Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill

Abstracts

Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP (horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O₂• –), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.

Agaricus blazei Murrill; antioxidant; oxidant species; chemiluminescence; polymorphonuclear neutrophils

Cogumelos apresentam propriedades nutricionais e medicinais bastante empregadas na manutenção da saúde humana e têm sido considerado por pesquisadores como possíveis fontes de seqÜestradores de radicais livres. Neste trabalho, as propriedades antioxidantes de extratos aquosos de Agaricus blazei Murill, produzidos por maceração e decocção, são demonstrados in vitro. A resistência à oxidação é demonstrada através de três mecanismos: i) inibição de processos oxidativos enzimáticos, with 100% de inibição da HRP (horseradish peroxidase) e MPO (myeloperoxidase); ii) inibição do estresse oxidativo celular, com 80% de inibição do oxidative burst de neutrófilos polimorfonucleares (PMN) e iii) ação direta sobre espécies reativas, com 62% e 87% de supressão do HOCl e ânion superóxido (O₂• –), respectivamente. A partir destes dados, concluise que o extrato aquoso deste cogumelo tem significativa capacidade antioxidante, indicando sua possível aplicação para propósitos nutracêuticos e medicinais.

Agaricus blazei Murril; antioxidantes; espécies oxidantes; quimiluminescência; neutrófilos polimorfonucleares

INTRODUCTION

Oxidations are essential reactions in the biological processes of many organisms. However, reactive oxygen species (ROS), which are continuously produced in vivo, are known to promote cell death and tissue damage (Calabrese et al. 2007, Halliwell and Gutteridge 1999). Aging and diseases, including atherosclerosis, diabetes, cancer and cirrhosis, have been linked to oxidative damage (Vellosa et al. 2007a, Morton et al 2000, Eastwood 1999, Vinson et al. 1995, Halliwell et al. 1995a).

There is an increasing emphasis on research aimed to identify and utilise antioxidants from natural sources (Ramarathnam et al. 1995). Antioxidants are of great interest because of their possible role in protecting the organism against damage by ROS (Halliwell et al. 1995b) such as superoxide anion radical (O₂• –), hydrogen peroxide (H₂O₂) and hydroxyl radical (HO^•). These species are by-products of normal metabolism that can attack biological molecules such as lipids, proteins, DNA and RNA, leading to cell or tissue injury associated with degenerative diseases (Jung et al. 1999).

Agaricus blazei Murrill (Agaricaceae), an edible mushroom, is native to the small Brazilian town of Piedade (Mizuno 1995, Heinemann et al. 1993). Today, A. blazei is consumed globally as food or in tea on account of its putative medicinal properties (Mshandete and Cuff 2007, Kaneno et al. 2004). Previous studies with isolated fractions of the A. blazei fruit bodies indicated that some samples exhibited antimutagenic, anticarcinogenic and immunostimulative activities (Mizuno et al. 1998, Kawagishi et al. 1990, Itoh et al. 1994, Osaki et al. 1994, Mizuno et al. 1990).

Natural products with antioxidant activities are used to aid the endogenous immune system. As a result, increasing interest has been expressed in the antioxidative roles of nutraceutical products (Pietta et al. 1998). Polyphenols and flavonoids are plant constituents with a probable effect on the organism's ability to scavenge free radicals (Vellosa et al. 2006). Studies by Barros et al. (2007), Ferreira et al. (2007) and others (Turkoglu et al. 2007, Oliveira et al. 2007, Cheung et al. 2003, Cheung and Cheung 2005, Lo and Cheung 2005, Yang et al. 2002) indicate a correlation between the mushrooms' antioxidant activity and their phenolic content.

The aim of this study is to evaluate the antioxidant properties of aqueous extracts of A. blazei, particularly the reduction in activity of oxidoredutase enzymes such as HRP, the scavenging effects on radicals such as superoxide radical and HOCl and the inhibition of the oxidative burst of activity of polymorphonuclear neutrophils (PMNs).

MATERIALS AND METHODS

The enzyme horseradish peroxidise (HRP) type VI, phorbol-12-myristate-13-acetate (PMA), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 5-thio-2-nitrobenzoic acid (TNB), phenazine methosulphate (PMS), nicotinamide adenine dinucleotide reduced (NADH), nitrobluetetrazolium (NBT), luminol and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) were purchased from Sigma-Aldrich Chemical Company, (St Louis, MO, USA). Hydrogen peroxide (30% solution) was purchased from Peroxides, Brazil (São Paulo, SP, Brazil). All of the reagents used for buffer preparation were analytical grade.

Apparatus

All chemiluminescence measurements were carried out using a luminescence photometer Luminometer 1251 (BioOrbit model, Finland) and were monitored on a computer workstation running the program Multiuse v 2.0. All assays were conducted on an HP 8453 Diode Array Spectrophotometer.

Mushroom Material

Agaricus blazei Murrill (A. blazei) fruiting bodies were collected at Valemar Ranch (São José do Rio Preto, SP, Brazil) and identified by Dr. Arailde Fontes Urben (EMBRAPA researcher).

Two types of A. blazei aqueous extracts were prepared. First, a DECOCTION extract was obtained by decocting 1 g of powdered mushroom in 100 mL of distilled water until the volume was reduced by half. Next, a MACERATION extract was obtained by macerating 1 g of powdered mushroom in 50 mL of distilled water. Material was refrigerated at 4°C for 60 minutes and then filtered and fractionated. The extract was used within 4 hours.

HRP/H₂O₂/Guaiacol Kinetics Activity Measurements

Both macerate and decoction extracts were subjected to the peroxidase colorimetric assay. All horseradish peroxidase activity assays were conducted by measuring the oxidation of guaiacol as substrate. H₂O₂ and HRP solutions were prepared in MilliQ water and their concentrations were determined spectrophotometrically using the molar absorption coefficients of ϵ_{230 nm} = 80 M⁻¹cm⁻¹ (Brestel 1985) and ϵ _{403 nm} = 1.02 × 10⁵ M⁻¹cm⁻¹ (Ohlsson and Paul 1976), respectively. Typical reaction mixtures contained 10⁻⁹ M HRP enzyme, 50 mM phosphate buffer, pH 7.0 and 25 mM guaiacol, at 25°C. The reaction was started by the addition of H₂O₂ and product formation was followed spectrophotometrically at 470 nm (Makinen and Tenovuo 1982, Gazarian and Lagrimini 1996). The initial reaction rate (v_o) was determined by the angular coefficient of the plot of absorbance at 470 nm versus time (seconds), extrapolated to time zero. All reactions were conducted in triplicate (Desser et al. 1972).

Chemiluminescence Assay of HRP Activity

Inhibition of HRP activity by A. blazei was measured by chemiluminescence. Luminol solutions were prepared in MilliQ water and their concentrations were determined spectrophotometrically using the molar absorption coefficient ϵ _{347 nm} = 7,636 M⁻¹cm⁻¹ (Allen and Loose 1976). Typical reaction mixtures contained 3×10⁻⁸ M HRP enzyme, 50 mM phosphate buffer, pH 7.0 and 5×10⁻⁶ M luminol, at 37 °C (Brestel 1985). All reactions were conducted in triplicate. The net reaction is illustrated in the following scheme:

where HRP, HRP-I and HRP-II are the ferric peroxidase and intermediate compounds of horseradish peroxidase; and L and L^• are luminol and its radical product of one electron oxidation, respectively. The radical product of luminol oxidation is then converted into 3-aminophthalate, resulting in light emission (Dodeigne et al. 2000). The integrated light emission was taken as the analytical readout.

MPO/H₂O₂/TNB Kinetic Activity Measurements

All myeloperoxidase (MPO) activity assays were conducted by measuring the oxidation of 5-thio-2-nitrobenzoic acid (TNB). TNB was prepared according to Ching et al. (1994). The concentration of TNB was determined by measuring the absorbance at 412 nm using ϵ = 13,600 M⁻¹ cm⁻¹.

Typical reaction mixtures contained 0.04 U/mg MPO enzyme, 50 mM phosphate buffer, pH 7.0, 0.07 mM TNB, 0.1 mM taurine, 50 µL of A. blazei, and 0.1 mM H₂O₂, at 25°C. The reaction was started by the addition of H₂O₂ and the TNB consumption was monitored spectrophotometrically at 412 nm. The initial reaction rate (v_o) was determined by the angular coefficient of the plot of the absorbance at 412 nm versus time (in seconds), extrapolated to time zero. All reactions were conducted in triplicate.

The antioxidant activity of the extract from macerated A. blazei was determined by comparing the results of three reactions:

The antioxidant activity was calculated as the percent inhibition of TNB oxidation to DTNB by the following equation:

where A, B and C correspond to the absorbance change observed for reactions A, B and C, respectively.

Chemiluminescence Assay Using PMNS

Primary polymorphonuclear neutrophils (PMNs) were obtained according to the following protocol. A 5% oyster glycogen solution (dissolved in 0.85% NaCl) was injected into the peritoneum of anesthetized rats. This work was submitted to the Ethics Committee of FCFAR / UNESP and authorized by the protocol CEP n° 65/2009. The animals were kept with food and liquid ad libitum and sacrificed 12 h after injection. Calcium-free Dulbecco's phosphate-buffered saline (PBS-D, 20 mL), containing 10 IU heparin per mL, was injected into the peritoneal cavity (Babior and Cohen 1981, Paino et al. 2005). The discharge was collected and centrifuged for 3 min at 2,000 g. The cell pellet was layered on Ficoll-Hypaque 1077 and centrifuged for 25 min at 2,000 g. PMN cells were collected, washed and kept in ice-cold PBS-D until required.

The effect of mushroom macerate on PMN activity was assayed by chemiluminescence (Parij et al. 1998). PMNs (1 × 10⁶ cells/mL), suspended in PBS-D buffer, pH 7.2, were stimulated by the addition of phorbol-12-myristate-13-acetate (PMA, 1×10⁻⁵ mM) in the presence of luminol (10⁻⁵ M). The chemiluminescence emission was measured in millivolts (mV) at 37°C in 1-s intervals for 1 hour. The ideal dose was determined by the addition of different volumes of A. blazei extracts. PMN activity was expressed as a percent reduction in the maximum chemiluminescence response of PMNs to PMA in a positive control reaction. Negative controls were performed by omitting PMA. The integrated light emission was taken as the analytical readout.

Assay of Superoxide Anion Radical (O₂• –) Suppression

Superoxide radicals, produced by reduced nicotinamide adenine dinucleotide (NADH) and phenazine methosulphate (PMS), reduce nitrotetrazolium blue chloride (NBT) to produce a formazan compound. The intensity of the colour is inversely proportional to the antioxidant concentration (Vellosa et al. 2007B, Kakkar et al. 1984). The assay was carried out in sodium pyrophosphate buffer (25 mM, pH 8.3) and the mixture contained 25 µL of 0.372 mM PMS, 75 µL of 0.6 mM NBT, 50 µL of 1.56 mM NADH, macerate mushroom extract (several volumes) and buffer to a final volume of 1 mL. Reactions were started by the addition of NADH. After a 90 seconds incubation at 25°C, 100 µL of glacial acetic acid and 900 µL of sodium pyrophosphate buffer were added. After vigorous homogenisation, the colour intensity of the mixture was measured at 560 nm. All reactions were conducted in triplicate.

Statistical Analysis

Data are reported as the mean ± SD. The Student t-test was used to determine the difference between test and control preparations, with the level of significance set at p < 0.05.

RESULTS AND DISCUSSION

The goal of this study was to evaluate the aqueous extract from A. blazei as a source of pharmacological agents against oxidative stress. The aqueous extracts used for most experiments were obtained by maceration at 25°C, as it was observed that extracts obtained by decoction had low activity (Figure 1). A possible explanation for this observation is that the high temperature (∼100°C) of the extraction induces polymerization of lower molar mass phenols, leading to a lower antioxidant capacity.

Figure 1
HRP/guaiacol/H₂O₂ assay in aqueous extract, macerated and decoction of A. blazei. The incubation mixture contained HRP/ 3×10⁻⁵ mM; H₂O₂ /3.0×10⁻¹ mM; Guaiacol /5.0×10⁻⁴ mM, (Mean ± SD; n=3).

Kinetics Enzymatic HRP/Guaiacol/H₂O₂ Assay

Job and Dunford (1976) have studied the oxidation of several phenols and aromatic amines through horseradish peroxidase (HRP). The one-electron oxidation of organic compounds (AH) by horseradish peroxidase may be represented as follows:

The HRP activity was studied by spectrophotometrically monitoring guaiacol oxidation, which generates a chromophore with an absorbance at 470 nm within 1 minute. Figure 1 presents the inhibitory effects of aqueous extracts of A. blazei, prepared by either maceration or decoction, on the HRP/guaiacol/H₂O₂ assay. HRP activity was completely inhibited in the presence of the macerate. The mechanism by which the extracts exert their antioxidant properties cannot be determined by this method alone; however, it is likely that the macerate extract provides phenolic compounds able to act on the enzyme system or to reduce the oxidised product.

Chemiluminescence Assay Using HRP and Agaricus Blazei Murill

The luminol-dependent chemiluminescent assay lacks specificity regarding the ROS generated upon neutrophil activation (Dodeigne et al. 2000); therefore, experiments were performed to determine the effect of A. blazei extracts on the activity of the HRP/H₂O₂ enzymatic system. Peroxidases catalyse the oxidation of luminol by hydrogen peroxide.

Figure 2 presents the inhibitory effects of aqueous macerated extracts from A. blazei on the HRP-catalysed, luminol-dependent chemiluminescence assay. Note that in the control reaction of luminol, HRP and H₂O₂, the light intensity is 2,000 mV; the addition of 100 µL of aqueous macerated extract reduces the light intensity to 18.5 mV and, hence, reduces oxidation by HRP.

Figure 2
Effect of the mushroom macerate extract over chemiluminescence intensity during luminol oxidation by hydrogen peroxide catalyzed by HRP. The incubation mixture contained 50 mM of Phosphate buffer, pH 7.0, 3×10⁻⁸ mM HRP, and 5×10⁻⁶ mM luminol. Chemiluminescence intensity was registered 5 min, by 37°C, after the start of the reaction.

Antioxidant Activity of Agaricus blazei murill by the MPO/H₂O₂/TNB System

The neutrophils' oxidative burst is the result of the assembly of the multi-enzyme NADPH-oxidase system that promotes the one-electron reduction of oxygen to superoxide anion radical (Babior 2000). Next, this species is reduced to hydrogen peroxide by superoxide dismutase. Finally, hydrogen peroxide is used by myeloperoxidase (MPO) to oxidise chloride to hypochlorous acid (HOCl) (Hampton et al. 1998, Podrez et al. 2000, Lapenna and Cuccurullo 1996). This highly oxidising molecule has been proposed to be the primary agent responsible for the antimicrobial action of PMNs. Figure 3 represents the antioxidant activity of aqueous extracts of A. blazei in the following reactions:

This assay is based on the ability of a substance (scavenger) to inhibit the oxidation of 5-thio-2-nitrobenzoic acid (TNB) to 5,5′-dithio-2-nitrobenzoic acid (DTNB) in the presence of the oxidants HOCl and chloramine-taurine (Tau-Cl, a species also formed in vivo), (Ching et al. 1994) generated in vitro by MPO and H₂O₂. The antioxidant activity of the macerated extract of A. blazei is expressed by the following reaction:

The antioxidant activity, calculated as the percent inhibition of TNB oxidation, was 62%.

Figure 3
Antioxidant activity of aqueous macerate extract of A. blazei on the system MPO/Taurine/H₂O₂/Cl⁻. (Mean ± SD; n=3). (Mean ± SD; n=3). The incubation mixture contained: MPO/0.04 U/mg, H₂O₂/2×10⁻⁵ mM, TNB/0.074 mM, Tau/0.1 mM, PBS-D buffer by Cl⁻, pH 7.4. Columns: A, MPO/Taurine/Catalase/TNB, by absorbance of 1.130; B, MPO/Taurine/H₂O₂/Catalase/TNB, by absorbance of 0.130; and C, MPO/Taurine/H₂O₂/Catalase/TNB/A. blazei, by absorbance of 0.749.

Luminol-Dependent Chemiluminescence Assay Using Polymorphonuclear Neutrophils (PMNS): Oxidative Burst Inhibition by Agaricus Blazei Murill

Luminol-dependent chemiluminescence (LDCL), commonly believed to result from the production of O₂• –, HOCl and H₂O₂, is observed during the oxidative burst of PMNs (Dahlgren and Karlsson 1987, Allen 2000).

The oxidative burst of PMNs was monitored using the soluble stimulant phormol-12-myristate-13-acetate (PMA). Studies by Dahlgren and Karlsson (1987) showed that LDCL produced by PMNs depends largely on the generation of HOCl by the MPO/H₂O₂/Cl⁻ system. Thus, the oxidation of luminol can occur either by the peroxidase reaction or by the direct reaction of luminol with HOCl.

PMN assays conducted in the presence of the A. blazei aqueous extracts demonstrate reduced LDCL on PMA-stimulated PMNs (Figure 4). This effect may be linked to the action of compounds in the A. blazei extract on the oxidative burst enzymes or to a direct reaction with HOCl and possibly other reactive oxygen species such as superoxide anion.

Figure 4
Effect of the mushroom macerate extract over luminol-dependent chemiluminescence assay using PMN. The incubation mixture contained PMN/1×10⁶ cells/mL stimulators by PMA/1×10⁻⁵ mM the absence end presence of 50µL of A. blazei.

Suppression of superoxide anion radical (O₂• –)

We have also evaluated the potential of macerated extracts of A. blazei to suppress superoxide anion formation using a non-enzymatic superoxide generation method (Kakkar et al. 1984). The radical is generated by reacting phenazine methosulphate with NADH, resulting in NBT reduction. We used this reaction to evaluate if the extract (Figure 5) was able to suppress superoxide anion formation in vitro. Note that in this reaction, NBT must be present in excess to evaluate the real potency of samples in suppressing superoxide anion (Vellosa et al. 2007B). The ability of A. blazei extracts to scavenge superoxide anion radical (O₂• –) (% inhibition = 51.8 ± 2.1%) may be expected to contribute to its possible anti-radical action.

Figure 5
Suppression of superoxide anion radical (O₂• –) by different volumes (µL) of A. blazei macerate extract. The incubation mixture contained: Phenazine Methosulphate – 168×10⁻⁶ mM; NBT – 30×10⁻⁶ mM; NADH – 78×10⁻⁶ mM; Sodium pyrophosfate buffer – 25 mM. (Mean ± SD; n=3).

CONCLUSION

The results indicate that A. blazei aqueous macerated extracts are capable of diminishing ROS levels by interfering with enzymatic ROS generators or by suppressing different reactive species. Evidence in support of these conclusions came from the inhibited formation of superoxide anion, the lessened extent of HRP-dependent luminol-dependent chemiluminescence, the inhibition of the oxidative-burst in neutrophils and the inhibition of HRP and MPO enzymatic oxidative kinetics. From these observations, it may be suggested that the A. blazei mushroom can be used as a possible pharmacological agent against oxidative stress and as a nutritional and pharmaceutical source of new therapeutic compounds, due to the mushroom's excellent antioxidant properties.

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support. The authors thank Regina H. C. Garcia and Edalcy G. Cerrano for providing most of the A. blazei used in this study.

REFERENCES

• Allen RC, Dale DC and Taylor Jr FB. 2000. Blood phagocyte luminescence: gauging systemic immune activation. Methods Enzymol 305: 591-609.
• Allen RC and Loose LD. 1976. Phagocitic activation of luminol-dependent chemiluminecence in rabbit alveolar and peritoneal macrophages. Biochem Biophys Res Commun 69: 245-253.
• Babior BM. 2000. Phagocytes and oxidative stress. Am J Med 109: 33-44.
• Babior M and Cohen HJ. 1981. Measurement of neutrophil function: phagocytosis, degranulation, the respiratory burst and bactericidal killing. In: Cline MJ (Ed), Methods in Hematology: Leucocyte Function, Churchill Livingstone, New York, p. 1-38.
• Barros L, Ferreira MJ, Queirós B, Ferreira ICFR and Baptista P. 2007. Total phenols, ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chem 100: 413-419.
• Brestel EP. 1985. Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence. Biochem Biophys Res Commun 126: 482-488.
• Calabrese V, Guagliano E, Sapienza M, Panebianco M, Calafato S, Puleo E, Pennisi G, Mancuso C, Butterfield DA and Stella AG. 2007. Redox regulation of cellular stress response in aging and neurodegenerative disorders: role of vitagenes. Neurochem Res 32: 757-773.
• Cheung LM and Cheung PCK. 2005. Mushroom extracts with antioxidant activity against lipid peroxidation. Food Chem 89: 403-409.
• Cheung LM, Cheung PCK and Ooi VEC. 2003. Antioxidant activity and total phenolics of edible mushroom extracts. Food Chem 81: 249-255.
• Ching TL, De Jong J and Bast A. 1994. A method for screening hypochlorous acid scavengers by inhibition of the oxidation of 5-thio-2-nitrobenzoic acid: application to anti-asthmatic drugs. Anal Biochem 218: 377-381.
• Dahlgren C and Karlsson A. 1999. Respiratory Burst in Human Neutrophils. Immunol Method 232: 3-14.
• Dodeigne C, Thumus L and Lejeune R. 2000. Chemiluminescence as diagnostic tool. A review. Talanta 51: 415-439.
• Eastwood MA. 1999. Interaction of dietary antioxidants in vivo: how fruits and vegetables prevent disease? QJM: Monthly Journal of the Association of Physicians 92: 527-530.
• Ferreira ICFR, Baptista P, Vilas-Boas M and Barros L. 2007. Free radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal. Food Chem 100: 1511-1516.
• Gazarian IG and Lagrimini LM. 1996. Purification and unusual kinetic properties of a tabacco anionic peroxidase. Phytochem 41: 1029-1034.
• Halliwell B, Aeschbach R, Loliger J and Aruoma OI. 1995b. The characterization of antioxidants. Food Chem Toxicol 33: 601-617.
• Halliwell B and Gutteridge JMC. 1999. Free Radicals in Biology and Medicine, 5th ed., Claredon Press: Oxford, 888 p.
• Halliwell B, Murcia MA, Chirico S and Aruoma OI. 1995a. Free radicals and antioxidants in food and in vivo: what they do and how they work. Crit Rev Food Sci Nutr 35: 7-20.
• Hampton MB, Kettle AJ and Winterbourn CC. 1998. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92: 3007-3017.
• Heinemann P. 1993. Agarici Austroamericani. VIII. Agariceae des regions intertropicales d'Amérique du Sud. Bull Jard Bot Nat Belg 62: 355-384.
• Itoh H, Amano H and Noda H. 1994. Inhibitory action of a (1-6)-β-glucan–protein complex (FIII-2-b) isolated from Agaricus blazei Murrill ([Himematsutake]) on meth a fibrosarcoma bearing mice and its antitumor mechanism. Jpn J Pharmacol 66: 265-271.
• Job D and Dunford B. 1976. Substituent effect on the oxidation of phenols and aromatic amines by horseradish peroxidase compound I. Eur J Biochem 66: 607-614.
• Jung HA, Park JC, Chung HY, Kim J and Choi JS. 1999. Antioxidant flavonoids and chlorogenic acid from the leaves of Eriobotrya japonica. Arch Pharm Res 22: 213-218.
• Kakkar P, Das B and Viswanathan PN. 1984. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem Biophys 21: 130-132.
• Kaneno R, Fontanari LM, Santos AS, Di-Stasi LC, Rodrigues-Filho E and Eira AF. 2004. Effects of extract from Brazilian sun-mushrron (Agaricus blazei) on the NK activity and lymphoproliferative responsiveness of ehrlich tumor-bearing mice. Food Chem Toxicol 42: 909-916.
• Kawagishi H, Kanao T, Inagaki R, Mizuno T, Shimura K, Ito H, Hagiwara T and Nakamura T. 1990. Formolysis of a potent antitumor (1-6)-beta-d-glucan–protein complex from Agaricus blazei fruiting bodies and antitumor activity of the resulting products. Carbohydr Polymers 12: 393-403.
• Lapenna D and Cuccurullo F. 1996. Hypochlorous acid and its pharmacological antagonism: an update picture. Gen Pharmacol 27: 1147-1154.
• Lo KM and Cheung PCK. 2005. Antioxidant activity of extracts from the fruiting bodies of Agrocybe aegerita var. alba. Food Chem 89: 533-539.
• Makinen KK and Tenovuo J. 1982. Observations on the use of guaiacol and 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as peroxidase substrate. Anal Biochem 126: 100-108.
• Mizuno M, Morimoto M, Minato K and Tsuchida H. 1998. Polysaccharides from Agaricus blazei stimulate lymphocyte T cell subsets in mice. Biosci Biotechnol Biochem 62: 434-437.
• Mizuno T. 1995. Bioactive biomolecules of mushrooms-food, function and medicinal effect of mushroom fungi. Food Rev Int 11: 7-21.
• Mizuno T, Hagiwara T, Nakamura T, Ito H, Shimura K, Sumiya T and Asakura A. 1990. Antitumor activity and some properties of water-soluble polysaccharides from [Himematsutake], the fruiting body of Agaricus blazei Murill. Agric Biol Chem 54: 2889-2896.
• Morton LW, Caccetta RAA, Puddey IB and Croft KD. 2000. Chemistry and biological effects of dietary phenolic compounds: relevance to cardiovascular disease. Clin Exp Pharmacol Physiol 27: 52-59.
• Mshandete AM and Cuff J. 2007. Proximate end nutrient composition of three types of indigenous edible wild mushrooms grown in Tanzania and their utilization prospects. Afr J Food Agric Nutr Dev 7 http://www.ajfand.net/Volume7/No6/Mshandete2615.pdf (Accessed 25 August 2011)
  » http://www.ajfand.net/Volume7/No6/Mshandete2615.pdf
• Ohlsson PI and Paul KG. 1976. The molar absortivity of horsehadish peroxidase. Acta Chem Scand Ser B 30: 373-377.
• Oliveira OMMF, Vellosa JCR, Fernandes AS, Buffa-Filho W, Hakime-Silva RA, Furlan M and Brunetti il. 2007. Antioxidant activity of Agaricus blazei. Fitoterapia 78: 263-264.
• Osaki Y, Kato T, Yamamoto K, Okubo J and Miyazaki T. 1994. Antimutagenic and bactericidal substances in the fruit body of a Basidiomycete Agaricus blazei. Yakugaku Zasshi 114: 342-350.
• Paino IMM, Ximenes VF, Fonseca LM, Kanegae MPP, Khalil NM and Brunetti IL. 2005. Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils. Braz J Med Biol Res 38: 543-551.
• Parij N, Nagy AM, Fondu P and Neve J. 1998. Effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of human neutrophils. Eur J Pharmacol 352: 299-305.
• Pietta P, Simonetti P and Mauri P. 1998. Antioxidant activity of selected medicinal plants. J Agric Food Chem 46: 4487-4490.
• Podrez EA, Abu-Soud HM and Hazen SL. 2000. Myeloperoxidase generated oxidants and atherosclerosis. Free Rad Biol Med 28: 1717-1725.
• Ramarathnam N, Osawa T, Ochi H and Kawakishi S. 1995. The contribution of plant food antioxidants to human health. Trends Food Sc Tech 6: 75-82.
• Turkoglu A, Duru ME, Mercan N, Kivrak I and Gezer K. 2007. Antioxidant and antimicrobial activities of Laetiporus sulphurous (Bull.) Murrill. Food Chem 101: 267-273.
• Vellosa JCR, Barbosa VF, Khalil NM, Santos VAFFM, Furlan M, Brunetti IL and Oliveira OMMF. 2007b. Profile of Maytenus aquifolium action over free radicals and reactive oxygen species. Braz J Pharm Sci 43: 447-453.
• Vellosa JCR, Barbosa VF and Oliveira OMMF. 2007a. Pesquisa de Produtos Naturais: Plantas e Radicais Livres. REF 4: 119-130.
• Vellosa JCR, Khalil NM, Formenton VA, Ximenes VF, Fonseca LM, Furlan M, Brunetti IL and Oliveira OMMF. 2006. Antioxidant activity of Maytenus ilicifolia root bark. Fitoterapia 77: 243-244.
• Vinson JA, Dabbagh YA, Serry MM and Jang J. 1995. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric Food Chem 43: 2800-2802.
• Yang JH, Lin HC and Mau JL. 2002. Antioxidant properties of several commercial mushrooms. Food Chem 77: 229-235.

Publication Dates

• Publication in this collection
  13 Aug 2013
• Date of issue
  Sept 2013

History

• Received
  29 Aug 2011
• Accepted
  22 Oct 2012