**3. Evaluation of several antioxidant species in indigenous edible mushrooms**

As described in previous chapter, enzymatic and non-enzymatic antioxidants were reported to be present in edible mushrooms, and having roles in balancing human metabolic processes related to oxidative stress [64]. Since mid of twentieth century, after Harman's "Free-Radical Theory of Ageing", an intensive research on involvement of free radicals and antioxidants in living processes has been performed [64]. They are commonly named *reactive species* the advanced knowledge allowed their classification in terms of intensity [65], as well as of the nature of active centers (either oxygen, nitrogen, carbon or sulfur species) [64].

Wild grown and cultivated mushroom species have been studied from the perspective of possible correlations between non-enzymatic and enzymatic antioxidants. Thus, for the first category several phytochemical characteristics have been determined: total phenolic content, total flavonoids, antioxidant activity, and four trace micronutrients (Zn, Fe, Mn, and Cu), and for enzymatic antioxidants data, activity of catalase (CAT) and peroxidase (POX) have been evaluated.

### **3.1 Analytical techniques**

Different instrumental analytical techniques were reported by authors [66] to identify and quantify antioxidants in edible mushrooms. Among these, high performance chromatography (HPLC) and gas chromatography (GC) using various detection devices, spectroscopic techniques such as nuclear magnetic resonance (RMN), Fourier transform infrared (FTIR), ultraviolet-visible (UV-VIS), as well as inductively coupled plasma mass spectrometry (ICP-MS) are among the most applied. According to available equipment, the mushroom samples were characterized mainly through the absorption spectroscopic methods UV-VIS and FTIR spectroscopy, and information on trace micro-nutrients was gathered through ICP-MS. Analytical process included the classic steps of (a) sampling, (b) sample treatment and/or preparation, and (c) qualitative and/or quantitative analysis. For the step (a), mushroom samples were collected from the natural habitat according with **Table 1** and then representative portions from each sample were taken for further treatment. For the step (b), several procedures have been used: (i) oven drying at 40°C for 48 h; (ii) grounding to

**23**

**Figure 1.**

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms…*

less than 2 mm, cap and stipe separately; (iii) extraction of analyte(s), for 4 h, at room temperature, under continuous mixing, in two type of solvents—redistilled water and hydroalcoholic 50% (v/v), dry matter (g) to solvent ratio (mL) was of 4:100; (iv) centrifugation of obtained extracts; (v) wet digestion with concentrated HNO3 and

Polyphenols are a class of compounds with structures containing at least one aromatic ring with at least one hydroxyl group bonded on it. They are classified according to the number of rings and to their functional groups bound in the structure, and thus we have: phenolic acids, flavonoids, stilbenes, and lignans, coumarins, tannins. Phenolic acids were reported to be the main polyphenolic compounds

Total phenolic content in mushrooms was reported to be successfully determined by Folin Ciocalteu method [67], and an adapted method was applied for the studied mushrooms [67]. The Folin Ciocalteu reagent is a mixture prepared by dissolving sodium tungstate (Na2WO4·2H2O) and sodium molybdate (Na2MoO4·2H2O) in water with hydrochloric acid and phosphoric acid. Hydrated lithium sulfate (Li2SO4·H2O) may be added to this mixture to prevent turbidity that may appear due to formation of some insoluble sodium salts [67]. The mixture is very stable if protected to reduction agents and light. Diluted reagent also needs to be protected to light. The chemical process, occurring at basic pH, is based on molybdenum reduction from +6 (yellow) to +4 (blue) after the oxidation of polyphenols in

Light absorption of a monochromatic radiation of 765 nm is measured with a UV-VIS spectrophotometer. Colored liquid samples were placed in 10 mm light path cuvettes and readings were made versus a blank sample prepared with all reagents as samples, but with extractant instead of mushrooms extract. A calibration curve with gallic acid as reference antioxidant was plotted before each measurement set,

Similar experimental procedures were applied for both aqueous and hydroalcoholic extracts, different samples dilutions were used so that the linear domain

Total polyphenols content were expressed as milligrams of gallic acid equivalents per mL of extract, and then reported to mushroom dry weight (mg GAE/g d.w.). All experiments were performed in triplicate and the means ± standard deviations (SD)

Flavonoids are antioxidant compounds whose structure has two benzene rings (A and B) and an oxygen containing pyran ring (C). Six subclasses of flavonoids are generally accepted for classification, as follows: flavonols, flavones, isoflavones, flavanones, anthocyanidins and flavonols [70–72], differentiated by the oxidation

*Reaction scheme for total polyphenols determinations by Folin Ciocalteu spectrometric procedure.*

of Beer-Lambert-Bouguer law and calibration range were reached.

H2O2, at ratios of 0.2 g dry mushroom sample to 50 mL solution.

*DOI: http://dx.doi.org/10.5772/intechopen.82578*

*3.1.1 Total content of polyphenols (TCP)*

samples, and may be described in **Figure 1** [68].

calibration range chosen was 0.01–0.08 mg/mL.

in mushrooms [37].

were reported [69].

*3.1.2 Total flavonoid content (TFC)*

less than 2 mm, cap and stipe separately; (iii) extraction of analyte(s), for 4 h, at room temperature, under continuous mixing, in two type of solvents—redistilled water and hydroalcoholic 50% (v/v), dry matter (g) to solvent ratio (mL) was of 4:100; (iv) centrifugation of obtained extracts; (v) wet digestion with concentrated HNO3 and H2O2, at ratios of 0.2 g dry mushroom sample to 50 mL solution.

## *3.1.1 Total content of polyphenols (TCP)*

*Food Engineering*

**mushrooms**

**3.1 Analytical techniques**

through an increased water retention capacity, water absorption capacity, foaming capacity, fat absorption capacity and a decreased bulk density and syneresis. These

Drying methods (convective drying, freeze-drying, vacuum microwave drying and a combination of convective predrying and vacuum microwave finish drying) were tested in order to evaluate their influence on the sensory profile and implicitly on the quality of the oyster mushrooms (*Pleurotus ostreatus* Jacq.) [62]. The total concentration of aroma/volatiles compounds of fresh mushroom was reduced significantly by all drying treatments. However, the combined treatment mentioned above leads to obtaining products with a sensory profile closer to the fresh mushrooms. Nonthermal plasma technology (NTPT) was investigated in order to better understanding of the mechanism of interaction of food bioactive compounds and plasma and consequently its successful adoption by industry. Reviewing the influence of NTPT on functional food components, Muhammad et al. [63] showed that the plasma activated water (PAW) has the effect of increasing the antioxidant activity and the concentration of ascorbic acid of button mushroom. The antioxi-

data indicated that mushroom flours can be very suitable in human diet.

dant activity was extended with increases in PAW processing time.

**3. Evaluation of several antioxidant species in indigenous edible** 

active centers (either oxygen, nitrogen, carbon or sulfur species) [64].

activity of catalase (CAT) and peroxidase (POX) have been evaluated.

Wild grown and cultivated mushroom species have been studied from the perspective of possible correlations between non-enzymatic and enzymatic antioxidants. Thus, for the first category several phytochemical characteristics have been determined: total phenolic content, total flavonoids, antioxidant activity, and four trace micronutrients (Zn, Fe, Mn, and Cu), and for enzymatic antioxidants data,

Different instrumental analytical techniques were reported by authors [66] to identify and quantify antioxidants in edible mushrooms. Among these, high performance chromatography (HPLC) and gas chromatography (GC) using various detection devices, spectroscopic techniques such as nuclear magnetic resonance (RMN), Fourier transform infrared (FTIR), ultraviolet-visible (UV-VIS), as well as inductively coupled plasma mass spectrometry (ICP-MS) are among the most applied. According to available equipment, the mushroom samples were characterized mainly through the absorption spectroscopic methods UV-VIS and FTIR spectroscopy, and information on trace micro-nutrients was gathered through ICP-MS. Analytical process included the classic steps of (a) sampling, (b) sample treatment and/or preparation, and (c) qualitative and/or quantitative analysis. For the step (a), mushroom samples were collected from the natural habitat according with **Table 1** and then representative portions from each sample were taken for further treatment. For the step (b), several procedures have been used: (i) oven drying at 40°C for 48 h; (ii) grounding to

As described in previous chapter, enzymatic and non-enzymatic antioxidants were reported to be present in edible mushrooms, and having roles in balancing human metabolic processes related to oxidative stress [64]. Since mid of twentieth century, after Harman's "Free-Radical Theory of Ageing", an intensive research on involvement of free radicals and antioxidants in living processes has been performed [64]. They are commonly named *reactive species* the advanced knowledge allowed their classification in terms of intensity [65], as well as of the nature of

**22**

Polyphenols are a class of compounds with structures containing at least one aromatic ring with at least one hydroxyl group bonded on it. They are classified according to the number of rings and to their functional groups bound in the structure, and thus we have: phenolic acids, flavonoids, stilbenes, and lignans, coumarins, tannins. Phenolic acids were reported to be the main polyphenolic compounds in mushrooms [37].

Total phenolic content in mushrooms was reported to be successfully determined by Folin Ciocalteu method [67], and an adapted method was applied for the studied mushrooms [67]. The Folin Ciocalteu reagent is a mixture prepared by dissolving sodium tungstate (Na2WO4·2H2O) and sodium molybdate (Na2MoO4·2H2O) in water with hydrochloric acid and phosphoric acid. Hydrated lithium sulfate (Li2SO4·H2O) may be added to this mixture to prevent turbidity that may appear due to formation of some insoluble sodium salts [67]. The mixture is very stable if protected to reduction agents and light. Diluted reagent also needs to be protected to light. The chemical process, occurring at basic pH, is based on molybdenum reduction from +6 (yellow) to +4 (blue) after the oxidation of polyphenols in samples, and may be described in **Figure 1** [68].

Light absorption of a monochromatic radiation of 765 nm is measured with a UV-VIS spectrophotometer. Colored liquid samples were placed in 10 mm light path cuvettes and readings were made versus a blank sample prepared with all reagents as samples, but with extractant instead of mushrooms extract. A calibration curve with gallic acid as reference antioxidant was plotted before each measurement set, calibration range chosen was 0.01–0.08 mg/mL.

Similar experimental procedures were applied for both aqueous and hydroalcoholic extracts, different samples dilutions were used so that the linear domain of Beer-Lambert-Bouguer law and calibration range were reached.

Total polyphenols content were expressed as milligrams of gallic acid equivalents per mL of extract, and then reported to mushroom dry weight (mg GAE/g d.w.). All experiments were performed in triplicate and the means ± standard deviations (SD) were reported [69].

### *3.1.2 Total flavonoid content (TFC)*

Flavonoids are antioxidant compounds whose structure has two benzene rings (A and B) and an oxygen containing pyran ring (C). Six subclasses of flavonoids are generally accepted for classification, as follows: flavonols, flavones, isoflavones, flavanones, anthocyanidins and flavonols [70–72], differentiated by the oxidation

**Figure 1.** *Reaction scheme for total polyphenols determinations by Folin Ciocalteu spectrometric procedure.* level of the C ring of the basic 4-oxoflavonoid (2-phenyl-benzo-γ-pyrone) nucleus. Presence of flavonoids in edible mushroom extracts has been confirmed by several authors [67, 69, 73], some of these reported also their molecular identification (i.e. myricetin, chrysin, catechin, resveratrol, quercetin, others). The antioxidant activity of flavonoids, as for polyphenolics in general, is mainly given by the presence and position of multiple hydroxyl groups in their molecules. Thus, it is considered that the primary mechanism of the radicals scavenging activity of flavonoids is hydrogen atom donation [70, 71].

Total flavonoid content in aqueous and hydro-alcoholic mushroom extracts was measured by the aluminum chloride colorimetric assay described in the literature [74], adapted for the working conditions [69]. Method's principle is based on Al3+ ions to form complex combinations with carbonyl group from C-4 carbon and hydroxyl group from C-3 or C-5 carbons from flavonoids structure (**Figure 2**). Further, aluminum can bond the orthodihydroxyl groups from A- and B-nucleus of flavonoids. The effect of the formation of these bonds results in coloration of the working solution in yellow due to resulted complex combinations.

Sample absorbances were measured in 10 mm cuvettes, at 510 nm, against redistilled water, and concentrations were calculated using the calibration curve drawn before each tests set, in the concentration range of 0.1–1 mg/mL of quercetin, used as reference flavonoid. Total flavonoids contents were expressed as mg quercetin equivalents per mL mushroom extract, and then converted to mushroom dry weight. Analytical data were collected on triplicate samples, mean values together with standard deviations were reported [69].

#### *3.1.3 Antioxidant activity (AA)*

Several chemical and biochemical assays can be used in order to evaluate the total antioxidant activity of mushrooms, and the 2,2-diphenyl-1-picrylhydrazyl DPPH• assay is one of the most frequently used [75–77]. Measurement principle is based on the fact that the antioxidant compounds from mushroom extracts release an electron or a hydrogen atom, and convert the DPPH• to a more stable, diamagnetic molecule, according to reaction below. DPPH• is a stable, long-lived organic nitrogen radical with a strong absorption around 517nm (**Figure 3**).

The antioxidant activity of the studied mushroom extracts was assessed using DPPH• method. For good tests results, fresh ethanolic DPPH solutions (20 mg/mL) were prepared daily by weighing the necessary amount of DPPH powder (usually kept at −20°C), and kept in dark until experiments end. Samples were prepared by mixing aliquots of mushroom extract with DPPH solution, kept in dark at room temperature for 30 min, then sample absorbances were read to spectrophotometer, where zero absorbance was considered the extractant used for extracts preparation.

**25**

lected [10].

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms…*

Reagent and sample blanks were prepared and measured for each test. Calculations

*AA* (%) = [*Areagent blank* –(*Aextract*–*Asample blank*)/*Areagent blank*] × 100 (1)

where *AA* is the global antioxidant activity of mushroom extract solutions, and *A* is the absorbance of the corresponding solution (as per subscripted text). As indicated by Eq. (1), results were calculated as % scavenging of DPPH at a fixed antioxidant concentration. A low absorbance of the tested sample indicates a high

To investigate the chemical functional groups of organic compounds in mushroom extracts, Fourier transform infrared spectroscopy was used. The chemical changes induced by extraction techniques as well as the various functional groups responsible for biological activities were detected in the mid-infrared absorption region using a Vertex 80v spectrometer (Bruker) equipped with a diamond attenuated total reflection crystal accessory [78]. The extracts were placed on the sample chamber of attenuated total reflection—Fourier transform infrared spectrometer without any preparation. The important absorption frequencies

Minerals Cu, Fe, Zn, Mn are included in mushroom food chain, and, in low concentrations, they are considered antioxidant micronutrients. This designation is justified by their capability to catalyze some reactions producing reactive oxygen species, and their enzyme activation properties [10]. Edible mushrooms were reported as metals bio-accumulators, however high levels of essential metals intake

Trace elements Cu, Fe, Zn and Mn were measured by ICP-MS technique in aqueous solutions obtained by wet digestion. Before each test were performed the system calibration using Certipur® Certified Reference Material ICP multielement standard IV (~1000 mg/L in 6.5% HNO3, Merck). The instrumental parameters were: 1.5 kW plasma power, with 1 L/min argon nebulizer flow and 10.75 L/min plasma argon flow respectively, and precise analytical data were col-

, as well as the fingerprint region of the

*DOI: http://dx.doi.org/10.5772/intechopen.82578*

were done according to equation:

*Reaction scheme for antioxidant activity determination by DPPH method.*

**Figure 3.**

free-radical-scavenging activity.

*3.1.4 Fourier transform infrared spectroscopy*

were noted in the range of 3600–600 cm<sup>−</sup><sup>1</sup>

*3.1.5 Inductive coupled plasma mass spectrometry (ICP-MS)*

could produce toxic effects when exceed certain values [78].

spectra [79].

#### **Figure 2.** *Flavonoids complex combinations with Al3+—quercetin example.*

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms… DOI: http://dx.doi.org/10.5772/intechopen.82578*

**Figure 3.**

*Food Engineering*

hydrogen atom donation [70, 71].

with standard deviations were reported [69].

an electron or a hydrogen atom, and convert the DPPH•

nitrogen radical with a strong absorption around 517nm (**Figure 3**).

netic molecule, according to reaction below. DPPH•

*Flavonoids complex combinations with Al3+—quercetin example.*

*3.1.3 Antioxidant activity (AA)*

DPPH•

DPPH•

level of the C ring of the basic 4-oxoflavonoid (2-phenyl-benzo-γ-pyrone) nucleus. Presence of flavonoids in edible mushroom extracts has been confirmed by several authors [67, 69, 73], some of these reported also their molecular identification (i.e. myricetin, chrysin, catechin, resveratrol, quercetin, others). The antioxidant activity of flavonoids, as for polyphenolics in general, is mainly given by the presence and position of multiple hydroxyl groups in their molecules. Thus, it is considered that the primary mechanism of the radicals scavenging activity of flavonoids is

Total flavonoid content in aqueous and hydro-alcoholic mushroom extracts was measured by the aluminum chloride colorimetric assay described in the literature [74], adapted for the working conditions [69]. Method's principle is based on Al3+ ions to form complex combinations with carbonyl group from C-4 carbon and hydroxyl group from C-3 or C-5 carbons from flavonoids structure (**Figure 2**). Further, aluminum can bond the orthodihydroxyl groups from A- and B-nucleus of flavonoids. The effect of the formation of these bonds results in coloration of the

Sample absorbances were measured in 10 mm cuvettes, at 510 nm, against redistilled water, and concentrations were calculated using the calibration curve drawn before each tests set, in the concentration range of 0.1–1 mg/mL of quercetin, used as reference flavonoid. Total flavonoids contents were expressed as mg quercetin equivalents per mL mushroom extract, and then converted to mushroom dry weight. Analytical data were collected on triplicate samples, mean values together

Several chemical and biochemical assays can be used in order to evaluate the total antioxidant activity of mushrooms, and the 2,2-diphenyl-1-picrylhydrazyl

The antioxidant activity of the studied mushroom extracts was assessed using

were prepared daily by weighing the necessary amount of DPPH powder (usually kept at −20°C), and kept in dark until experiments end. Samples were prepared by mixing aliquots of mushroom extract with DPPH solution, kept in dark at room temperature for 30 min, then sample absorbances were read to spectrophotometer, where zero absorbance was considered the extractant used for extracts preparation.

method. For good tests results, fresh ethanolic DPPH solutions (20 mg/mL)

 assay is one of the most frequently used [75–77]. Measurement principle is based on the fact that the antioxidant compounds from mushroom extracts release

to a more stable, diamag-

is a stable, long-lived organic

working solution in yellow due to resulted complex combinations.

**24**

**Figure 2.**

*Reaction scheme for antioxidant activity determination by DPPH method.*

Reagent and sample blanks were prepared and measured for each test. Calculations were done according to equation:

$$AA \text{ (\%)} = \left[ A\_{\text{reangent blank}} - (A\_{\text{extract}} - A\_{\text{sample blank}}) / A\_{\text{reangent blank}} \right] \times 100 \tag{1}$$

where *AA* is the global antioxidant activity of mushroom extract solutions, and *A* is the absorbance of the corresponding solution (as per subscripted text). As indicated by Eq. (1), results were calculated as % scavenging of DPPH at a fixed antioxidant concentration. A low absorbance of the tested sample indicates a high free-radical-scavenging activity.

#### *3.1.4 Fourier transform infrared spectroscopy*

To investigate the chemical functional groups of organic compounds in mushroom extracts, Fourier transform infrared spectroscopy was used. The chemical changes induced by extraction techniques as well as the various functional groups responsible for biological activities were detected in the mid-infrared absorption region using a Vertex 80v spectrometer (Bruker) equipped with a diamond attenuated total reflection crystal accessory [78]. The extracts were placed on the sample chamber of attenuated total reflection—Fourier transform infrared spectrometer without any preparation. The important absorption frequencies were noted in the range of 3600–600 cm<sup>−</sup><sup>1</sup> , as well as the fingerprint region of the spectra [79].

#### *3.1.5 Inductive coupled plasma mass spectrometry (ICP-MS)*

Minerals Cu, Fe, Zn, Mn are included in mushroom food chain, and, in low concentrations, they are considered antioxidant micronutrients. This designation is justified by their capability to catalyze some reactions producing reactive oxygen species, and their enzyme activation properties [10]. Edible mushrooms were reported as metals bio-accumulators, however high levels of essential metals intake could produce toxic effects when exceed certain values [78].

Trace elements Cu, Fe, Zn and Mn were measured by ICP-MS technique in aqueous solutions obtained by wet digestion. Before each test were performed the system calibration using Certipur® Certified Reference Material ICP multielement standard IV (~1000 mg/L in 6.5% HNO3, Merck). The instrumental parameters were: 1.5 kW plasma power, with 1 L/min argon nebulizer flow and 10.75 L/min plasma argon flow respectively, and precise analytical data were collected [10].

#### *3.1.6 Peroxidases*

Peroxidases are one of the classes of enzymes involved in the antioxidant defense mechanisms, together with superoxide dismutase, catalases, and others [10]. Experimental evaluation of peroxidase (POX) relies on its property to oxidize in the presence of hydrogen peroxide or other peroxide compounds (i.e. aromatics).

Oxidation of guaiacol by peroxidases in the presence of H2O2 is generally used for the colorimetric assay, absorbances measurements are performed at 420 nm, the chemical process involved is presented in **Figure 4**.

For accurate POX determination, fresh mushrooms are used to obtain the extracts that are further measured. Final results were reported as POX units per gram of mushroom. The unit of POX activity was defined as the oxidation of one micromole H2O2 per minute at 25°C (pH = 7.0).

#### *3.1.7 Catalases*

Catalases (CAT) are intracellular antioxidant enzymes present in edible mushrooms [10]. They are oxidoreductases, as they use hydrogen peroxide both as a receptor of electrons and as an electrons donor, decomposing it according to reaction presented in **Figure 5**.

Evaluation of catalase activity involves contacting a weighted amount of fresh mushroom with a measured volume of hydrogen peroxide at room temperature, allowed to stand for several minutes. The not-converted amount of hydrogen peroxide is then determined by titration with potassium permanganate in acid medium. Results are reported as CAT units per mushroom gram, while the unit of CAT activity is defined as the amount of enzyme decomposing one micromole H2O2 per minute at 25°C.

#### **3.2 Analytical data and results interpretation**

Total phenolic content of studied mushrooms (aqueous and hydroalcoholic extracts) is mentioned in **Figure 6**. As mentioned before, data were converted to milligrams of gallic acid equivalents (GAE) per gram of dried weight (d.w.). Experimental findings show certain differences between values for hydroalcoholic extracts and those prepared with water as solvent. On the other hand, in general for studied mushrooms, no significant differences between the two anatomic parts, as

**27**

respectively (excepting *Boletus edulis*).

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms…*

distinctly tested, cap and stipe. However, exceptions are observed, and will be further discussed. Concentration values found range between 9.28 ± 0.03 mg GAE/g d.w. (cap of *Cantharellus cibarius*) and 69.65 ± 0.23 mg GAE/g d.w. (cap of *Agaricus campestris*), average values being established for the cultivated species (**Figure 6**). Considering both solvents and mushroom species, *Agaricus campestris* (cap) registered the highest difference between the content of phenolic compounds in the samples prepared in hydroalcoholic extractant instead of water, while the smallest one was determined in the case of *Macrolepiota procera* (stipe), who showed a slight preference for water. Intermediate differences were established for caps of *Russula vesca*, *Russula alutacea* and *Agaricus bisporus* white respectively, where the hydroalcoholic extractant was favorable to a better polyphenols extraction, while for *Boletus edulis* (stipe) water was a more convenient extractant for extraction of phenolic compounds. Differences between anatomic parts were found to *Pleurotus ostreatus* (cultivated), *Russula alutacea*, *Boletus edulis* and *Macrolepiota procera*, higher total polyphenolic content was measured in caps than in stipes. An opposite behavior was found to *Cantharellus cibarius* mushroom hydroalcoholic extract, where TCP values were higher in stipe than in cap, and by 4.93 times of the case of *Agaricus campestris*. For aqueous extracts, closer values of TCP in caps and stipes were found. From the perspective of their origin, experimental findings for TCP showed lower average values for cultivated mushrooms than wild species group, regardless of the extractant type (15.03 mg GAE/g d.w. for water extracts and 20.14 mg GAE/g d.w. for hydro-alcoholic extracts). Also, no significant differences between caps and stipes for aqueous extracts, for both cultivated and wild species, average values for TCP were 13.54 mg GAE/g d.w. for cultivated and 13.05 mg GAE/g d.w. for wild ones

*Total content of polyphenols in studied mushrooms, aqueous and hydroalcoholic extracts, cap and stipe* 

Once the total flavonoid content (TFC) is considered, slight differences from the above mentioned findings were found. Thus, as may be observed in **Figure 7**, for some species (caps or stipes), flavonoids extraction in hydroalcoholic extractant was better than in water. TFC values measured in the hydroalcoholic extracts of *Russula alutacea* (cap), *Cantharellus cibarius* (stipe), *Russula vesca* (cap) and *Pleurotus ostreatus*—wild growing (cap), were higher than in their aqueous extracts. Aqueous extracts TFC exhibited values ranging between 0.22 ± 0.02 mg QE/g

*DOI: http://dx.doi.org/10.5772/intechopen.82578*

*Decomposition of hydrogen peroxide catalyzed by catalase.*

**Figure 5.**

**Figure 6.**

*[mg GAE/g d.w.]*

**Figure 4.** *Oxidation of guaiacol to tetraguaiacol, reaction catalyzed by peroxidase.*

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms… DOI: http://dx.doi.org/10.5772/intechopen.82578*

$$\text{2H}\_2\text{O}\_2 \xrightarrow{\text{catalase}} \text{2H}\_2\text{O} + \text{O}\_2$$

**Figure 5.**

*Food Engineering*

*3.1.6 Peroxidases*

*3.1.7 Catalases*

per minute at 25°C.

reaction presented in **Figure 5**.

Peroxidases are one of the classes of enzymes involved in the antioxidant defense

Oxidation of guaiacol by peroxidases in the presence of H2O2 is generally used for the colorimetric assay, absorbances measurements are performed at 420 nm, the

For accurate POX determination, fresh mushrooms are used to obtain the extracts that are further measured. Final results were reported as POX units per gram of mushroom. The unit of POX activity was defined as the oxidation of one

Catalases (CAT) are intracellular antioxidant enzymes present in edible mushrooms [10]. They are oxidoreductases, as they use hydrogen peroxide both as a receptor of electrons and as an electrons donor, decomposing it according to

Evaluation of catalase activity involves contacting a weighted amount of fresh mushroom with a measured volume of hydrogen peroxide at room temperature, allowed to stand for several minutes. The not-converted amount of hydrogen peroxide is then determined by titration with potassium permanganate in acid medium. Results are reported as CAT units per mushroom gram, while the unit of CAT activity is defined as the amount of enzyme decomposing one micromole H2O2

Total phenolic content of studied mushrooms (aqueous and hydroalcoholic extracts) is mentioned in **Figure 6**. As mentioned before, data were converted to milligrams of gallic acid equivalents (GAE) per gram of dried weight (d.w.). Experimental findings show certain differences between values for hydroalcoholic extracts and those prepared with water as solvent. On the other hand, in general for studied mushrooms, no significant differences between the two anatomic parts, as

mechanisms, together with superoxide dismutase, catalases, and others [10]. Experimental evaluation of peroxidase (POX) relies on its property to oxidize in the presence of hydrogen peroxide or other peroxide compounds (i.e. aromatics).

chemical process involved is presented in **Figure 4**.

micromole H2O2 per minute at 25°C (pH = 7.0).

**3.2 Analytical data and results interpretation**

*Oxidation of guaiacol to tetraguaiacol, reaction catalyzed by peroxidase.*

**26**

**Figure 4.**

*Decomposition of hydrogen peroxide catalyzed by catalase.*

#### **Figure 6.**

*Total content of polyphenols in studied mushrooms, aqueous and hydroalcoholic extracts, cap and stipe [mg GAE/g d.w.]*

distinctly tested, cap and stipe. However, exceptions are observed, and will be further discussed. Concentration values found range between 9.28 ± 0.03 mg GAE/g d.w. (cap of *Cantharellus cibarius*) and 69.65 ± 0.23 mg GAE/g d.w. (cap of *Agaricus campestris*), average values being established for the cultivated species (**Figure 6**).

Considering both solvents and mushroom species, *Agaricus campestris* (cap) registered the highest difference between the content of phenolic compounds in the samples prepared in hydroalcoholic extractant instead of water, while the smallest one was determined in the case of *Macrolepiota procera* (stipe), who showed a slight preference for water. Intermediate differences were established for caps of *Russula vesca*, *Russula alutacea* and *Agaricus bisporus* white respectively, where the hydroalcoholic extractant was favorable to a better polyphenols extraction, while for *Boletus edulis* (stipe) water was a more convenient extractant for extraction of phenolic compounds.

Differences between anatomic parts were found to *Pleurotus ostreatus* (cultivated), *Russula alutacea*, *Boletus edulis* and *Macrolepiota procera*, higher total polyphenolic content was measured in caps than in stipes. An opposite behavior was found to *Cantharellus cibarius* mushroom hydroalcoholic extract, where TCP values were higher in stipe than in cap, and by 4.93 times of the case of *Agaricus campestris*. For aqueous extracts, closer values of TCP in caps and stipes were found. From the perspective of their origin, experimental findings for TCP showed lower average values for cultivated mushrooms than wild species group, regardless of the extractant type (15.03 mg GAE/g d.w. for water extracts and 20.14 mg GAE/g d.w. for hydro-alcoholic extracts). Also, no significant differences between caps and stipes for aqueous extracts, for both cultivated and wild species, average values for TCP were 13.54 mg GAE/g d.w. for cultivated and 13.05 mg GAE/g d.w. for wild ones respectively (excepting *Boletus edulis*).

Once the total flavonoid content (TFC) is considered, slight differences from the above mentioned findings were found. Thus, as may be observed in **Figure 7**, for some species (caps or stipes), flavonoids extraction in hydroalcoholic extractant was better than in water. TFC values measured in the hydroalcoholic extracts of *Russula alutacea* (cap), *Cantharellus cibarius* (stipe), *Russula vesca* (cap) and *Pleurotus ostreatus*—wild growing (cap), were higher than in their aqueous extracts. Aqueous extracts TFC exhibited values ranging between 0.22 ± 0.02 mg QE/g

**Figure 7.**

*Total flavonoids content in studied mushrooms, aqueous and hydroalcoholic extracts, cap and stipe [mg quercetin/g d.w.].*

d.w. (stipe of *Pleurotus ostreatus* cultivated) and 26.51 ± 0.04 mg QE/g d.w. (cap of *Boletus edulis*), while TFC values for hydroalcoholic extracts were in the range of 0.12 ± 0.04 mg QE/g d.w. (stipe of *Russula vesca*) and 20.77 ± 0.06 mg QE/g d.w. (stipe of *Cantharellus cibarius*).

Similarities with total phenolics were found for total flavonoids detected, for comparisons made between mushroom species of different origin. Thus, experimental data showed that TFC average values in cultivated species were lower than in wild grown ones. Compared data for flavonoids found in caps and stipes showed that for both cultivated and wild species higher flavonoids content were noticed in cap for both extracts. Several exceptions have been noticed from this behavior: hydroalcoholic extracts of *Agaricus bisporus* brown (cultivated), and aqueous extract of *Agaricus campestris* and alcoholic extracts of *Cantharellus cibarius* and *Macrolepiota procera* respectively (wild species). One may conclude that total flavonoids content varied depending on the mushroom species and used extractant, polar solvents dissolving more flavonoids [69].

Analytical data for antioxidant activity of studied edible mushrooms extracts (cap and stipe), evaluated through DPPH method as previously described, showed some high and low limits. Thus, for aqueous extracts it was found that, *Agaricus bisporus brown* (cap) had the strongest DPPH radical-scavenging activity of 88.64%, while the lowest value of 25.72% was found in *Macrolepiota procera* (cap). When water-ethanol 50% (v/v) was used as extraction solvent, limit values were 74.93% for *Boletus edulis* (cap) and 13.61% respectively for *Russula alutacea* (stipe). Also, notable differences were found between analytical data recorded on cap and stipe of studied species [69]. Example of hydroalcoholic extracts is relevant: while most of mushroom species showed higher *AA*% values in caps than in stipes corresponding to same species, several exceptions were observed for *Cantharellus cibarius*, *Macrolepiota procera* and *Agaricus bisporus* brown where slight higher values were found in stipes than in caps. With regards to this phytochemical parameter (*AA*), a general behavior was noticed for studied mushrooms. Thus, notable differences between analytical data recorded for various species and when using the two extractant types.

By infrared spectroscopy several chemical functional groups that may be responsible for the antioxidant character of mushrooms, as quantified by classes of compounds or as a whole with the above mentioned ultraviolet-visible spectroscopic methods. Significant characteristic frequencies were observed in the range of 3600–600 cm<sup>−</sup><sup>1</sup> and fingerprint region, and were assigned to different organic compounds with ▬OH functional groups. As a relevant example, obtained results indicated that hydroalcoholic mushroom extracts may contain active functional groups as alcohols, esters and aldehydes [10].

**29**

**Figure 9.**

**Figure 8.**

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms…*

Quantification of micronutrients in studied mushrooms showed, as may be observed in **Figure 8**, they are rather rich in Mn, Fe, Cu, and Zn, metals having a significant role in enzymatic systems activation. One may exemplify with data for species like *Boletus edulis* with Mn content of 130.73 mg/kg, and *Macrolepiota* 

Enzymatic antioxidants peroxidase and catalase determinations in indigenous mushroom species were previously reported [10, 57], also **Figure 9** shows both enzymes activities. Significant variations were found for both studied enzymes. Higher values of catalase activity were found in species as *Agaricus bisporus white* and *brown* and *Russula vesca*, while species like *Boletus edulis* and *Pleurotus ostreatus wild* showed lower values. Measured values for catalase activity were in the range of 3.58–14.67 μmols H2O2/g/min. Also, highest values of peroxidase activity were found in mushroom species like *Russula alutaceea* and *Macrolepiota procera*, while

From the origin perspective, it was found that *Pleurotus ostreatus* cultivated had a 2.49 times higher catalase activity than the same wild species, while peroxidase activities for both wild and cultivated *Pleurotus ostreatus* were similar. Some correlations between metallic nutrients content enzymatic activities of mushrooms have been reported [10], and next chapter, through a chemometric approach will highlight further correlations between enzymatic and non-enzymatic antioxidant

*DOI: http://dx.doi.org/10.5772/intechopen.82578*

*procera*, with Fe content of 715.14 mg/kg [10].

lowest values of this enzyme were found in *Chantarellus cibarius*.

*Micronutrients concentrations in studied mushroom samples: a) Mn; b) Fe; c) Cu; d) Zn.*

*Peroxidase and catalase activity in studied mushroom species.*

species, as were determined for studied mushrooms.

*Correlation between Enzymatic and Non-Enzymatic Antioxidants in Several Edible Mushrooms… DOI: http://dx.doi.org/10.5772/intechopen.82578*

Quantification of micronutrients in studied mushrooms showed, as may be observed in **Figure 8**, they are rather rich in Mn, Fe, Cu, and Zn, metals having a significant role in enzymatic systems activation. One may exemplify with data for species like *Boletus edulis* with Mn content of 130.73 mg/kg, and *Macrolepiota procera*, with Fe content of 715.14 mg/kg [10].

Enzymatic antioxidants peroxidase and catalase determinations in indigenous mushroom species were previously reported [10, 57], also **Figure 9** shows both enzymes activities. Significant variations were found for both studied enzymes. Higher values of catalase activity were found in species as *Agaricus bisporus white* and *brown* and *Russula vesca*, while species like *Boletus edulis* and *Pleurotus ostreatus wild* showed lower values. Measured values for catalase activity were in the range of 3.58–14.67 μmols H2O2/g/min. Also, highest values of peroxidase activity were found in mushroom species like *Russula alutaceea* and *Macrolepiota procera*, while lowest values of this enzyme were found in *Chantarellus cibarius*.

From the origin perspective, it was found that *Pleurotus ostreatus* cultivated had a 2.49 times higher catalase activity than the same wild species, while peroxidase activities for both wild and cultivated *Pleurotus ostreatus* were similar. Some correlations between metallic nutrients content enzymatic activities of mushrooms have been reported [10], and next chapter, through a chemometric approach will highlight further correlations between enzymatic and non-enzymatic antioxidant species, as were determined for studied mushrooms.

**Figure 8.** *Micronutrients concentrations in studied mushroom samples: a) Mn; b) Fe; c) Cu; d) Zn.*

**Figure 9.** *Peroxidase and catalase activity in studied mushroom species.*

*Food Engineering*

**Figure 7.**

*[mg quercetin/g d.w.].*

(stipe of *Cantharellus cibarius*).

polar solvents dissolving more flavonoids [69].

groups as alcohols, esters and aldehydes [10].

d.w. (stipe of *Pleurotus ostreatus* cultivated) and 26.51 ± 0.04 mg QE/g d.w. (cap of *Boletus edulis*), while TFC values for hydroalcoholic extracts were in the range of 0.12 ± 0.04 mg QE/g d.w. (stipe of *Russula vesca*) and 20.77 ± 0.06 mg QE/g d.w.

*Total flavonoids content in studied mushrooms, aqueous and hydroalcoholic extracts, cap and stipe* 

Similarities with total phenolics were found for total flavonoids detected, for comparisons made between mushroom species of different origin. Thus, experimental data showed that TFC average values in cultivated species were lower than in wild grown ones. Compared data for flavonoids found in caps and stipes showed that for both cultivated and wild species higher flavonoids content were noticed in cap for both extracts. Several exceptions have been noticed from this behavior: hydroalcoholic extracts of *Agaricus bisporus* brown (cultivated), and aqueous extract of *Agaricus campestris* and alcoholic extracts of *Cantharellus cibarius* and *Macrolepiota procera* respectively (wild species). One may conclude that total flavonoids content varied depending on the mushroom species and used extractant,

Analytical data for antioxidant activity of studied edible mushrooms extracts (cap and stipe), evaluated through DPPH method as previously described, showed some high and low limits. Thus, for aqueous extracts it was found that, *Agaricus bisporus brown* (cap) had the strongest DPPH radical-scavenging activity of 88.64%, while the lowest value of 25.72% was found in *Macrolepiota procera* (cap). When water-ethanol 50% (v/v) was used as extraction solvent, limit values were 74.93% for *Boletus edulis* (cap) and 13.61% respectively for *Russula alutacea* (stipe). Also, notable differences were found between analytical data recorded on cap and stipe of studied species [69]. Example of hydroalcoholic extracts is relevant: while most of mushroom species showed higher *AA*% values in caps than in stipes corresponding to same species, several exceptions were observed for *Cantharellus cibarius*, *Macrolepiota procera* and *Agaricus bisporus* brown where slight higher values were found in stipes than in caps. With regards to this phytochemical parameter (*AA*), a general behavior was noticed for studied mushrooms. Thus, notable differences between analytical data

recorded for various species and when using the two extractant types.

By infrared spectroscopy several chemical functional groups that may be responsible for the antioxidant character of mushrooms, as quantified by classes of compounds or as a whole with the above mentioned ultraviolet-visible spectroscopic methods. Significant characteristic frequencies were observed in the range

compounds with ▬OH functional groups. As a relevant example, obtained results indicated that hydroalcoholic mushroom extracts may contain active functional

and fingerprint region, and were assigned to different organic

**28**

of 3600–600 cm<sup>−</sup><sup>1</sup>
