]. c CO2 density [35]. d Same letters indicated no significant differences at level of 5% (p < 0.05).

0.18 0.16

Extract (g)

0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

0

50

100

150

200

250

300

350

400

450

Time (min) Fig. 1. Overall extraction curve of Agaricus brasiliensis extract obtained by SFE with pure CO2 at 20 MPa, 313.15 K and 12 ± 2 g O2 /min.

The soluble compounds in the Mac from the mushroom A. brasiliensis were fractionated and the higher yields were obtained for water (0.77%) and Hx (0.46%). The yields of the fractions of DCM and EtAc were 0.15% and 0.06%, respectively. Kitzberger [34] also obtained higher yields for the solvents Hx and water in the fractionation the soluble compounds from Mac of Shiitake mushroom. The HD yield was of 0.004 ± 0%, lower than most other results. In HD, the water boiling point causes vapor formation, carrying volatile compounds from the sample, and the extract is characterized as essential oil, different from other techniques that solubilize various families of components (more complex mixtures of solutes), resulting in higher yields. Therefore, because of the low yield provided by HD, we can affirm that the mushroom A. brasiliensis has only a small amount of volatile compounds. 3.1.1. SFE with pure CO2 The kinetic study was performed in order to define the extraction time. The overall extraction curve (OEC) obtained by SFE with pure CO2 at 20 MPa, 313.15 K and 12 ± 2 g CO2 /min is presented in Fig. 1. The shape of the extraction curve indicates that at different stages of the extraction, different mechanisms control the mass transfer. At the beginning of the extraction convection is the main mass transfer mechanism, since there is available solute over the

313.15 K

0.4

323.15 K

0.2

333.15 K

0.0 X0 (%)d

51

5

10

15

20

25

30

35

Pressure (MPa) Fig. 2. Crossover isotherms for the global yield of Agaricus brasiliensis extracts obtained by SFE with pure CO2 .

particle’s surface. When this solute is depleted, compounds from inside the particles begin to be extracted, so diffusion becomes the controlling mechanism. In the extractions with pure supercritical CO2 , the evaluation of the global yield obtained under different conditions of temperature and pressure indicates the effect of solubility of the mushroom’s compounds in the solvent, and, consequently the influence of solubility on the process yield. The global yield results for SFE are also presented in Table 1 for the different conditions of temperature and pressure studied. The highest yield for the mushroom A. brasiliensis extract was 1.19 ± 0.01%, obtained at 323.15 K and 30 MPa, where the solvent density is of 871 kg/m3 . The lowest yield was 0.5 ± 0.1%, obtained at 333.15 K and 10 MPa, with solvent density of 295 kg/m3 . These results indicate that the amount of oil extracted is related to solvent power, which is function of its density and, therefore, to the process temperature and pressure. The CO2 solvation power depends on its density, which increases with pressure at constant temperature and decreasing with temperature at constant pressure [14]. At all the tested temperatures it is possible to observe the increase in X0 when pressure increases from 10 to 20 MPa. This behavior is explained by the increase of solvent density with enhancing pressure, increasing the solvation power of CO2 [13]. The effect of temperature on extraction yield, at constant pressure, can occur by two mechanisms: (a) the increase in process temperature increases the solubility due to increased vapor pressure of the solute; and (b) on the other hand, the temperature increase reduces the solubility due to the decrease in the solvent’s density. These two opposite effects may result in the crossover of the isotherms, in a phenomenon known as retrogradation [20,26]. In the present work this crossover behavior is not completely characterized. Nevertheless, the SFE yields presented in Table 1 and Fig. 2 suggest that crossover could happen. At the lowest tested pressure, 10 MPa, we clearly notice a negative effect of temperature on X0 , which is caused by the reduction of solvent’s density. Otherwise, at higher pressures (20 and 30 MPa) the effect of temperature on SFE yield is not observed, indicating that solvent’s density and solute vapor pressure have opposite effects that annulate each other. Possibly, at pressures above this range, a positive effect of increasing temperature could be detected, if the effect of solute vapor pressure overcame that of solvent density. Considering that at higher pressures a supercritical fluid is less compressible than near its critical point, a reduced effect of solvent’s density over 30 MPa would be realistic. Based on the present results, it can only be affirmed that isotherm crossover can occur at pressures above 20 MPa. Very similar results were also obtained by Andrade et al. [36] in SFE from coffee grounds. The isotherm crossover was also detected for SFE of Shiitake mushroom between 15 and 20 MPa [37].

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S. Mazzutti et al. / J. of Supercritical Fluids 70 (2012) 48–56

Fig. 3. Global yield (X0 ) of mushroom Agaricus brasiliensis extracts obtained by supercritical fluid extraction (SFE) with ethanol as co-solvent.

The analysis of variance for the SFE with CO2 indicates that the pressure effect and the interaction between pressure and temperature effect were significant at a level of 5% (p < 0.05) for the mushroom A. brasiliensis extraction yield. Otherwise, the temperature effect was not significant on yield results, probably because of the opposing influence of temperature. 3.1.2. SFE with CO2 plus co-solvent (CS) EtOH is a food grade modifier often used as co-solvent for SFE, in order to improve the technique efficiency by increasing the recovery of polar compounds. EtOH was selected to be used as a co-solvent based on the global yield obtained in the low-pressure extractions and also by the biological activity shown by the extracts (results in Section 3.2). The use of a co-solvent implies the addition of one step to the extraction process (removal of solvent from the final extract). Therefore, the amount of co-solvent used must be reduced in order to minimize operational costs with its evaporation. The effect of fractions of 2.5%, 5.0% and 10.0% (w/w) of co-solvent EtOH on the extraction yield was investigated for SFE at 323.15 K and 20 MPa. The global yield results for SFE with co-solvent are presented in Fig. 3, which shows an increase in X0 values with enhancing EtOH fraction for all amounts of co-solvent used. Results from Fig. 3 show an increase in yield up to 380% compared with pure CO2 , reaching the value of 4.2% (w/w) for extraction using 10% EtOH. Kitzberger et al. [37] verified that the extraction yield of SFE from Shiitake increased from 0.57%, with pure CO2 , to 3.81% when 15% of EtOH was used as cosolvent. This behavior can be explained by the increase in the solubility of polar compounds in the mixture EtOH/CO2 , compared to the solubility in pure CO2 , which is essentially a non-polar solvent. Furthermore, not only the solubilities of certain components increase with the use of co-solvent, but also the number of components solubilized by the solvent, which reduces the process selectivity and increases the global yield. 3.2. Antioxidant potential Determination of the antioxidant capacity of food should take into account the overall concentrations and compositions of diverse antioxidants, because the total antioxidant capacity is due to the combined activities of diverse antioxidants, including phenolics. Various methods, based on different mechanisms, must be used in parallel to evaluate the antioxidant capacity of compounds, since different methods can give very different results [38]. Table 2 shows the antioxidant activity results according to analyses of TPC and DPPH, achieved for the samples of mushroom A. brasiliensis extracts obtained from different extraction methods (Sox, Mac, HD and SFE). The results were compared to the synthetic product BHT, used as standard sample. The values of TPC from the extracts obtained by SFE showed no variation with changes in pressure or temperature, and presented

values between 14.1 and 31.8 mg EAC/g. For the LPE methods, the best results were obtained with ethanol as solvent, for the maceration and Soxhlet extraction methods, reaching 74 ± 4 mg EAC/g and 46 ± 2 mg EAC/g, respectively. The values of TPC from mushroom extracts obtained by LPE and SFE methods were lower than the verified for the synthetic antioxidant BHT, which was of 423 mg EAC/g. According to Table 2, the highest AA% values (determined by the DPPH method) for LPE methods were obtained with ethanol as solvent, for the maceration and Soxhlet extracts. The highest AA% values for SFE were obtained at 333.15 K and 30 MPa. Literature shows 323.15 K as the optimum temperature for phenolic compounds extraction from vegetable matrixes [39]. Among the results from Table 2 it can be noticed that SFE 323.15 K/10 MPa and SFE 323.15 K/20 MPa were the best conditions for AA%, corroborating the literature data. However, at 30 MPa, the AA% increases with temperature. Concentration of sample at which the inhibition percentage reaches 50% is its EC50 value. The lowest EC50 values were observed at 323.15 K. EC50 value is negatively related to the antioxidant activity, as it expresses the amount of antioxidant needed to decrease its radical concentration by 50%. The lower the EC50 value, the higher is the antioxidant activity of the tested sample. Silva et al. [40] measured the antioxidant activity of different extracts of the mushroom A. blazei and evaluated the oxidative stability of soybean oil added with the mushroom extract. The results showed that the methanol:water extract resulting from 6 h of extraction presented the highest antioxidant activity (28.9%). Fig. 4 shows the total phenolic content (TPC) and antioxidant activity (AA%) evaluated by free radical scavenging activity (DPPH) of mushroom A. brasiliensis extracts obtained by SFE with ethanol as co-solvent. According to Fig. 4, the values of TPC and AA% increase with EtOH fraction of co-solvent used. The best results of TPC and AA% were found for the extract obtained using 10.0% ethanol as cosolvent. Important substances that show antioxidant activity are polar components and, since CO2 is a non-polar solvent, it does not favor the solubilization of such components. The enrichment in co-solvent fractions improves the extractions of compounds with antioxidant activity due to proportional changes in the solvent mixture characteristics. The results of TPC and AA% of the extracts of A. brasiliensis showed a moderate antioxidant potential. However, other methods for determination of antioxidant activity should be applied to a more conclusive evaluation. 3.3. Antimicrobial activity (MIC) The MIC results are presented in Table 3 for mushroom A. brasiliensis extracts obtained by different extraction techniques (Sox, Mac, HD and SFE), tested against Gram-positive bacteria: S. aureus, B. cereus. All the MIC results against Gram-negative bacteria (E. coli and P. aeruginosa) were over 2000 ␮g/mL, excepting the DCM extract, that presented MIC of 1500 ␮g/mL against E. coli. Natural products can be classified as antimicrobial agents based on the MIC values of its extracts [23,41]. This classification is: strong inhibitors for MIC values lower than 500 ␮g/mL; moderate inhibitors for MIC between 600 and 1500 ␮g/mL; weak inhibitors for MIC above 1600 ␮g/mL. The potential of various natural products with biological activity can be detected with this classification. The results of A. brasiliensis extracts obtained from LPE and SFE were very effective against the Gram-positive bacteria. Extracts obtained from SFE with CO2 at 20 MPa and 313.15 K, 10 MPa and temperatures of 323.15 K and 333.15 K and Sox with the solvents Hx, DCM and EtAc showed the lower MIC values against S. aureus. These low MIC values can be classified as strong inhibitors. Strong inhibitors extracts of B. cereus were obtained from SFE with CO2 at 30 MPa and 333.15 K, 10 MPa and temperatures of 323.15 K and 333.15 K and Sox with the solvents Hx and DCM.

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Table 2 Antioxidant activity for mushroom Agaricus brasiliensis extracts measured by different methods. Extraction

Soxhlet

Maceration SFE 313.15 K/10 MPa SFE 323.15 K/10 MPa SFE 333.15 K/10 MPa SFE 313.15 K/20 MPa SFE 323.15 K/20 MPa SFE 333.15 K/20 MPa SFE 313.15 K/30 MPa SFE 323.15 K/30 MPa SFE 333.15 K/30 MPa BHT *

Solvent

TPC (mg EAC/g)*

EC50 (␮g/mL)*

AA% (500 ␮g/mL)*

Hexane Dichloromethane Ethyl acetate Ethanol Water Ethanol CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 –

12.6f ± 0.5 27cd ± 2 46b ± 2 47b ± 5 27cd ± 2 74a ± 4 32c ± 4 22d ± 3 14ef ± 3 24d ± 2 22d ± 2 24.6cd ± 0.5 21.2de ± 0.5 19df ± 1 24d ± 1 423

2647f ± 72 1632h ± 27 1172i ± 69 1053ij ± 30 1033j ± 21 700k ± 4 6952a ± 5 5187c ± 24 7001a ± 3 4462e ± 14 3543e ± 3 5765b ± 11 4622d ± 8 2379g ± 64 2527f ± 84 89.7

15.2e ± 0.2 18d ± 3 25.5c ± 0.5 30.2b ± 0.2 25c ± 0.2 37.2a ± 0.3 4.64i ± 0.06 7.42h ± 0.07 4.89i ± 0.3 6.6h ± 0.2 9.9g ± 0.1 8.0h ± 0.1 9.6g ± 0.2 10.0g ± 0.4 13.0f ± 0.2 113

Same letters indicate no significant differences at level of 5% (p < 0.05).

Fig. 4. (a) Total phenolic content (TPC) and (b) antioxidant activity evaluated by free radical scavenging activity (DPPH) of mushroom Agaricus brasiliensis extracts obtained by supercritical fluid extraction (SFE) with ethanol as co-solvent.

Table 3 Minimum inhibition concentration (MIC) values of mushroom Agaricus brasiliensis extract determined by the microdilution method for S. aureus and B. cereus. Extraction

Solvent

MIC (␮g/mL) S. aureus

Soxhlet

Maceration SFE 313.15 K/10 MPa SFE 323.15 K/10 MPa SFE 333.15 K/10 MPa SFE 313.15 K/20 MPa SFE 323.15 K/20 MPa SFE 333.15 K/20 MPa SFE 313.15 K/30 MPa SFE 323.15 K/30 MPa SFE 333.15 K/30 MPa SFE 323.15 K/20 MPa SFE 323.15 K/20 MPa SFE 323.15 K/20 MPa

Hexane Dichloromethane Ethyl acetate Ethanol Water Ethanol CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 + 2.5% EtOH CO2 + 5% EtOH CO2 + 10% EtOH

500 375 250 >2000 >2000 >2000 1000 375 250 500 750 2000 >2000 1000 1000 1500 1000 1250

B. cereus 500 312 1000 >2000 >2000 2000 1000 500 375 2000 1000 1000 >2000 1500 500 1500 1500 1500

The results of A. brasiliensis extracts obtained from LPE and SFE presented low effectiveness against the Gram-negative bacteria. The MIC values obtained classify the extracts as weak inhibitors, excepting the extract from Sox with DCM, which is classified as moderate inhibitor. The higher resistance of the Gram-negative bacteria could be due to the complexity of the cell wall of this group of microorganisms. Indeed, the external membrane of Gramnegative bacteria renders highly hydrophilic surfaces whereas the negative charge of the surface of the Gram-positive wall may reduce their resistance to antibacterial compounds [41,43].

The use of co-solvent in SFE (EtOH) only increased the process yield, compared with CO2 extraction, but produce extracts with reduced microbial inhibition. This suggests that the most powerful inhibitors for the microorganisms tested are non-polar compounds that can be recovered with pure CO2 , as organic acids. Michielin et al. [23] and Benelli et al. [26] described low MIC results for S. aureus obtained by the supercritical extracts from Cordia verbenacea and orange pomace, respectively, comparing with the other extraction procedures tested. Kitzberger et al. [26] verified shiitake mushroom extracts obtained with supercritical fluids were effective against the growth of Micrococcus luteus and B. cereus and not efficient against S. aureus and E. coli. The different extraction techniques employed produced extracts with moderate and strong antimicrobial potential. However, supercritical extraction allows the recovery of extracts in shorter times of extraction, and using smaller amounts of solvent. The extracts of the mushroom A. brasiliensis obtained by different extraction methods were tested against three types of Candida fungi: C. albicans, C. parapsilosis and C. krusei. For C. parapsilosis and C. krusei no inhibition zone was detected with any extract. The tests with C. albicans presented inhibition zone only for the supercritical extracts obtained at 20 MPa and 323.15 K and 20 MPa and 333.15 K with a 9 mm and 9.5 mm halo, respectively, indicating a weak inhibition power. Therefore, the antifungal analysis indicates higher efficiency of the supercritical extracts compared with the low-pressure extracts for the C. albicans. 3.4. Chemical profile The composition results are presented in Table 4, with the name of the compounds and the relative composition (integrated composition) for the extracts obtained by supercritical fluid extraction

54

Table 4 Relative composition profile, in % peak area, of mushroom Agaricus brasiliensis extracts obtained by low-pressure extractions (LPE) and supercritical fluid extraction (SFE). Identified component

TR (min)

Relative area (%) SFE + EtOH

LPE Sox

10 MPa

10 MPa

10 MPa

20 MPa

20 MPa

20 MPa

30 MPa

30 MPa

30 MPa

20 MPa/323.15 K

Hx

313.15 K

323.15 K

333.15 K

313.15 K

323.15 K

333.15 K

313.15 K

323.15 K

333.15 K

2.5%

SFE

3.56 4.28 5.21 6.39 6.86 7.50 7.80 8.42 10.53 13.13 14.15 15.44 16.62 16.80 16.81

0.29 0.73

1.88

4.67

0.76

2.55 1.96

0.76 1.13

0.57 0.84 1.92 19.18

8.72 1.06 1.70 57.67

8.37 1.75 2.94 41.41

0.23 0.06 0.15 0.43 0.93 0.39 0.66 0.17 0.75 3.75

0.42 1.36

1.75 0.22 0.68 22.88 0.11

0.94

0.29

1.40

0.65 1.30

0.94 0.96

1.31 1.15 0.79

10.58

5%

1.57 54.10

1.21 0.66 0.81 32.73

53.99

0.48 0.67 21.44

10%

0.09

0.32

0.58 0.25

12.43

15.20

4.61

59.09

44.24

10.80

2.15

EtAc

0.51

1.10

9.10

10.78

9.98

70.49

39.04

68.89

5.89

4.24

1.44 1.37

3.16 1.87

1.05

2.36 14.41

17.84

17.13

13.08

23.49

18.81

30.20

3.83 0.68

5.39

6.40

7.69

8.67

5.40

2.15

17.02 18.51 18.61

7.2 0.98

20.02 22.53 23.84 24.10 24.12 24.16 24.49 24.52

0.93

3.15

27.44 0.76 0.64 0.61 0.65 1.41 0.92 1.24

0.80 0.73

2.31 1.34

2.33 1.33

S. Mazzutti et al. / J. of Supercritical Fluids 70 (2012) 48–56

2-Pentyl furan Hexanoic acid Acid carbonic, dihexyl ester Octanoic acid Benzoic acid (E,E) 2,4-Decadienal 2,4-Decadienal Pentadecane 9-Oxononanoic acid Tetradecanoic acid Pentadecanoic acid n-Hexadecanoic acid Heptanoic acid Oleic acid 9,12-Octadecadienoic acid Octadecanoic acid. 2-(2-hydroxyetoxy)ethyl ester Octadecanoic acid Eicosanoic acid bis(2-ethylhexyl) ester Hexanedioic acid Docosanoic acid 9(11)-Dehydroergosteryl benzoate Anthraergostatetraenol Ergosterol 5,6-Dihydroergosterol 7,22-Ergostadienol ␣-Ergostenol ␥-Ergostenol

DCM

S. Mazzutti et al. / J. of Supercritical Fluids 70 (2012) 48–56

(SFE) and low-pressure (LPE) extraction. The major identified components, in terms of % area peak, are n-hexadecanoic acid (palmitic acid) and 9,12-octadecadienoic acid (linoleic acid). Coelho et al. [17] obtained extracts from the mushroom A. blazei by SFE with CO2 at 40 MPa and 243.15 K and the major identified components were also palmitic acid and oleic acid. Environmental factors involved in sample collection, such as seasonality, climate, and maturity stage are important factors in research involving plants and mushrooms. The production of secondary metabolites is a function of plant interaction versus environment in response to chemical and biological factors. This factor can explain the difference in the results of biological activity and chemical profile of different extracts of the same species but collected in different places and periods [43]. Linolenic acid is an unsaturated fatty acid not synthesized in the body, and among unsaturated fatty acids, ␣-linolenic acid and the r-linolenic acid are known as able to reduce the levels of serum cholesterol, triglycerides, and LDL-cholesterol, decreasing the risk of arteriolosclerosis, cancer, and allergic diseases [45]. The presence of linoleic acid in the extracts’ composition suggests this component as the main responsible for the antimicrobial activity, although the biological activity can also be attributed to a synergistic effect among different components [46]. The antibacterial activity of fatty acids is probably due to the ability of these compounds to disrupt the membranes of bacterial cells and cause lysis of the cells [47]. Although palmitic and linoleic acids were the most abundant compounds among those identified by GC, no trends were detected concerning the effects of pressure and temperature on composition. When comparing the co-solvent extracts with the pure CO2 extracts, we notice that the number of identified compounds was much lower. This may be due to the extraction of non-volatile compounds, as phenolics, when ethanol was used. Such compounds might have been preferentially solubilized instead of the minor compounds detected in the pure CO2 extracts. The extracts obtained by SFE showed more fatty acids than those obtained by Soxhlet. Hexanoic acid, octanoic acid, tetradecanoic acid, pentadecanoic acid, oleic acid, eicodecanoic acid and 9-oxononanoic acid were some of the identified components in the SFE extracts. It has been observed that the conventional lowpressure processes, such as Soxhlet, had reduced ability to extract functional compounds mainly because: (i) these methods are not selective. These processes often need further stages of fractionation to obtain the desired compound. Consequently, loss of compounds can occur along these fractionation steps, and (ii) the high temperature used in the Soxhlet method can degrade thermally sensible compounds. Other important compound detected in the LPE extract was ergosterol, the precursor of vitamin D and the intermediate of cortisone and hormone flavone. Furthermore, it was reported that ergosterol isolated from fungi exhibited pharmacological activities, including enzyme inhibition to cyclooxygenase, antioxidation and anti-tumor activity, through in vitro and in vivo experiments [48]. Facing the detected presence of ergosterol, the examination of the antitumor activity of extracts of A. brasiliensis is interesting and might lead to promising results. No correlation was found between the chemical profiles analyzed by GC–MS and the antioxidant activities of the extracts. This suggests that the antioxidant compounds of the extracts are not detectable by this technique. Since the total phenolic content of most extracts was high, and phenolics are often non-volatile, it seems that these compounds are strongly responsible for the antioxidant activities, although they were not identified. Other analytical techniques, such as high or ultra high performance liquid chromatography, would certainly be useful to quantify phenolics and relate them to the antioxidant activities of the extracts.

55

4. Conclusions Supercritical CO2 extraction of mushroom A. brasiliensis was carried out and the effects of pressure, temperature and ethanol concentration on the global yield of the extracts were studied. The best conditions in the studied range to obtain high yields resulted to be 30.0 MPa and 323.15 K, where SFE yield was of 1.19% using pure CO2 as solvent. When ethanol was added as co-solvent, the extraction yields were even higher, reaching 4.2% with 10% of ethanol, due to the recovery of polar compounds. Conventional extraction techniques, such as Soxhlet, maceration and hydrodistillation were performed for comparative purposes. Soxhlet presented the highest yields, 57% and 37%, when using water and ethanol as solvent, respectively. Hydrodistillation technique showed the lowest extraction yields, revealing the non-volatile characteristic of the extractable compounds. Preliminary evaluations of the antioxidant potential of the mushroom extracts have demonstrated moderate antioxidant activity. However, other techniques for determining the antioxidant activity should be used. Since GC–MS was not able to identify such compounds, no relation was detected between the chemical profile and the antioxidant activities. The antibacterial activity presented by LPE and SFE indicated great potential of the extracts as inhibitors of the microorganism growth for Grampositive bacteria, but not against Gram-negative, probably due to cell wall properties. The extracts presented weak inhibition power against Candida species. The major identified components were palmitic acid and linoleic acid. The SFE technique is suitable to obtain functional compounds from a natural source, contributing to increase the aggregate value of food products. Further investigations concerning the antitumor activity of extracts from mushroom A. brasiliensis should be performed, and other analytical techniques should be applied to detect and quantify the phenolic compounds of this mushroom. Acknowledgment The authors wish to acknowledge CAPES for the financial support. References [1] F. Firenzuoli, L. Gori, G. Lombardo, The medicinal mushroom Agaricus blazei murrill: review of literature and pharmaco-toxicological problems, eCAM 5 (2008) 3–15. [2] S.P. Wasser, M.Y. Didukh, M.A.L. Amazonas, E. Nevo, P. Stamets, A.F. Eira, Is a widely cultivated culinary-medicinal Royal Sun Agaricus (the Himematsutake mushroom) indeed Agaricus blazei Murill? International Journal of Medical Mushroom 4 (2002) 267–290. [3] M.L. Largeteau, R.C. Llarena-Hernández, C. Regnault-Roger, J.M. Savoie, The medicinal Agaricus mushroom cultivated in Brazil: biology, cultivation and non-medicinal valorization, Applied Microbiology and Biotechnology 92 (2011) 897–907. [4] S. Bernardshaw, E. Johnson, G. Hetland, An extract of the mushroom Agaricus blazei Murill administered orally protects against systemic Streptococcus pneumoniae infection in mice, Scandinavian Journal of Immunology 62 (2005) 393–398. [5] A.C. Silva, M.C. Oliveira, P.V. Del Ré, N. Jorge, Utilizac¸ão de extrato de cogumelo como antioxidante natural em óleo vegetal, Ciência e Agrotecnologia 33 (2009) 1103–1108. [6] A.A. Soares, C.G.M. Souza, F.M. Daniel, G.P. Ferrari, S.M.D. Costa, R.M. Peralta, Antioxidant activity and total phenolic content of Agaricus brasiliensis (Agaricus blazei Murril) in two stages of maturity, Food Chemistry 112 (2009) 775–781. [7] S.-J. Huang, J.-L. Mau, Antioxidant properties of methanolic extracts from Agaricus blazei with various doses of ␥-irradiation, LWT 39 (2006) 707–716. [8] Y.-W. Kim, K.-H. Kim, H.-J. Choi, D.-S. Lee, Anti-diabetic activity of b-glucans and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei, Biotechnology Letters 27 (2005) 483–487. [9] T. Takaku, Y. Kimura, H. Okuda, Isolation of an antitumor compound from Agaricus blazei Murill and its mechanism of action, Journal of Nutrition 131 (2001) 1409–1413. [10] C.-H. Yu, S.-F. Kan, C.-H. Shu, T.-J. Lu, L. Sun-Hwang, P.S. Wang, Inhibitory mechanisms of Agaricus blazei Murill on the growth of prostate cancer in vitro and in vivo, Journal of Nutritional Biochemistry 20 (2009) 753–764.

56

S. Mazzutti et al. / J. of Supercritical Fluids 70 (2012) 48–56

[11] C.-F. Kim, J.-J. Jiang, K.-N. Leung, K.-P. Fung, C.B.-S. Lau, Inhibitory effects of Agaricus blazei extracts on human myeloid leukemia cells, Journal of Ethnopharmacology 122 (2009) 320–326. [12] T. Mizuno, Medical properties and clinical effects of culinary-medicinal mushroom Agaricus blazei Murrill (Agaricomycetideae), International Journal of Medicinal Mushrooms 4 (2002) 299–312. [13] M.S. Mantovani, M.F. Bellini, J.P.F. Angeli, R.J. Oliveira, A.F. Silva, L.R. Ribeiro, ␤Glucans in promoting health: Prevention against mutation and cancer, Reviews in Mutation Research 658 (2008) 154–161. [14] G. Brunner, Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Process, vol. 4, Steinkopff, Darmstadt, 1994. [15] C. Pereira, M. Meireles, Supercritical fluid extraction of bioactive compounds: fundamentals, applications and economic perspectives, Food and Bioprocess Technology 3 (2010) 340–372. [16] M.I. Abdullah, J.C. Young, D.E. Games, Supercritical fluid extraction of carboxylic and fatty acids from Agaricus spp. mushrooms, Journal of Agricultural and Food Chemistry 42 (1994) 718–722. [17] J.P. Coelho, L. Casquinha, A. Velez, A. Karmali, Supercritical CO2 extraction of secondary metabolites from Agaricus blazei. Experiments and modeling, Alicerces 2 (2009) 1–7. [18] L.H.C. Carlson, Size Meter, LCP/EQA/UFSC, Florianópolis, 2001. [19] C. Zetzl, G. Brunner, M.A.A. Meireles, Standardized low-cost batch SFE units for university education and comparative research, in: Proceedings of the 6th International Symposium on Supercritical Fluids, vol. 1, Versailles, 2003, pp. 577–581. [20] E.M.Z. Michielin, L.F.V. Bresciani, L. Danielski, R. Yunes, S.R.S. Ferreira, Composition profile of horsetail (Equisetum giganteum L.) oleoresin: comparing SFE and organic solvents extraction, Journal of Supercritical Fluids 33 (2005) 131–138. [21] A.O.A.C., Official Methods of Analysis, 18th ed., Association of Official Agricultural Chemists, Maryland, 2005. [22] J.A. Byers, Catálogo Phenomenex, 2009, available from: http://www. phenomenex.com/phen/Doc/z366.pdf. [23] E.M.Z. Michielin, A.A. Salvador, C.A.S. Riehl, A. Smânia Jr., E.F.A. Smânia, S.R.S. Ferreira, Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods, Bioresource Technology 100 (2009) 6615–6623. [24] N. Mezzomo, B.R. Mileo, M.T. Friedrich, J. Martínez, S.R.S. Ferreira, Supercritical fluid extraction of peach (Prunus persica) almond oil: process yield and extract composition, Bioresource Technology 101 (2010) 5622–5632. [25] L.L. Mensor, F.S. Menezes, G.G. Leitão, A.S. Reis, T.C. Santos, C.S. Coube, S. Leitão, Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method, Phytotherapy Research 15 (2001) 27–30. [26] P. Benelli, C.A.S. Riehl, A. Smânia Jr., E.F.A. Smânia, S.R.S. Ferreira, Bioactive extracts of orange (Citrus sinensis L. Osbeck) pomace obtained by SFE and low pressure techniques: mathematical modeling and extract composition, The Journal of Supercritical Fluids 55 (2010) 132–141. [27] J.A.J. Rossi, V.L. Singleton, Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents, American Journal Enology Viticulture 16 (1965) 144–158. [28] A. Smânia Jr., F.D. Monache, E.F. Smânia, M.L. Gil, L.C. Benchetrit, F.S. Cruz, Antibacterial activity of a substance produced by the fungus Pycnoporus sanguineus (Fr.) Murr, Journal of Ethnopharmacology 45 (1995) 177–181. [29] H. Ávila, E. Smânia, F. Monache, A. Smânia Jr., Structure activity relationship of antibacterial chalcones, Bioorganic and Medicinal Chemistry 16 (2008) 9790–9794.

[30] E.F.A. Smânia, F.D. Monache, A. Smânia Jr., R.A. Yunes, R.S. Cuneo, Antifugal activity of sterols and triterpenes isolated from Ganoderma annulare, Fitoterapia 74 (2003) 375–377. [31] National Committee for Clinical Laboratory Standards, Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts: Approved Guideline M44-A, Clinical and Laboratory Standards Institute, 2004, available from: http://www.clsi.org/source/orders/free/m44-a2.pdf. [32] National Institute of Standards and Technology, NIST Standard Reference Database 1A, NIST/EPA/NIH Mass Spectral Library with Search Program: (Data Version: NIST 08, Software Version 2.0f), available from: http://www.nist.gov/srd/nist1a.htm. [33] M. Markom, M. Hasan, W.R.W. Daud, H. Singh, J.M. Jahim, Extraction of hydrolysable tannins from Phyllanthus niruri Linn.: effects of solvents and extraction methods, Separation and Purification Technology 52 (2007) 487–496. [34] C.S.G. Kitzberger, Obtenc¸ão de Extrato de Cogumelo Shiitake (Lentinula edodes) com CO2 a Alta Pressão, 2005, available from: http://www.tede.ufsc.br/teses/PEAL0048.pdf. [35] S. Angus, B. Armstrong, K.M. De Reuck, International Thermodynamic Tables of the Fluid State: Carbon Dioxide, Pergamon Press, Oxford, 1976. [36] K.S. Andrade, R.T. Gonc¸alvez, M. Maraschin, R.M. Ribeiro-do-Valle, J. Martínez, S.R.S. Ferreira, Supercritical fluid extraction from spent coffee grounds and coffee husks: antioxidant activity and effect of operational variables on extract composition, Talanta 88 (2012) 544–552. [37] C.S.G. Kitzberger, A. Smânia Jr., R.C. Pedrosa, S.R.S. Ferreira, Antioxidant, antimicrobial activities of shiitake (Lentinula edodes) extracts obtained by organic solvents and supercritical fluids, Journal of Food Engineering 80 (2007) 631–638. [38] J. Tabart, C. Kevers, J. Pincemail, J.O. Defraigne, J. Dommes, Comparative antioxidant capacities of phenolic compounds measured by various test, Food Chemistry 113 (2009) 1226–1233. [39] M. Pinelo, J. Sineiro, M.J. Núnez, Mass transfer during continuous solid–liquid extraction of antioxidants from grape byproducts, Journal of Food Engineering 77 (2006) 57–63. [40] A.C. Silva, M.C. Oliveira, P.V. Del Ré, N. Jorge, Utilizac¸ão de extrato de cogumelo como antioxidante natural em óleo vegetal, Ciência e Agrotecnologia 33 (2009) 1003–1008. [41] Y. Wang, H. He, J. Yang, Y. Di, X. Hao, New monoterpenoid coumarins from Clausena anisum-olens, Molecules 13 (2008) 931–937. [43] E.W. Koneman, S.E. Allen, W.M. Janda, P.D. Schreckenberger, W.C. Winn, Diagnóstico Microbiológico Texto y Atlas Color, 5th ed., Panamericana, Buenos Ayres, 1999. [45] J.-Y. Lee, Y.-S. Kim, D.-H. Shin, Antimicrobial synergistic effect of linolenic acid and monoglyceride against Bacillus cereus and Staphylococcus aureus, Journal of Agricultural and Food Chemistry 50 (2002) 2193–2199. [46] P.P. Alvarez-Castellanos, C.D. Bishop, M.J. Pascual-Villalobos, Antifungal activity of the essential oils of flower heads of garland chrysanthemum (Chrysanthemum coronarium) against agricultural pathogens, Phytochemistry 57 (2001) 99–102. [47] A. Hinton, K.D. Ingram, Use of oleic acid to reduce the population of the bacterial flora of poultry skin, Journal of Food Protection 9 (2000) 1282–1286. [48] C.-H. Shu, K.-J. Lin, Effects of aeration rate on the production of ergosterol and blazeispirol A by Agaricus blazei in batch cultures, Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 212–216.