Electron spin resonance analysis of different mushroom parts and their hydroxyl radical scavenging activities assessed by spin trapping method

Abstract

In this study, we analyzed the different parts of mushrooms using electron spin resonance (ESR) to identify radical species, and we examined their hydroxyl radical scavenging activities. The four kinds of mushrooms investigated in this study, Agrocybe cylindracea (Shakkiko1 and Shakkiko2), Grifola gargal, and Coprinus comatus were separated into caps, stipes, and bases of stipes. ESR spectra of all specimens consisted of signals originating from organic free radicals, Fe³⁺, Mn²⁺, and Cu²⁺ species. The cap showed the highest Fe content. The different mushroom body parts revealed different hydroxyl radical scavenging activity values, among which the C. comatus stipes were the highest. The radical scavenging activities of the evaluated specimens did not correlate with the Fe contents; however, a positive correlation with the Cu contents was observed.

Introduction

Mushrooms have been used as food and food-flavoring materials in soups and sauces for centuries because of their unique and subtle flavor. Recently, they have also garnered attention as functional foods and sources of physiologically beneficial medicines that are devoid of undesirable side effects (Cohen et al., 2014; Feeny et al., 2014). Mushrooms have been used in several therapies in light of their anticancer, antiviral, immunostimulatory, and hypolipidemic activities (Breene, 1990; Chang and Buswell, 1996). Chen et al. (2010) reported that many of the biological functions of mushrooms have been attributed to their free radical scavenging and antioxidant activities. Oxidation is essential to several living organisms, as it produces energy to fuel biological processes. However, the uncontrolled production of oxygen-derived free radicals is involved in the onset of multiple diseases such as cancer, rheumatoid arthritis, and atherosclerosis, as well as in degenerative processes associated with aging (Halliwell and Gutteridge, 1984; Hisaka and Osawa, 2014).

Mushrooms are a fungus with a distinctive fleshy fruiting body, which can be morphologically divided into cap, stipe, and base of the stipe (Kalač, 2009). Several studies have reported the differences between the cap and stipe in chemical components, antioxidant properties, and anti-inflammatory activities (Li et al., 2010; Park et al., 2017; Vetvicka et al., 2018). We reported that Agrocybe cylindracea, Grifola gargal (Kanno et al., 2013), and Coprinus comatus (Kanno et al., 2020) have antioxidant effects. Nakazawa et al. (2018) reported that several mushroom-derived ingredients, which possess antitumor, immunomodulatory, antiviral, and antimicrobial activities, have been isolated and identified, such as phenolic compounds, polysaccharides, peptides, and proteins (Lindequist et al., 2005; Giavasis, 2014; Li and Shah, 2016). These compounds have been characterized using a variety of analytical methods such as gas chromatography (GC), high-performance liquid chromatography (HPLC), and Fourier-transform infrared spectroscopy (FT-IR).

However, few publications report the use of electron spin resonance (ESR) spectroscopy in characterizing and identifying paramagnetic chemical species such as organic radicals or transition metallocomplexes in mushrooms. ESR studies in the food science research field have been used to study the anti-oxidation behavior of foods and to detect organic radicals produced after food irradiation (Ukai and Shimoyama, 2003; Kikuchi et al., 2011; Akram et al., 2012; Faure and Nystrom, 2016; Hougaard et al., 2016; Nakazawa et al., 2018). Therefore, we analyzed the different parts of mushrooms using ESR to identify radical species in this study. Importantly, we also assessed their hydroxyl radical scavenging activities, aiming to discover potential correlations between the hydroxyl radical scavenging activities and mineral contents in the mushrooms.

Materials and Methods

Reagents    Spin trapping reagent, 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline-N-oxide (CYPMPO), was purchased from Radical Research (Hino, Japan). All other chemicals used in this study were obtained from Wako Pure Chemical Co. (Tokyo, Japan) and were of the highest grade available.

Samples    A. cylindracea (Shakkiko1 and Shakkiko2) was obtained from the Aichi Prefectural Forestry Research Institute, Aichi, Japan). As the medium of A. cylindracea, Cryptomeria japonica, Quercus serrata, and wheat bran were mixed at a ratio of 5: 5: 3, respectively, and A. cylindracea was cultivated according to the method reported by Ishikawa et al. (2019). G. gargal (Iwade-GG010 strain) was obtained from the Iwade Research Institute of Mycology Group, Mie, Japan). G. gargal was cultivated according to the method described by Harada et al. (2015a). Mycelium of C. comatus (Coprino:Iwade-51 strain) was obtained from the Iwade Research Institute of Mycology Group and subsequently cultivated using composted bark. We used the same raw materials as those reported by Harada et al. (2015b). Each mushroom was cleaned to remove any residual compost, segmented (into the fruiting body, cap, stipe, and base of stipe), and then freeze-dried. As for A. cylindracea (Shakkiko1), the weights of the cap, stipe, and the base of stipe, as a proportion of the weight of the fruiting body, were 45.0, 35.6, and 19.4%, respectively. For A. cylindracea (Shakkiko2), the weights of the cap, stipe, and the base of stipe, as a proportion of the weight of the fruiting body, were 48.4, 42.5, and 9.1%, respectively. For G. gargal, the weights of the cap, stipe, and the base of stipe, as a proportion of the weight of the fruiting body, were 38.1, 32.8, and 29.0%, respectively. As for C. comatus, the weights of the cap, stipe, and the base of stipe, as a proportion of the weight of the fruiting body, were 48.5, 44.1, and 7.4%, respectively.

ESR analysis    A portion of each part was cut into approximately 2–4 mm long fine pieces using a ceramic knife. Each ex vivo sample (weight of 16 ± 0.5 mg) was placed into an ESR quartz tube for conventional X-band measurements, which was then sealed in air. These samples were used for the ESR analysis of paramagnetic chemical species relevant to mushrooms such as transition metallocomplexes. The remaining portions of each sample in each part were powdered.

For the ESR measurements at room temperature and in a wide range of static magnetic fields, enabling us to identify the signals due to transition metallocomplexes in the sample, an ESR tube was inserted into the cylindrical ER4122SHQE Super-High-Q cavity of a CW ELEXSYS E500 X-band ESR spectrometer (Bruker BioSpin Corporation, MA, USA). The instrumental conditions were as follows: microwave frequency = 9.30293 GHz, microwave power = 2.0 mW, modulation amplitude = 1.0 mT, number of scans = 4.

Hydroxyl radical scavenging activity    Three grams of the powdered sample was mixed with 30 mL of distilled water (at 25 °C and 100 rpm for 60 min) to extract the sample in distilled water. After centrifugation at 10 000 rpm for 10 min, the supernatants were filtered using filter paper (No. 2, Advantec, Tokyo, Japan). The extract was adjusted to 30 mL with distilled water and frozen at –70 °C for further analysis. The extract-derived solution samples were used for ESR analysis of the hydroxyl radical scavenging activities relevant to mushrooms.

The hydroxyl radical scavenging activity was measured using the method described previously by Kameya et al. (2011). A light illuminator (Cat. No. RUVF-203SR, Radical Research) was used to produce hydroxyl radicals from H₂O₂. The light source was a 200 W Hg-Xe arc lamp (Sanei Electronics, Tokyo, Japan), which primarily delivered UV light within a wavelength range of 200–400 nm. The illuminator was equipped with a quartz fiber optical guide, and its far end was mechanically fitted to the hole in front of the ESR cavity. The illuminator was equipped with a computer-controlled mechanical shutter, and the illumination period ranged from 0.1 to 100 s with 0.01 s of precision. The illumination condition was optimized so that H₂O₂ was efficiently photo-decomposed to generate hydroxyl radicals. Using the attenuated Hg-Xe arc lamp light, 50 µL of a phosphate buffer solution (100 mM, pH 7.4) containing 50 µL of 1% H₂O₂, 40 µL of 20 mM CYPMPO, and 30 µL of 10 mM diethylenetriaminepentaacetic acid (DTPA) was illuminated for 5 s. The hydroxyl radical scavenging activity was expressed as an equivalent concentration to ascorbic acid (mM/mg sample).

ESR analysis for the hydroxyl radical scavenging activity assay    For the ESR measurements of the hydroxyl radicals generated in the extract-derived solution samples, an X-band ESR spectrometer (JES-RE-1X, JEOL, Akishima, Japan) equipped with 100-kHz field modulation (Ukai et al., 2009) was used. We used the Win-Rad operation software (Radical Research) for ESR signal acquisition. The spectrometer settings were as follows: i) resonance field: 335.5 mT, ii) field modulation width: 0.1 mT, iii) microwave power: 6 mW, iv) field scan width/rate: ± 3.5 mT/min, time constant: 0.1 s. ESR spectra were collected at room temperature. We applied the method described by Kohri et al. (2009) to analyze our data.

Mineral analysis    We prepared a hydrochloric acid solution for mineral analysis according to Suzuki et al. (1987). Briefly, the powdered sample (0.3 g) was mixed with 30 mL of 10% hydrochloric acid and then heated for 120 min in a water bath at 80 °C with occasional stirring. After centrifugation at 10 000 rpm for 10 min, the supernatant was filtered using quantitative filter paper (No. 6, Advantec, Tokyo, Japan). The 10% hydrochloric acid solution was used for the Fe analysis. The powdered sample (0.3 g) was mixed with 30 mL of 3% hydrochloric acid and extracted by stirring at 25 °C and 100 rpm for 60 min. After centrifugation at 10 000 rpm for 10 min, the supernatant was filtered using quantitative filter paper (No. 6, Advantec). This 3% hydrochloric acid solution was used for the Cu analysis. The powdered sample (0.6 g) was mixed with 20 mL of 1% hydrochloric acid and extracted using sonication for 10 min. The sample was further extracted by stirring at 25 °C and 100 rpm for 60 min. After centrifugation at 10 000 rpm for 10 min, the supernatant was filtered using quantitative filter paper (No. 6, Advantec). We used a 1% hydrochloric acid solution for Mn analysis. A blank solution was also prepared using a similar experimental procedure.

The mineral contents were determined using an atomic absorption spectrometer (AAS) (AA-6200, Shimadzu, Kyoto, Japan) with an air-acetylene burner flame source. Mineral analysis was performed as previously described (Mallikariuna et al., 2013). Briefly, an aliquot of the ash solution was aspirated into the instrument for the determination of Fe, Cu, and Mn.

The AAS calibration was performed using the working standard prepared from commercially available mineral standard solutions (1 000 µg/mL). The most appropriate wavelength, hollow cathode lamp current, gas mixture flow rate, slit width, and other AAS instrument parameters for metals/minerals were selected according to the users' manual of the instrument, and background correction was applied during the mineral determination. The concentrations of all the minerals were expressed as mg/g dry weight of the sample.

Statistical analysis    All data are expressed as mean values with their associated standard deviations (S.D.). Statistical analyses were performed using BellCurve for Excel (BellCurve, Tokyo). The results of the hydroxyl radical scavenging activities and mineral contents were analyzed using one-way ANOVA and the Tukey–Kramer test. The correlations between the hydroxyl radical scavenging activities and mineral contents were assessed using correlation coefficient analysis.

Results and Discussion

ESR spectra    The conventional X-band CW-ESR spectra, observed at room temperature for each part of the mushroom fruiting bodies, are shown in Fig. 1. The spectra consist of four signal components, indicated as P₁ to P₄. The signal indicated as P₂ and appearing around g = 4.0 was attributed to spin-sextet-state Fe³⁺ species (Nakazawa et al., 2018). The broad signal around g = 2.2 (P₃) with the contribution from hyperfine structures arises from spindoublet Cu²⁺ species in relatively high-symmetric (axial) ligand environments. Furthermore, we detected a sextet of the hyperfine lines attributable to Mn²⁺ species (P₄). The sharp signal appearing at g = 2.0 (P₁) is due to organic free radicals (spin-doublet) resulting from biochemical reactions. The main features of the observed spectrum are similar to those of the X-band ESR spectrum of whole dried fruiting bodies (in powdered form) for certain mushrooms (Kanno et al., 2014). In this study, no complete spectral simulations for the solid state were attempted to derive the spin Hamiltonian parameters for magnetic properties such as the g-, fine structure, and hyperfine structure tensors of metallocomplexes. Noticeably, the relative intensities of the observed ESR signals depend on the mushroom, indicating that metallo-ion binding sites are differently distributed in the mushroom, and further detailed material isolations are required for any biological relevance to metallocomplexes.

Fig. 1.

ESR spectra for each part of the mushroom fruit bodies.

Hydroxyl radical scavenging activity of water extracts from different parts of mushrooms    Recent studies have shown that certain mushrooms possess antioxidant activities (Yang et al., 2002; Cheung et al., 2003; Puuttaraju et al., 2006; Asatiani et al., 2007). However, few studies have reported on the hydroxyl radical scavenging activity of mushrooms. Hydroxyl radicals are highly reactive and therefore have potential cell damage capacity. Table 1 shows the hydroxyl radical scavenging activity of the water extracts from the different mushroom parts. All tested mushrooms showed potential hydroxyl radical scavenging activities, with C. comatus exhibiting the highest values ranging from 84.4 ± 12.9 to 101.7 ± 37.0 µmol ascorbic acid eq. / g. This result might be explained by the higher ergothioneine content in C. comatus, as ergothioneine is an antioxidant (Kanno et al., 2020). C. comatus is also known to have antioxidant effects (Li et al., 2010). We believe that further studies comparing the hydroxyl radical scavenging activities of other mushrooms are necessary.

Table 1. Hydroxyl radical scavenging activity of water extracts from different mushroom parts.

	Hydroxyl radical scavenging activity (µmol ascorbic acid eq. / g)
	Agrocybe cylindracea (Shakikko1)	Agrocybe cylindracea (Shakikko2)	Grifola gargal	Coprinus comatus
Cap	34.0 ± 3.4 ab	27.7 ± 5.6 a	13.8 ± 3.1 b	84.4 ± 12.9 a
Stipe	43.5 ± 2.5 a	19.2 ± 4.3 a	9.9 ± 2.4 b	101.7 ± 37.0 a
Base of Stipe	25.4 ± 5.5 b	2.9 ± 0.3 b	81.5 ± 10.1 a	89.3 ± 15.1 a

There is meaningfulness in the level of significance of 5% of significant difference between the different alphabet.

The water extract from the base of the stipe of G. gargal had significantly (p < 0.05) higher hydroxyl radical scavenging activity when compared with that of the cap and stipe. In the case of A. cylindracea (Shakkiko1), the stipe showed the highest hydroxyl radical scavenging ability compared to that of the cap and base of the stipe. In A. cylindracea (Shakkiko2), the cap showed the highest hydroxyl radical scavenging ability compared with that of the base and base of the stipe. We found no specific difference in the scavenging ability among the mushrooms under study.

Contents of three transition elements from different parts of mushrooms    Mushrooms contain important minerals that are required for normal functioning of the human body (Gbolagade et al., 2006). Minerals and trace elements play a key role in biomedical activities associated with this food, since they have a multitude of known and unknown biological functions such as the maintenance of intracellular oxidative balance (Stohs and Bagchi, 1995; Shoham and Youdim, 2000). The abundance of three transition elements (Fe, Cu, and Mn) in different mushroom segments is shown in Table 2.

Table 2. Contents of three transition elements in four species of mushrooms.

		Agrocybe cylindracea (Shakikko1)	Agrocybe cylindracea (Shakikko2)	Grifola gargal	Coprinus comatus
		(µg / g)	(µg / g)	(µg / g)	(µg / g)
	Cap	53.4 ± 0.6 a	34.8 ± 1.0 a	43.5 ± 1.8 a	49.0 ± 1.2 a
Fe	Stipe	35.7 ± 1.0 b	19.3 ± 2.3 c	13.8 ± 2.1 c	20.7 ± 2.2 c
	Base of Stipe	32.4 ± 3.4 b	29.7 ± 1.3 b	24.1 ± 0.4 b	28.6 ± 1.1 b
	Cap	45.2 ± 0.6 a	40.1 ± 0.4 a	5.5 ± 0.4 a	129.7 ± 0.8 a
Cu	Stipe	40.6 ± 0.1 c	28.5 ± 0.1 c	5.2 ± 0.3 a	66.3 ± 0.2 b
	Base of Stipe	43.7 ± 0.8 b	31.4 ± 0.3 b	5.7 ± 1.2 a	43.2 ± 0.5 c
	Cap	4.69 ± 0.06 b	5.41 ± 0.07 b	6.77 ± 0.02 b	5.39 ± 0.01 a
Mn	Stipe	4.65 ± 0.07 b	4.75 ± 0.02 c	6.56 ± 0.06 c	3.25 ± 0.01 c
	Base of Stipe	6.27 ± 0.02 a	12.69 ± 0.10 a	10.00 ± 0.05 a	3.66 ± 0.05 b

There is meaningfulness in the level of significance of 5% of significant difference between the different alphabet.

The investigated mushroom caps showed the highest Fe content among the other parts, while the different parts showed various Fe contents. The cap contained the highest Cu content in each mushroom, except for G. gargal. However, the base of the stipe contained the highest Mn content, except for C. comatus. These results were consistent with the findings of ESR spectroscopy for solid-state mushrooms. We note that the results in this study show no direct relevance to any biological functions due to the transition metal elements in ex vivo mushrooms, although Fe-transferrins or their homologs have been identified by ESR spectroscopy in ex vivo mushrooms, combined with advanced quantum chemical calculations (Nakazawa et al., 2018).

Relationship between hydroxyl radical scavenging activity and contents of transition elements    In this study, we identified a relationship between hydroxyl radical scavenging activity and the contents of three transition elements (Fig. 2). We found that the hydroxyl radical scavenging activity of the evaluated specimens did not correlate with the Fe content, and there was a negative correlation with the Mn content. However, we found a positive correlation between hydroxyl radical scavenging activity and the Cu content. We also evaluated the correlation between the hydroxyl radical scavenging activity and the contents of the transition elements. The hydroxyl radical scavenging activity was significantly correlated with the Cu (RS 0.583, p < 0.05) and Mn (RS –0.566, p < 0.05) contents.

Fig. 2.

Comparison of the hydroxyl radical scavenging activity with the transition element contents.

Fe and Cu metal ions are the main catalysts for free radical-generating reactions in the human body. Trace elements are generally attached to and transported by proteins such as ceruloplasmin, transferrin, metallothionein, and transmanganin, which transport Cu²⁺–Cu⁺, Fe³⁺, Zn²⁺, and Mn²⁺ ions, respectively. Ceruloplasmin reportedly exhibits antioxidant effects (Shahabi et al., 2010; Vlasova et al., 2019), suggesting that ceruloplasmin might also contribute to the radical scavenging activity, albeit partially. We believe that further investigations of these protein metallo-protein functionalities relevant to radical scavenging activities are necessary. Moreover, in order to examine the components that are related to the hydroxyl radical scavenging activity in mushrooms, further research should investigate the active sites of copper-binding proteins as well as the degree of contribution by ergothioneine to hydroxyl radical scavenging activity.

In conclusion, different parts of the four kinds of mushrooms A. cylindracea (Shakkiko1 and Shakkiko2), G. gargal, and C. comatus in this study were analyzed using X-band ESR spectroscopy to identify radical species, and their hydroxyl radical scavenging activities were examined. The ESR spectra of all the specimens of ex vivo mushrooms consisted of a signal of approximately g = 2.0 (attributed to organic free radicals), a weak signal at around g = 4.0 from Fe³⁺ species in its spin-sextet state, a sextet signal centered at around g = 2.0 from Mn²⁺, and a broad signal at around g = from Cu²⁺ species. All the mushroom samples contacting the water extract for spin trapping-ESR spectroscopic assays were found to possess hydroxyl radical scavenging activity. C. comatus showed the highest reactivity to hydroxyl radicals among the mushrooms studied. The contents of the three transition elements were examined in different parts of the mushrooms. The cap contained the highest Fe content. The radical scavenging activity of the evaluated specimens did not correlate with the Fe content, and there was a negative correlation with the Mn content. However, we found a positive correlation between the hydroxyl radical scavenging activity and the Cu content. Our findings suggest that chemical identification of the binding site of Cu²⁺ species in mushrooms enables further investigation of the effects of antioxidants in mushrooms.

Acknowledgements    This work was supported by JSPS KAKENHI Grant Number JP19K02305.

Abbreviations

ESR

electron spin resonance

CYPMPO

5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline-N-oxide

DTPA

diethylenetriaminepentaacetic acid

AAS

Atomic absorption spectra

GC

gas chromatograph

HPLC

high performance liquid chromatography

FT-IR

Fourier-transform infrared spectroscopy

References

•  Akram,  K.,  Ahn,  J.J., and  Kwon,  J.H. (2012). Identification and characterization of gamma-irradiated dried lentinus edodes using ESR, SEM, and FTIR analyses. J. Food Sci., 77, C690-C696.
•  Asatiani,  M.D.,  Elisashvili,  V.I.,  Wasser,  S.P.,  Reznick,  A.Z., and  Nevo,  E. (2007). Free-radical scavenging activity of submerged mycelium extracts from higher basidiomycetes mushrooms. Biosci. Biotechnol. Biochem., 71, 3090-3092.
•  Breene,  W.M. (1990). Nutritional and medicinal value of specialty mushrooms. J. Food Protect., 53, 883-894.
•  Chang,  S.T. and  Buswell,  J.A. (1996). Mushroom nutriceuticals. World J. Microbiol. Biotechnol., 12, 473-476.
•  Chen,  X.H.,  Xia,  L.X.,  Zhou,  H.B., and  Qiu,  G.Z. (2010). Chemical composition and antioxidant activities of Russula griseocarnosa sp. nov. J. Agric. Food Chem., 58, 6966-6971.
•  Cheung,  L.M.,  Cheung,  P.C.K., and  Ooi,  V.E.C. (2003). Antioxidant activity and total phenolics of edible mushroom extracts. Food Chem., 81, 249-255.
•  Cohen,  N.,  Cohen,  J.,  Asatiani,  M.D.,  Varshney,  V.K.,  Yu,  H.T.,  Yang,  Y.C.,  Li,  Y.H.,  Mau,  J. L., and  Wasser,  S. P. (2014). Chemical composition and nutritional and medicinal value of fruiting bodies and submerged cultured mycelia of culinary – medicinal higher basidiomycetes mushrooms. Int. J. Med. Mushrooms, 16, 273-291.
•  Faure,  A.M. and  Nystrom,  L. (2016). Effect of apotransferrin, lactoferrin and ovotransferrin on the hydroxyl radical mediated degradation of beta-glucan. Food chem., 204, 1-16.
•  Feeney,  M.J.,  Miller,  A.M., and  Roupas,  P. (2014). Mushrooms – biologically distinct and nutritionally unique: exploring a “third food kingdom”. Nutr. Today, 49, 301-307.
•  Gbolagade,  J.,  Ajayi,  A.,  Oku,  I., and  Wankasi,  D. (2006). Nutritive value of common wild edible mushrooms from southern nigeria. Global J. Biotech. Biochem., 1, 16-21.
•  Giavasis,  I. (2014). Bioactive fungal polysaccharides as potential functional ingredients in food and nutraceuticals. Current Opinion in Biotechnology, 26, 162-173.
•  Halliwell,  B. and  Gutteridge,  J.M.C. (1984). Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet, 323, 1396-1397.
•  Harada,  E.,  Morizono,  T.,  Sumiya,  T., and  Meguro,  S. (2015a). Production of andean-patagonic edible mushroom Grifola gargal on wood-based substrates. Mycoscience, 56, 616-621.
•  Harada,  E.,  D'Alessandro-Gabazza,  C. N.,  Toda,  M.,  Morizono,  T.,  Chelakkot-Govindalayathil,  A.-L.,  Roeen,  Z.,  Urawa,  M.,  Yasuma,  M.,  Yano,  Y.,  Sumiya,  T., and  Gabazza,  E. C. (2015b). Amelioration of atherosclerosis by the new medicinal mushroom Grifola gargal singer. J. Med. Food, 18, 872-881.
•  Hisaka,  S. and  Osawa,  T. (2014). Lipid hydroperoxide – derived adduction to amino – phospholipid in biomembrane. Subcell Biochem., 77, 41-48
•  Hougaard,  A.B.,  Pindstrup,  H.,  Arneborg,  N.,  Andersen,  M.L., and  Skibsted,  L.H. (2016). Free radical formation by Lactobacillus acidophilus NCFM is enhanced by antioxidants and decreased by catalase. Food Res. Int., 79, 81-87.
•  Ishikawa,  K.,  Takeuchi,  Y., and  Katou  M. (2019). Studies on cultivation techniques to lengthen a stipe of Agrocybe cylindracea using wrapping materials. Chubu Shinrin Kenkyu, 67, 73-74. (in Japanese)
•  Kalač,  P. (2009). Chemical composition and nutritional value of European species of wild growing mushrooms: A review. Food Chemistry, 113, 9-16.
•  Kameya,  H. and  Ukai,  M. (2011). New ESR detection method of hydroxyl radical scavenging activity of blue berry. J. Materials Sci. Engineering, 1, 347-351
•  Kanno,  T.,  Kawamura,  S.,  Harada,  E.,  Kameya,  H.,  Ukai,  M., and  Osawa,  T. (2013). Radical scavenging ability by ESR spin trapping and oxygen radical absorbance capacity of hot water extracts from mushrooms. J. jpn. Soc. Food Sci. Technol. (Nippon Shokuhin Kagaku Kogaku Kaishi). 60, 173-178. (in Japanese)
•  Kanno,  T.,  Yamamoto,  K.,  Harada,  E.,  Kameya,  H.,  Ukai,  M., and  Osawa,  T. (2014). Electron spin resonance analysis of several mushrooms. Mushroom Science and Biotechnology (Nihon Kinoko Gakkaishi). 22, 19-23. (in Japanese)
•  Kanno,  T.,  Kazumura,  K.,  Niwa,  T.,  Harada,  E.,  Miyake,  Y., and  Osawa,  T. (2020). Evaluation of anti-inflammatory properties of hot water extract from Coprinus comatus using HL-60 and 3T3-L1 cells. Jpn. J. Food Chem. Safety, 27, 53-59.
•  Kikuchi,  M.,  Shimoyama,  Y.,  Ukai,  M., and  Kobayashi,  Y. (2011). ESR detection procedure of irradiated papaya containing high water content. Radiat. Phys. Chem 80, 664-667.
•  Kohri,  S.,  Fujii,  H.,  Oowada,  S.,  Endoh,  N.,  Sueishi,  Y.,  Kusakabe,  M.,  Shimmei,  M., and  Kotake,  Y. (2009). An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals. Analytical Biochem., 386, 167-171.
•  Li,  B.,  Lu,  F.,  Suo,  X.,  Nan,  H., and  Li,  B. (2010). Antioxidant properties of cap and stipe from Coprinus comatus. Molecules, 15, 1473-1486.
•  Li,  S. and  Shah,  N.P. (2016). Characterization, antioxidative and bifidogenic effects of polysaccharides form Pleurotus eryngii after heat treatments. Food Chem., 197, 240-249.
•  Lindequist,  U.,  Niedermeyer,  T.H.J., and  Jülich,  W.-D. (2005). The pharmacological potential of mushrooms. Evidence-Based Complementary and Alternative Medicine, 2, 285-299.
•  Mallikarjuna,  S.E.,  Ranjini,  A.,  Haware,  D.J.,  Vijayalakshmi,  M.R.,  Shashirekha,  M.N., and  Rajarathnam,  S. (2013). Mineral composition of four edible mushrooms. Journal of Chemistry, 805284, 1-5.
•  Nakazawa,  S.,  Kanno,  T.,  Sugisaki,  K.,  Kameya,  H.,  Matsi,  M.,  Ukai,  M.,  Sato,  K., and  Takui,  T. (2018). Fe-transferrins or their homologues in ex-vivo mushrooms as identified by ESR spectroscopy and quantum chemical calculations: A full spin-Hamiltonian approach for the ferric sextet state with intermediate zero-field splitting parameters. Food Chem. 266, 24-30.
•  Park,  Y.J.,  Jung,  E.S.,  Singh,  D.,  Lee,  D.E.,  Kim,  S.,  Lee,  Y.W.,  Kim,  J.-G., and  Lee,  C.H. (2017). Spatial (cap & stipe) metabolomic variations affect functional components between brown and white beech mushrooms. Food Res. Int., 102, 544-552.
•  Puttaraju,  N.G.,  Venkateshaiah,  S.U.,  Dharmesh,  S.M.,  Urs,  S.M.N., and  Somasundaram,  R. (2006). Antioxidant activity of indigenous edible mushrooms. J. Agric. Food. Chem., 54, 9764-9772.
•  Shahabi,  L.S.,  Zadeh,  S.A., and  Zadeh,  N.R. (2010). Serum level and antioxidant activity of ceruloplasmin in preeclampsia. Pakistan Journal of Biological Sciences, 13, 621-627.
•  Shoham,  S. and  Youdim,  M.B. (2000). Iron involvement in neural damage and microgliosis in models of neurodegenerative diseases. Cell Mol. Biol., 46, 743-760.
•  Stohs,  S.J. and  Bagchi,  D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321-336.
•  Suzuki,  H.,  Kimiko,  A., and  Minami,  H. (1987). Extraction conditions of hydrochloric acid for atomic absorption spectrometric determination. J. Cookery Sci. Jpn. (Nippon Chouri Kagaku Kaishi), 20, 72-79. (in Japanese)
•  Ukai,  M. and  Shimoyama,  Y. (2003). Free radicals in irradiated pepper: an electron spin resonance study. Appl. Magn. Reson., 24, 1-11.
•  Ukai,  M.,  Kameya,  H.,  Makamura,  H., and  Shimoyama,  Y. (2009). Radical scavenging activities of plant food of alkyloxy and superoxide radicals, Food Sci. Technol. Res., 15, 619-624.
•  Vetvicka,  V.,  Gover  O.,  Hayby,  H.,  Danay  O.,  Ezov,  N.,  Hadar,  Y., and  Schwartz,  B. (2018). Spatial distribution of glucan type and content between caps and stalks in Pleurotus eryngii: impact on the anti-inflammatory functionality. Int. J. Mol. Sci., 19, 1-13.
•  Vlasova,  I.I.,  Sokolov,  A.V.,  Kostevich,  V.A.,  Mikhalchik,  E.V., and  Vasilyev,  V.B. (2019). Myeloperoxidase-induced oxidation of albumin and ceruloplasmin: role of tyrosines. Biochemistry (Moscow), 84, 652-662.
•  Yang,  J.H.,  Lin,  H.C., and  Mau,  J.L. (2002). Antioxidant properties of several commercial mushrooms. Food Chem., 77, 229-235.