Mushrooms Do Not Contain Flavonoids

ManuscriptView metadata, citation and similar papers at core.ac.uk brought to you by CORE

Click here to view linked References provided by Digital.CSIC

1 2 3 4 Mushrooms do not contain flavonoids 5 6 7 8 9 10 Alicia Gil-Ramíreza*, Cristina Pavo-Caballeroa, Esther Baezaa, Nieves Baenasb, 11 12 13 Cristina Garcia-Viguerab, Francisco R. Marína, Cristina Soler-Rivasa 14 15 16 17 18 a 19 Department of Production and Characterization of Novel Foods. CIAL-Research Institute in Food 20 21 Science (UAM-CSIC). Madrid. Spain. 22 23 b 24 Department of Food Science and Technology. CEBAS-CSIC, Murcia, Spain 25 26 27 28 29 *Corresponding author: [email protected]/[email protected] 30 31 Running title: Mushrooms do not contain flavonoids. 32 33 34 Keywords: mushrooms, flavonoids, chalcone synthase, chalcone isomerase, phenylalanine 35 36 ammonia-lyase. 37 38 39 Chemical compounds studied in this article: Quercetin (PubChem CID: 5280343); Hesperidin 40 41 42 (PubChem CID: 3594); Luteolin (PubChem CID: 5280445); Luteolin-7-O-glucoside (PubChem 43 44 CID: 5280637); Gallic acid (PubChem CID: 370); Caffeic acid (PubChem CID: 689043); L-3,4- 45 46 47 dihydroxyphenylalanine (PubChem CID: 6047) and ergosterol (PubChem CID: 444679). 48 49 50 Abbreviations: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone 51 52 53 flavanone isomerase (CHI). 54 55 56 57 58 59 60 61 62 63 1 64 65 ABSTRACT

1 2 A literature and database review and several experiments were carried out because of the perturbing 3 4 increase and exponential spreading of a major mistake thought scientific publications. Up to October 2015, 5 6 more than 136 reports ensured that mushrooms contain flavonoids. However, 91% of the publications used 7 8 9 an unspecific colorimetric method that was developed to quantify them in plants. However, no sequences 10 11 encoding chalcone synthase or chalcone isomerase (key enzymes involved in the flavonoid biosynthetic 12 13 pathway) were found in genomic databases even in completely sequenced mushrooms indicating that they 14 15 16 do not have the enzymes needed for their production. Fruiting bodies obtained from substrates containing 17 18 onion wastes and mycelia grown on media supplemented with specific flavonoids showed no flavonoid 19 20 content after HPLC DAD/MS determinations indicating that they were also unable of absorbing them. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 2 64 65 1. Introduction

1 2 Flavonoids are already for decades intimately associated with plant kingdom, particularly with land plants 3 4 (Harborne, 1994; Robards & Antolovich, 1997; Winkel, 2006) and it is widely accepted by the scientific 5 6 community that flavonoids are plant secondary metabolites. Although edible mushrooms belong to a 7 8 9 completely different kingdom than plants, since 2005 an exponential increase in the number of publications 10 11 has been noticed measuring these compounds that are almost restricted to the vegetal word in 12 13 mushrooms. The fast spreading of this mistake is alarming since many other publications and even reviews 14 15 16 are giving it for granted and justifying fungal bioactivities due to the action of these compounds 17 18 (Fernandes, Antonio, Oliveira, Martins, & Ferreira, 2012; Guillamón et al., 2010; Jayakumar, Thomas, Sheu, 19 20 & Geraldine, 2011). 21 22 23 Flavonoids are compounds derived from the secondary metabolism in plants from precursors originated 24 25 via two different pathways of primary metabolism, phenylpropanoid derivatives and malonyl-CoA. 26 27 28 Malonyl-CoA is synthesised by carboxylation of acetyl-CoA, a central intermediate in the tricarboxylic acid 29 30 cycle. Coumaroyl-CoA is synthesised from the amino acid phenylalanine by three enzymatic steps initiated 31 32 by phenylalanine ammonia-lyase (PAL) within the so called phenylpropanoid pathway, involved in the 33 34 35 biosynthesis of many other compounds besides flavonoids (lignans, coumarins, stilbenes etc.). Flavonoid 36 37 synthesis starts by condensation of 4-coumaroyl-CoA with three molecules of malonylCoA yielding 38 39 naringenin chalcone. This reaction is catalyzed by chalcone synthase (CHS). The generated chalcone is 40 41 42 subsequently isomerised by chalcone flavanone isomerase (CHI) to yield a flavanone. From these central 43 44 intermediates the pathway diverges into several side branches, each yielding a different class of 45 46 47 flavonoids, isoflavones, anthocyanins etc. (Winkel, 2006). 48 49 Nowadays, flavonoid biosynthesis is considered as a classical example of a pathway that evolved gradually 50 51 by genetic change through specialization of a duplicate gene and also through the evolution of the 52 53 54 regulatory control on such gene. The climax of complexity may be exemplified in anthocyanin synthesis 55 56 where six sequential biochemical reactions enabled by six different enzymes are involved (Winkel, 2006). 57 58 Land plants are believed to have evolved from organisms like green algae (Charales) and although extensive 59 60 61 surveys were conducted, flavonoids were not found in algae (Markham, 1988). Furthermore, chalcone 62 63 3 64 65 synthase (the entry point to flavonoid byosintesis) was not described in green algae (Charales) but it was in

1 liverworts (Marchantiophyta) and mosses (Bryophyta) until more complex organisms such as flowering 2 3 plants (Angiosperma). The complexity of the flavonoid pathways and the synthetized compounds fit with 4 5 6 this evolutionary model (Rausher, 2006). 7 8 Mushrooms share with plants similarities in the biosynthetic pathways to generate phenolic compounds as 9 10 precursors of melanins. The melanogenesis starts with the shikimate pathway derived from the Krebs cycle 11 12 13 (as initial steps of the phenylpropanoid pathway) when the fungal hyphae are damaged or infected (Soler- 14 15 Rivas, Jolivet, Arpin, Olivier, & Wichers, 1999). Then, phenolic compounds are substrates of polyphenol 16 17 oxydases or peroxidases that catalyse oxidative reactions yielding diphenols, quinones and further 18 19 20 intermediate complexes that spontaneously generate GHB-, DOPA-derivative melanins or other specific 21 22 compounds. 23 24 25 The reported presence of flavonoids in mushrooms could be due to the facility of these organisms to 26 27 absorb many nutrients and compounds from the substrate where they grow or from neighbouring plants by 28 29 spreading their hyphae or forming mycorrhizae. Certain flavonoids are released by some plants to regulate 30 31 32 these symbiotic plant-microbe interactions defining the species tolerated to grow on their roots 33 34 (Grotewold, 2006). Although in taking of these compounds might negatively affect fungal growth because 35 36 flavonoids are frequently produced by plant as protection against fungal infections indeed, they were 37 38 39 widely reported as antifungal compounds (Grotewold, 2006). 40 41 Thus, in this work a detailed review of the literature was carried out to elucidate how this mistake might 42 43 44 have appeared and evolved until its fast spreading acceptance. The methodological mistake was 45 46 reproduced in order to explain the causes of the pitfall and other experiments and searches were carried 47 48 out to confirm that mushrooms cannot synthetize flavonoids neither absorb them from neighbouring 49 50 51 plants. 52 53 2. Experimental Section 54 55 56 2.1. Biological material 57 58 Mushroom strains used in this investigation were Lentinus edodes, Pleurotus ostreatus, Pleurotus eryngii, 59 60 61 Pleurotus pulmonarius, Agaricus bisporus, Agaricus blazei, Amanita ponderosa, Amanita caesarea, Lactarius 62 63 4 64 65 deliciosus, Craterellus cornucopioides, Cantharellus cibarius, Lyophyllum shimeji, Agrocybe aegerita,

1 Auricularia judea, Calocybe gambosa, Ganoderma lucidum, Hydnum repandum, Lepiota procera, Grifola 2 3 frondosa, Pholiota nameko, Hericium erinaceus, Boletus edulis, and Morchella conica. Fruiting bodies from 4 5 6 wild mushrooms were purchased on season from the local market in Madrid (Spain). The cultivable strains 7 8 were grown at CTICH (Centro Tecnológico de Investigación del Champiñón de La Rioja, Autol, Spain). 9 10 Fruiting bodies were frozen at -20C and freeze-dried. Dehydrated samples were ground into fine powder 11 12 13 as described in Gil-Ramírez et al. (2013) and stored at -20C until further use. 14 15 Commercial fully grown spawn of Pleurotus ostreatus K8 was kindly donated by Micelios Fungisem S.A. 16 17 18 (Autol, La Rioja, Spain) and was used to obtain fruiting bodies or mycelium subcultures. 19 20 2.2. Reagents 21 22 Malt extract agar (MEA), sodium hydroxide, quercetin, hesperidin, gallic, cafeic and formic acids, sodium 23 24 25 nitrite, L-3,4-dihydroxyphenylalanine (L-DOPA) and Folin-Ciocalteu’s reagent were purchased from Sigma- 26 27 Aldrich (Madrid, Spain). Luteolin and luteolin-7-O-glucoside were obtained from Extrasynthese (Lyon Nord, 28 29 France). AlCl · H2O was purchased at Panreac (Barcelona, Spain), ergosterol hydrate at Alfa Aesar Gmbh & 30 3 31 32 CoKG (Karlsruhe, Germany) and methanol (HPLC grade) at LAB-SCAN (Gliwice, Poland). 33 34 2.3. Systematic review of the literature 35 36 37 A quantitative and systematic search of the literature was carried out using the key words ‘mushroom’ and 38 39 ‘flavonoid’ in the Web of ScienceTM (Thomson Reuters) by selecting ‘all years’ (system start searching from 40 41 1864 up to now). The search included listed records up to October 2015. 42 43 44 Records were reviewed one by one and selected only if the abstract or title was indicating that flavonoids 45 46 (as total flavonoids or as specific identified flavonoid) were determined in mushrooms. Selected references 47 48 were listed extracting the publication year, Impact factor (IF) of the journal that year (except for those 49 50 51 articles published in 2015 where the indicated IF is from 2014), name of flavonoid identified (or total 52 53 flavonoid determination), methodology utilized for their determination and detection device used, 54 55 56 mushroom species analysed, type of extract (where indicated) and other considerations (see the 57 58 Supplementary Table 1). 59 60 61 62 63 5 64 65 2.4. In silico search for enzymes involved in the flavonoid biosynthesis

1 A protein search was carried out using Pfam (http://pfam.xfam.org/) and Blast® (Basic local alignment 2 3 search tool) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) looking for specific sequences of enzymes involved in 4 5 6 the biosynthesis of phenolic compounds and flavonoids. The selected key enzymes were phenylalanine 7 8 ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone isomerase (CHI). The taxonomic distribution of 9 10 the enzymes through the Eukaryote organisms was monitored using the Sunburst species distribution of 11 12 13 Pfam. Afterwards, the CHS amino acid sequence (393 aa) from Vitis vinifera was obtained from Genebank 14 15 (ref. seq. NP_001267879.1) (http://www.ncbi.nlm.nih.gov/genbank/) and utilized in Blastp as sequence to 16 17 search for homologies (alignments) within model organisms, different plant and fungi families and certain 18 19 20 specific species. 21 22 2.5. Lab scale production of fruiting bodies on flavonoid rich substrates 23 24 25 Specific substrate mixtures were prepared in triplicate as described by Ruiz-Rodriguez, Soler-Rivas, Polonia, 26 27 & Wichers (2010) with a few modifications: wheat straw and onion waste (leftovers after peeling onions) 28 29 were chopped (2 – 5 cm) and left overnight soaking up tap hot water. The excess of water was drained on a 30 31 32 sieve during 20 min. Afterwards, the soaked lignocellulosic substrates were mixed and homogenized with 33 34 0% (control) and 14% (w/w) onion waste. Homogenized substrates (500 g) were placed in plastic bags and 35 36 sterilized in autoclave (120C) during 15 minutes. 37 38 39 The sterilized substrates were inoculated with 10% (w/w) of fully colonized spawn and left incubated in 40 41 42 dark at 24C. When substrates were fully colonized temperature was changed to 16C and high humidity in 43 44 the presence of light, in a day/night cycle to induce fructification. One hole (approx. 2 cm diam.) was made 45 46 47 in one side of each bag. After few days (depending on substrate) pinheads were eye visible. Mushrooms 48 49 obtained from the first flush were harvested, weighed and lyophilized. Afterwards, dried fruiting bodies 50 51 were ground in a mortar with liquid nitrogen and stored at -20C for further analysis. 52 53 54 2.6. Mycelium cultivation on Petri dishes supplemented with flavonoids 55 56 Mycelia obtained from mother cultures generated from one grain of the P. ostreatus spawn were 57 58 inoculated on Petry plates containing MEA medium (50 g L-1 of malt extract agar, Sigma-Aldrich, (Madrid, 59 60 61 Spain)) and sterilized cellophane circles at the top of the semi-solid medium following the procedure 62 63 6 64 65 described by Ruiz-Rodríguez, Polonia, Soler-Rivas, & Wichers (2011). To test the effect of flavonoids or a

1 phenolic compound, MEA medium was supplemented with increasing concentrations of luteolin, luteolin-7- 2 3 O-glucoside (Extrasynthese, Lyon, France), quercetin, hesperidin or caffeic acid (Sigma-Aldrich) (from 0.25 4 5 6 up to 2 or 4 mg/mL depending on their saturation level) dissolved in ethanol (or MilliQ water for those 7 8 water-soluble). Plates were inoculated in triplicate and incubated at 25C. 9 10 2.7. Determination of fungal growth 11 12 13 Several cultivation parameters were evaluated during P. ostreatus cultivation on control 14 15 substrates (containing only wheat straw) and on substrates including onion waste. The incubation 16 17 18 time required before pinning (pinhead appearance) and for harvesting of the first flush was 19 20 21 recorded. Yield, biological efficiency and productivity were monitored as described by Ruiz-Rodriguez 22 23 et al. (2010). 24 25 26 Mycelial growth (minor and major diameters) was measured once a day until mycelia reached the plates 27 28 edge (Ruiz-Rodríguez et al., 2011). Growth was later expressed as cm/day using the slope at the linear 29 30 growth phase of the fungi. Mycelial biomass was also quantified by weighting the produced mycelia 31 32 33 scratched from the cellophane when they reached the plate edges. Afterwards, fresh mycelia were frozen, 34 35 freeze-dried and weighted again to calculate their dry weight. They were later ground in a mortar with 36 37 liquid nitrogen and stored at -20C for further analysis. 38 39 40 2.8. Determination of total phenol content 41 42 The total phenol concentration was determined by the Folin-Ciocalteu’s phenol reagent method according 43 44 45 to the procedure of Ramírez-Anguiano, Santoyo, Reglero, & Soler-Rivas (2007) using directly the powdered 46 47 fruiting bodies or mycelia (10 mg) prepared as previously described. Gallic acid was used as standard for 48 49 quantification. 50 51 52 2.9. Spectrophotometric determination utilized by literature as method to measure ´total flavonoid´ 53 54 The colorimetric determination was carried out as described by Choi, Lee, Chun, Lee, & Lee (2006) since it 55 56 was one of the first recorded publications that determined flavonoids in mushrooms using a modified 57 58 59 protocol previously utilized to measure flavonoids in mulberry (Zhishen, Mengcheng, & Jianming, 1999). 60 61 Briefly, powdered fruiting bodies (100 mg) were mixed with 10 ml methanol and stirred for 1h at room 62 63 7 64 65 temperature. Afterwards, solution was centrifuged (35000 rpm 15 min) and resulting supernatant filtered

1 and dried until dryness. Two hundred fifty microliters of obtained mushroom extracts (diluted up to 15 2 3 mg/mL with methanol) were mixed with 1.25 mL of distilled water and 75 L of a 5% NaNO2 solution. After 4 5 6 5 min, 150 L of a 10% AlCl3 · H2O was added. After 6 min, 500 L of 1M NaOH and 275 L of distilled water 7 8 were added to the mixture. The solution was mixed and the intensity of red-pink color was measured at 9 10 415 and 510 nm. Quercetin was used as standard for quantification and ergosterol (ergosta-5,7,22-trien-3β- 11 12 13 ol, 0.7 mg/mL) and L-Dopa (L-3,4-dihydroxyphenylalanine, 0.1 mg/mL) were solubilized in ethanol and 14 15 treated as mushroom extracts but not only its absorbance at those wavelengths were recorded but the 16 17 18 complete spectrum from 300 to 600 nm to compare with results from Pękal & Pyrzynska (2014). 19 20 2.10. Flavonoid detection and quantification by HPLC-DAD 21 22 The presence of flavonoids in mushrooms was determined by HPLC in two different labs using two 23 24 25 different devices and methods. 26 27 In the first one, powdered fruiting bodies or mycelia were mixed with methanol and treated as previously 28 29 described for the spectrophotometrical methods. Dried extracts were dissolved in 100 L methanol and 30 31 32 filtered (0.45 μm PVDF filter (Millex HV13, Millipore, Bedford, MA, USA)) before HPLC analysis. 33 34 Mushroom extracts (10 L) were injected in a Varian liquid chromatograph, model 920-LC Galaxy, with a 35 36 37 diode array (PAD) and fluorescence detectors (range scanned= 200-600 nm). Reverse phase 38 39 chromatographic separation was performed with an AquaTM C18 analytical column (250 x 4.6 mm i.d. with 40 41 an average particle size of 5 μm) after a precolum AquaTM C18 (4 x 3 mm) both from Phenomenex 42 43 44 (Madrid, Spain). As solvent (A) 2% formic acid (v/v) and (B) acetonitrile was used with the following 45 46 gradient profile: from 0 to 20 min, 0 to 30% B, from 20 to 25 min, 30 to 60% B, from 25 to 26 min, 60% B, 47 48 49 from 26 to 27, 60 to 100% B and 27 to 35 min washing with 100% B. The flow rate was 1 mL/min at 35C. 50 51 The absorbance changes were recorded in the vis-UV DAD and simultaneously at 2 wavelengths for the 52 53 different types of flavonoids: 280 nm for flavanones, 350 nm for flavones but the complete spectral range 54 55 56 was visualized to identify any possible flavonoid-related spectrum. 57 58 59 60 61 62 63 8 64 65 2.11. Flavonoid identification and quantification by HPLC-MS

1 The presence of flavonoids in mushrooms was also evaluated at the other lab by an HPLC–DAD– 2 3 ESI/MS device using as starting material the same mushroom samples that above indicated. Firstly, 4 5 6 powdered mushrooms (50 mg) were extracted with 1 mL of the extracting mixture MeOH:H2O:HCOOH 7 8 (25:24:1), homogenized using a vortex and sonicated in an ultrasonic bath for 60 min. Then, samples were 9 10 kept overnight at 4°C and sonicated again for 60 min. The supernatant for analysis was separated using a 11 12 13 centrifuge (9500 × g, 5 min) (model EBA 21, Hettich Zentrifugen) and filtered through a 0.45 µm PVDF 14 15 filter (Millex HV13, Millipore, Bedford, MA, USA) before the analyses were performed. 16 17 A chromatographic separation was performed using an HPLC system equipped with an Agilent 1200 series 18 19 20 (Agilent Technologies, Waldbronn, Germany) and coupled to a mass detector in series. The HPLC system 21 22 consisted of a binary capillary pump (G1376A), a degasser (G1379B), an autosampler (G1377A), a sample 23 24 25 cooler (control temperature 4C) (G1330B), and a photodiode array detector (G1315D) controlled by 26 27 Chemstation software (v.B.0103-SR2). The mass detector was a Bruker, model UltraHCT (Bremen, 28 29 Germany) ion trap spectrometer equipped with an electrospray ionisation (ESI) system and controlled by 30 31 32 Bruker Daltonics Esquire software (v.6.1). 33 34 The separation was performed using C18 Kinetex 100A (150 × 4.6 mm i.d.) column, 5 μm particle size. The 35 36 flow rate was 0.8 mL/min. Chromatographic separation was achieved using a binary gradient consisting of 37 38 39 (A) 1% formic acid and (B) acetonitrile as HPLC grade solvents. The elution profile started with 10% B, 40 41 reaching 30% B at 20 minutes, 60% B at 25 min and maintained up to 26 min. The injection volume was 8 42 43 44 µL. Chromatograms were recorded from 240 to 400 nm (set 340 nm for flavonoids). 45 46 The mass spectrometer settings were as follows: the pressure and flow rate of the dryer gas were set at 47 48 11 psi and 5 L/min respectively. The full-scan mass spectrum was from m/z 300 to 1400. Collision-induced 49 50 51 fragmentation experiments were performed in the ion trap using He as collision gas, with voltage ramping 52 53 cycles from 0.3 to 2 V. The heated capillary and voltage were maintained at 350C and 4275 V, respectively. 54 55 56 Other parameters for the detection were as follows: skimmer, 40 V; capillary exit, 108.5 V; octopole 1 DC, 57 58 12 V; octopole 2 DC, 1.7 V; octopole RF, 138.8 Vpp; lens 1, −5 V; lens 2, −60 V. The mass spectrometry data 59 60 were acquired in the negative ionization mode for flavonols. 61 62 63 9 64 65 3. Results

2 3.1. Systematic literature review 3 4 After the literature search, a total of 136 scientific works were found determining specific or total 5 6 flavonoids in mushrooms, although a few more (including reviews i.e. Fernandes et al. (2012); Guillamón et 7 8 9 al. (2010); Jayakumar et al. (2011)) were noticed where flavonoid presence was assumed because it was 10 11 described elsewhere. Attempts to detect flavonoids were carried out already in 2001 using HPLC (Mattila, 12 13 et al., 2001) however, authors did not detect them. It was in 2005 when a report was firstly published 14 15 16 indicating their presence (Y. M. Choi, Ku, Chang, & Lee, 2005)(see the Supplementary Table 1). The work 17 18 was included in a low-indexed journal but at the following year, the same group managed to publish a 19 20 similar work in a highly indexed journal (high for the area of Food Science and Technology)(Choi et al. 21 22 23 2006). Authors utilized a colorimetric method that was previously utilized to detect flavonoids in mulberry 24 25 (Zhishen et al. 1999) and referred the determination of ‘total flavonoids’ as ‘free and bound flavonoids’. 26 27 28 The same procedure was then followed in 2007 by several groups to detect flavonoids in mushrooms 29 30 pointing as ‘method of reference’ that utilized for mulberry or another used for propolis analysis (Park, 31 32 Koo, Ikegaki, & Contado, 1997). Despite others, three of these works were published in journals with IF 33 34 35 (Impact factor) >3 and 3 others in journals with IF>2 (Figure 1) promoting an exponential use of the 36 37 colorimetric method to determine flavonoids in mushrooms. The increase was such that from 5 works 38 39 published in 2008 it doubled the number in 2009 and raised up to 18 publications in 2010 and 2012 and 40 41 42 even 22 in 2013 and 2014. Although most of the reports through all these years were published in journals 43 44 with IF<1 (35%), many of them were also published in journals with IF>2 (21%) or IF>3 (11%) and even 4 of 45 46 47 them were published in journals with IF>4 (3%) in 2008, 2009 and 2012. 48 49 When a close look was given to the methodologies utilized to determine flavonoids in mushrooms, 91% of 50 51 the reports were based on colorimetric determinations utilizing AlCl3 (or Al(NO3)3) as reactive agent and a 52 53 54 spectrophotometer. Only 9.3% of the reports used more advanced systems such as HPLC (UV/DAD only 55 56 two used MS as detection devices) (Leal et al. 2013; Liu, Jia, Kan, & Jin, 2013), UPLC-ESI-MS/MS (Özyürek, 57 58 Bener, Güçlü, & Apak, 2014) and surprisingly, one manuscript indicated that these polyphenols and other 59 60 61 triterpenic acids were determined by GC-MS (Kalogeropoulos, Yanni, Koutrotsios, & Aloupi, 2013). 62 63 10 64 65 Moreover, the amounts of flavonoids found in the mushrooms also differed in the range of g/g dw (Babu

1 & Rao, 2013; Karaman, Stahl, Vulić, Vesić, & Čanadanović-Brunet, 2014) or mg/g (Kumari, Upadhyay, & 2 3 Atri, 2013; Yongcai, Xin, & Yaung-Iee, 2013) levels of 67.3 mg/g (Zhang et al. 2015) or 349.6 mg/g (Jeong et 4 5 6 al. 2013) meaning that 6.7 or 34.9% of the mushroom dry weight were flavonoids. These levels were found 7 8 in extracts obtained using different solvents including all polarities such as water, ethanol, methanol, 9 10 chloroform, ethyl acetate, butanol, petroleum ether, acetone, mixtures of indicated solvent and other 11 12 13 specific extracts (when it is well known that many flavonoids are virtually insoluble in some of the listed 14 15 solvents). 16 17 18 In addition to the ´flavonoid method´ some of the works also included a colorimetric method to determine 19 20 ‘anthocyanins’(Tevfik, Cihan, Oznur, & Ibrahim, 2011) or ‘anthocyanidins’ (Sharma & Gautam, 2015) in 21 22 mushrooms such as i.e. Agaricus arvensis or Cantharellus cibarius, although their fruiting bodies did not 23 24 25 show the red-violet-blue discoloration characteristic from berries and other food products containing 26 27 these pigments. Mushroom colors are due to different compounds depending on the considered species 28 29 but they have completely different molecular structures than antocyanins such as i.e. red 30 31 32 terphenylquinones related to orange-red grevillins, yellow-orange pulvic or pulvinic acids and 33 34 styrylpyrones, red-violet betacyanins or variegatic, xerocomic or atromentic acids or anthraquinones 35 36 (Velísek & Cejpek, 2011). 37 38 39 3.2. Systematic search in molecular databases for flavonoid-related gene homologies 40 41 In plants, the flavonoid biosynthesis required the presence of many enzymes but three of them play an 42 43 44 essential role: the phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone isomerase 45 46 (CHI)(Winkel, 2006). The presence of these proteins through the Eukaryote organisms was investigated 47 48 using the taxonomical distribution of Pfam ´Sunburst´ species distribution. Results indicated that the 3 49 50 51 enzymes were mostly distributed through the Viridiplantae kingdom (Table 1), particularly the chalcone 52 53 synthase and isomerase where respect. 93 and 87% of the known sequences were found in plant species 54 55 being the rest still uncategorized. 56 57 58 PAL-like proteins were found in 12 fungal species belonging to the phylla Basidomycota and Ascomycota 59 60 where the macrofungi and most of the edible mushrooms are included (Table 1). In fact commonly 61 62 63 11 64 65 consumed mushrooms such as Agaricus bisporus (white button mushroom) and Pleurotus eryngii (king

1 trumpet mushroom) expressed at least one sequence that match with the lyase aromatic family but no 2 3 protein related to the PF02797 or PF02431 families (where respect. CHS and CHI are included) have been 4 5 6 described so far. 7 8 Another attempt to try to find sequences encoding flavonoid-related enzymes was carried out using BLAST 9 10 as search tool. The CHS sequence from Vitis vinifera obtained from Genebank was used as example to look 11 12 13 for homologies within other Eukaryote organisms such as berries, citrus etc. as well as many fungal 14 15 species. CHS sequences within plant species showed high homologies (Table 2) and most of them were 16 17 identified as chalcone synthases including only one or several isoforms. However, homology with fungal 18 19 20 sequences dropped down to 17 – 3% in completely sequenced mushrooms such as A. bisporus and 21 22 Pleurotus ostreatus and no significant similarities were found in other edible mushrooms (although their 23 24 25 genomes are still not completely sequenced). However, the sequence of the proteins that slightly matched 26 27 grape CHS was not identified as CHS or any known enzyme, they were only hypothetical proteins. 28 29 Homologies with the V. vinifera chalcone synthase were also investigated through the known sequences of 30 31 32 all the mushroom genera where flavonoids were reported using methods different than the colorimetric 33 34 determination (Lactarius, Morchella, Lepista, Chantarelle, Pholliota, Tremella, Boletus, Amanita, Russula, 35 36 Gyromitra, Suillus, Cynomorium, Marasmius, Craterellus, Helvela, Clitocibe, Catathelasma, Stropharia, 37 38 39 Laccaria, Auricularia, Calocybe, Hygrophorus and truffles genus such as Tuber and Terfezia sp.) and no 40 41 significant similarities were found. 42 43 44 3.3. Spectrophotometric determinations and HPLC analysis of mushroom fruiting bodies 45 46 The total phenolic content of several mushroom species was determined using a commonly utilized 47 48 colorimetric method (Figure 2). Values ranged from 1 to more than 14 mg/g mushroom powder depending 49 50 51 on the strain being Boletus edulis the specie with the highest phenol content followed by others such as 52 53 Amanita ponderosa, Agrocybe aegerita or Calocybe gambosa. These values were in concordance with 54 55 other authors (Barros, Dueñas, Ferreira, Baptista, & Santos-Buelga, 2009; Del Signore, Romeo, & Giaccio, 56 57 58 1997; Ramírez-Anguiano et al. 2007). 59 60 61 62 63 12 64 65 Similarly, the protocol used by several publications to determine ¨total flavonoid¨ was applied to the same

1 mushroom species for comparison. Except for Lactarius deliciosus, most of the determined values were 2 3 similar or lower than the phenolic compounds ranging from 0.1 to 6.7 mg/g. The profile did not correlate 4 5 6 with the phenolic content since for instance Amanita cesarea showed similar phenolic composition than P. 7 8 ostreatus or Hericium erinaceus but it doubled the values obtained using the method utilized to determine 9 10 ‘total flavonoid’ in mushrooms. 11 12 13 On the other hand, when an ergosterol standard solution was prepared and treated as indicated to 14 15 determine ‘total flavonoids’ it positively reacted with the protocol showing absorbance at both 415 and 510 16 17 nm (Figure 3). Ergosterol is the major sterol from fungal hyphae but its structure is completely different 18 19 20 from a flavonoid. Other compounds typical for mushrooms such as L-Dopa showed similar spectrum than 21 22 those indicated by Pękal & Pyrzynska (2014) for some flavonoids, confirming that o-diphenols might also 23 24 25 induce false flavonoid-positives. 26 27 Moreover, when the selected mushroom species were prepared following typical methods to extract 28 29 flavonoids in plants (as described in M&M) and injected in HPLC-DAD/MS, no flavonoid peaks were 30 31 32 detected or identified in any of the 24 tested species. 33 34 3.4. Flavonoid absorption by fruiting bodies from the cultivation substrate 35 36 Pleurotus ostreatus was cultivated in wheat straw as control and straw supplemented with onion waste in 37 38 39 order to test whether the latter mycelium could partially absorb any of the flavonoids present in the onion 40 41 waste during its growth. Addition of the flavonoid-containing waste slightly increased 1 day the time for 42 43 44 pinning (inducing 1 day delay in harvesting) but did not reduce significantly fruiting bodies yield (15.7% 45 46 w/w) nor quality (90.4-85.6% water content). No significant differences were noticed in the levels of total 47 48 phenols (2.6 - 3.4 mg/g) neither in the values determined with the method utilized to measure ‘total 49 50 51 flavonoids’ in mushrooms cultivated with or without onion supplementation (respect. 1.8 - 2.2 mg/g). 52 53 The flavonoids present in the onion waste were extracted and analysed by HPLC/DAD-MS together with 54 55 the substrate after mycelial colonization and the fruiting bodies obtained from both control and 56 57 58 supplemented substrates. Some of the peaks detected in substrate (Figure 4a) were identified as 59 60 flavonoids (quercetin 3,7 or 3,4’ diglucoside, quercetin 3-glucoside, isorhamnetin 3-diglucoside and 61 62 63 13 64 65 isorhamnetin 3-glucoside) and they practically disappeared after mushrooms were grown in it, leaving only

1 traces of some of them detected, such as quercetin 3- glucoside (tR: 16,02 min) (Figure 4b). Their 2 3 disappearance from the substrate during the mycelial growth might be explained by transformation into a 4 5 6 different chemical (i.e. degradation) or by fungal absorption. However, the latter possibility is unlikely since 7 8 the HPLC chromatograms obtained after fruiting bodies analysis showed no peaks at any of the 9 10 11 wavelengths used to monitor flavonoids nor spectrum or mass which might resemble them (Figure 4c). 12 13 3.5. Flavonoid absorption by mycelium from culture medium 14 15 Pleurotus ostreatus mycelium was grown on Petri plates supplemented with increasing concentrations of 16 17 18 several flavonoids (quercetin, hesperidin, luteolin and luteolin-7-glucoside) and caffeic acid (as control) in 19 20 order to enhance or force their absorption if that was possible. Quercetin or hesperidin addition up to 21 22 2mg/ml did not influence significantly mycelial growth, the slightly extended lag phase (4 days) and the 23 24 25 growth reduction noticed (from 1.2 to approx. 0.5 cm/day in the exponential phase) was mostly due to the 26 27 ethanol utilized to solubilize the flavonoids because it was also observed in control samples were only 28 29 30 ethanol was added (0.4 cm/day). Ethanol (with or without flavonoid addition) induced larger biomass 31 32 production and water retention capacity by the mycelium since they showed higher fresh and dry weights 33 34 than the control. 35 36 37 Additions of luteolin or its glucosylated derivative to the cultivation medium resulted in inhibition of the 38 39 mycelial growth correlating with the flavonoids concentration utilized. The inhibition was more drastic 40 41 when the aglycone was used (Figure 5a) that when luteolin-7-glucoside was applied at similar 42 43 44 concentrations. However, addition of the simple phenolic compound did not significantly influenced 45 46 mycelial growth or biomass production except for a slight reduction noticed when applied at the highest 47 48 49 concentration tested (4mg/mL) (Figure 5b). 50 51 No significant differences were found when the total phenolic compounds were determined in the mycelia 52 53 obtained from plates supplemented with any of the selected flavonoids or concentrations. Control samples 54 55 56 with or without ethanol contained approx. 4.4 – 4.8 mg/g dw. Only caffeic acid addition induced a 57 58 significant increase (up to 7.3 mg/g dw) but only when a low concentration was applied (0.25 mg/mL). 59 60 However, when the samples were injected in HPLC-DAD and developed using a solvent gradient that 61 62 63 14 64 65 differently eluted the selected flavonoids and caffeic acid, none of the compounds were detected in the

1 mycelia grown at any of the selected flavonoid concentrations. 2 3 4 5 6 4. Discussion 7 8 9 The literature review carried out indicated that, at the present, approximately 136 scientific publications 10 11 are worldwide available ensuring that they can determine flavonoids in mushrooms. Moreover, during the 12 13 citations compilation many other articles were found (more than 50) were authors indicated in their 14 15 16 introduction section or under specific sub-headings (in reviews) that mushrooms contain flavonoids 17 18 because previous reports demonstrated their presence. However, in a more detailed observation of the 19 20 methodologies utilized to demonstrate their presence, almost 91% of the publications utilized an 21 22 23 unspecific colorimetric method developed to determine them in plants or plant products. The method 24 25 involved the measurement at 410 – 430 nm (some authors used other wavelengths such as 510 nm) of a 26 27 28 colorimetric reaction that take place after addition of AlCl3 which selectively reacts only with certain 29 30 flavonoids depending on the reaction medium utilized. The analytical method was proposed in the 60s 31 32 (Christ & Müller, 1960) and it was already indicated that was not accurate enough to determine ‘total 33 34 35 flavonoids’ neither in plants (Pękal & Pyrzynska, 2014). The method is based on the nitration of any 36 37 aromatic ring bearing a catechol group with its 3 or 4 positions unsubstituted or sterically blocked. After 38 39 addition of Al(III), a yellow complex is formed that turn red in the presence of NaOH and can be quantified 40 41 42 at 510 nm. However, if AlCl3 is added in neutral or acidic media (without NaNO2) it should only be used to 43 44 determine flavonols (queretin, rutin, galangin etc.) and luteolin. In the presence of NaNO2 in alkaline 45 46 47 medium seems to react with rutin, luteolin and catechins but, also with other phenolic acids bearing 48 49 catecholic moieties such as chlorogenic acid, in fact in the past, this method was used for the 50 51 determination of o-diphenols (Barnum, 1977). Most of the protocols followed by authors that indicated 52 53 54 the presence of flavonoids in mushrooms used NaNO2 and therefore, the more unspecific test. Moreover, 55 56 mushrooms contain not only chlorogenic acid but many other hydroxycinnamic acids (Puttaraju, 57 58 Venkateshaiah, Dharmesh, Urs, & Somasundaram, 2006), o-diphenols (i.e. glutaminyl-3,4- 59 60 61 dihydroxybenzene) and molecules including catechol residues such as pigments (i.e. variegatorubin, 62 63 15 64 65 hispolon, 3,4´,4-trihydroxypulvinone, inoscavin A etc) or melanin-precursors (i.e. 5,6-dihydroxyindole,

1 leucodopachrome)(Soler-Rivas et al. 1999; Velísek & Cejpek, 2011), even ergosterol (with a completely 2 3 different chemical structure from a flavonoid or a phenolic compound) showed a positive response in the 4 5 6 AlCl3-test. This molecule is only partially soluble in methanol however; it might also contribute together 7 8 with the other phenolic compounds to the absorbance determined as ‘total flavonoids’. This might also 9 10 explain the lack of linear correlation noticed between the amount of total phenolic compounds and the 11 12 13 absorbances quantified as ‘total flavonoids’. 14 15 Nevertheless, 13 publications identified specific flavonoids by HPLC/UPLC/GC using UV, DAD or MS as 16 17 detection devices (although in one of them no device or method was described, HPLC was assumed 18 19 20 because authors were able of quantifying quercetin (Zhishen et al. 1999)). These reports indicated the 21 22 presence of compounds such as quercetin, luteolin, myricetin, naringenin, naringin, hesperetin, rutin, 23 24 25 morin, kaempferol, chrysin and derivatives, genistein, apigenin or catechin in commonly consumed 26 27 mushrooms i.e. P. ostreatus, A. bisporus, B. edulis, C. cibarius, L. deliciosius, and other less popular (see the 28 29 Supplementary Table 1). The presence of these compounds in mushrooms could be due to two 30 31 32 possibilities: that mushroom can synthetize them or absorb them from neighbouring plants or from those 33 34 that establish symbiotic interactions via mycorrhizae (although not all the mushrooms species establish 35 36 them). 37 38 39 In order to synthetize flavonoids, mushrooms should contain in their genome at least a copy of the three 40 41 key enzymes within their biosynthetic pathway, phenylalanine ammonia-lyase (PAL), chalcone synthase 42 43 44 (CHS) and chalcone isomerase (CHI) and nowadays only PAL has been reported in mushrooms according to 45 46 genomic databases. No gene encoding CHS or CHI have been identified so far suggesting that they were 47 48 not yet studied or not found because they were neither detected in those mushrooms that are, at the 49 50 51 present, completely sequenced (Agaricus bisporus and Pleurotus ostreatus). These results indicate that 52 53 mushrooms can generate phenolic compounds using their PAL but they cannot further transform them 54 55 into flavonoids because the lacked the necessary enzymes. 56 57 58 Fungi evolved separately from plants long before the enzymatic mechanism specifically designed to 59 60 produce most of the primitive flavonoids appeared. Moreover, although it is possible that, initially, 61 62 63 16 64 65 flavonoids were a metabolic by-product, nature found function for them in plants. For instance, they are

1 signals for pollen tube formation, for bacteria attraction (nitrification), pest and plagues repellents etc. But, 2 3 what do they do in fungi? Is there any single report informing about their role in fungal biology? 4 5 6 Then, if they do not synthetize them, the second possibility might be that they could absorb them from 7 8 their substrate or from plants. However, when P. ostreatus was cultivated on onion waste containing 9 10 flavonoids the obtained fruiting bodies lacked quercetin- or isorhamnetin-derivatives or any other 11 12 13 flavonoid. Therefore, the flavonoids described at least in P. ostreatus (i.e. Huang, Cai, Xu, & Zhao (2012), 14 15 Palacios et al. (2011) or Sapozhnikova, Byrdwell, Lobato, & Romig (2014)) cannot be explained by an 16 17 absorption mechanism and as all fungi share a similar biology, these conclusions could be extended to the 18 19 20 rest of species, or at least to those 24 strains analysed in is study since no trace of flavonoid was found 21 22 using HPLC. 23 24 25 When P. ostreatus mycelium was forced to grow on semi-synthetic media with increasing concentrations of 26 27 certain flavonoids no absorption was also noticed on the contrary, some of them inhibited the fungal 28 29 growth confirming the fungicide properties that have been described for flavonoids in many other 30 31 32 publications (Orhan, Özçelik, Özgen, & Ergun, 2010; Winkel, 2006). In several reports flavonoids were found 33 34 in the range of micrograms per gram mushroom (dw) however in many others, they were present in mg/g 35 36 reaching levels of 349.6 mg/g flavonoids (Jeong et al. 2013), where it seems unlikely that mushrooms could 37 38 39 absorb that amount of toxic compounds (35% dw) without significant changes in the fruiting bodies quality 40 41 and metabolism. If this was the case, mushrooms could have an extremely interesting commercial 42 43 44 application as flavonoid source since in plants, they are present in large concentrations only under specific 45 46 physiological conditions such as in the earliest developmental stages (Jourdan, McIntosh, & Mansell, 1985). 47 48 When citrus fruits such as sour orange are still immature (diameter < 10 mm), values of i.e. neohesperidin 49 50 51 are still below 30% dry matter and they are being used as raw material for industrial extraction processes 52 53 (Ortuño et al. 1997). 54 55 56 5. Conclusions 57 58 Edible mushrooms cannot synthesise flavonoids because they do not have the main enzymes involved in 59 60 61 their metabolic pathway and no significant absorption was noticed from fruiting bodies cultivated in 62 63 17 64 65 flavonoid-enriched substrates or from mycelia grown of flavonoid-supplemented lab media. Thus,

1 spectrophotometric methods for ´total flavonoids´ determination should not be applied to fungal samples. 2 3 The flavonoids identified in several mushroom species using advance identification devices such as DAD 4 5 6 and MS might be due to sample contaminations or other pitfalls within the utilized protocols. Scientists 7 8 should be more careful when planning experiments to avoid the exponential spreading of large mistakes 9 10 such as considering fungi as plants. 11 12 13 14 15 Conflict of interest 16 17 18 The authors declare that they have no conflict of interest. 19 20 21 Acknowledgments 22 23 24 This review was supported by AGL2010-21537 and AGL2014-56211-R projects financed by the national R+D 25 26 program from the Spanish Ministry of Science and Innovation and ALIBIRD-C M S2009/AGR-1469 regional 27 28 program from the Community of Madrid (Spain). 29 30 31 Appendix 32 33 34 Supplementary Table 1 35 36 References 37 38 39 Babu, D., & Rao, G. N. (2013). Antioxidant properties and electrochemical behavior of cultivated 40 41 commercial Indian edible mushrooms. Journal of Food Science and Technology, 50, 301-308. 42 43 44 Barnum, D. W. (1977). Spectrophotometric determination of catechol, epinephrine, dopa, dopamine and 45 46 other aromatic vic-diols. Analytica Chimica Acta, 89, 157-166. 47 48 49 Barros, L., Dueñas, M., Ferreira, I. C. F. R., Baptista, P., & Santos-Buelga, C. (2009). Phenolic acids 50 51 determination by HPLC–DAD–ESI/MS in sixteen different Portuguese wild mushrooms species. Food 52 53 and Chemical Toxicology, 47, 1076-1079. 54 55 56 Choi, Y., Lee, S. M., Chun, J., Lee, H. B., & Lee, J. (2006). Influence of heat treatment on the antioxidant 57 58 activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom. Food Chemistry, 59 60 99, 381-387. 61 62 63 18 64 65 Choi, Y. M., Ku, J. B., Chang, H. B., & Lee, J. S. (2005). Antioxidant activities and total phenolics of ethanol

1 extracts from several edible mushrooms produced in Korea. Food Science and Biotechnology, 14, 2 3 700-703. 4 5 6 Christ, B., & Müller, K. H. (1960). Zur serienmäßigen Bestimmung des Gehaltes an Flavonol-Derivaten in 7 8 Drogen. Archiv der Pharmazie, 293, 1033-1042. 9 10 Del Signore, A., Romeo, F., & Giaccio, M. (1997). Content of phenolic substances in basidiomycetes. 11 12 13 Mycological Research, 101, 552-556. 14 15 Fernandes, Â., Antonio, A. L., Oliveira, M. B. P. P., Martins, A., & Ferreira, I. C. F. R. (2012). Effect of gamma 16 17 and electron beam irradiation on the physico-chemical and nutritional properties of mushrooms: A 18 19 20 review. Food Chemistry, 135, 641-650. 21 22 Gil-Ramírez, A., Clavijo, C., Palanisamy, M., Ruiz-Rodríguez, A., Navarro-Rubio, M., Pérez, M., Marín, F. R., 23 24 25 Reglero, G., & Soler-Rivas, C. (2013). Study on the 3-hydroxy-3-methyl-glutaryl CoA reductase 26 27 inhibitory properties of Agaricus bisporus and extraction of bioactive fractions using pressurised 28 29 solvent technologies. Journal of the Science of Food and Agriculture, 93, 2789-2796. 30 31 32 Grotewold, E. (2006). The science of flavonoids. New York: Springer-verlag New York. 33 34 Guillamón, E., García-Lafuente, A., Lozano, M., D´Arrigo, M., Rostagno, M. A., Villares, A., & Martínez, J. A. 35 36 (2010). Edible mushrooms: Role in the prevention of cardiovascular diseases. Fitoterapia, 81, 715- 37 38 39 723. 40 41 Harborne, J. B. (1994). The flavonoids–Advances in research since 1986 London, U.K.: Chapman & Hall. 42 43 44 Huang, X. D., Cai, J. X., Xu, L.-X., & Zhao, I. Z. (2012). Study on antioxidant effects in vitro and in vivo of total 45 46 flavonoids from Inonotus obliquus. Journal of food Science and Biotechnology, 31, 1062-1068. 47 48 Jayakumar, T., Thomas, P. A., Sheu, J. R., & Geraldine, P. (2011). In-vitro and in-vivo antioxidant effects of 49 50 51 the oyster mushroom Pleurotus ostreatus. Food Research International, 44, 851-861. 52 53 Jeong, S.-C., Koyyalamudi, S. R., Hughes, J. M., Khoo, C., Bailey, T., Marripudi, K., Park, J. P., Kim, J. H., & 54 55 Song, C.-H. (2013). Antioxidant and Immunomodulating Activities of Exo-and Endopolysaccharide 56 57 58 Fractions from Submerged Mycelia Cultures of Culinary-Medicinal Mushrooms. 15, 251-266. 59 60 61 62 63 19 64 65 Jourdan, P. S., McIntosh, C. A., & Mansell, R. L. (1985). Naringin Levels in Citrus Tissues : II. Quantitative

1 Distribution of Naringin in Citrus paradisi MacFad. Plant Physiology, 77, 903-908. 2 3 Kalogeropoulos, N., Yanni, A. E., Koutrotsios, G., & Aloupi, M. (2013). Bioactive microconstituents and 4 5 6 antioxidant properties of wild edible mushrooms from the island of Lesvos, Greece. Food and 7 8 Chemical Toxicology, 55, 378-385. 9 10 Karaman, M., Stahl, M., Vulić, J., Vesić, M., & Čanadanović-Brunet, J. (2014). Wild-growing lignicolous 11 12 13 mushroom species as sources of novel agents with antioxidative and antibacterial potentials. 14 15 International Journal of Food Sciences and Nutrition, 65, 311-319. 16 17 Kumari, B., Upadhyay, R. C., & Atri, N. S. (2013). Evaluation of Nutraceutical Components and Antioxidant 18 19 20 Potential of North Indian Wild Culinary-Medicinal Termitophilous Mushrooms. 15, 191-197. 21 22 Leal, A. R., Barros, L., Barreira, J. C. M., Sousa, M. J., Martins, A., Santos-Buelga, C., & Ferreira, I. C. F. R. 23 24 25 (2013). Portuguese wild mushrooms at the “pharma–nutrition” interface: Nutritional 26 27 characterization and antioxidant properties. Food Research International, 50, 1-9. 28 29 Liu, J., Jia, L., Kan, J., & Jin, C.-h. (2013). In vitro and in vivo antioxidant activity of ethanolic extract of white 30 31 32 button mushroom (Agaricus bisporus). Food and Chemical Toxicology, 51, 310-316. 33 34 Markham, K. R. (1988). Distribution of flavonoids in the lower plants and its evolutionary significance. In J. 35 36 B. Harborne (Ed.), The flavonoids. Advances in research since 1980. (pp. 427-468). London: 37 38 39 Chapman and Hall. 40 41 Mattila, P., Könkö, K., Eurola, M., Pihlava, J.-M., Astola, J., Vahteristo, L., Hietaniemi, V., Kumpulainen, J., 42 43 44 Valtonen, M., & Piironen, V. (2001). Contents of Vitamins, Mineral Elements, and Some Phenolic 45 46 Compounds in Cultivated Mushrooms. Journal of Agricultural and Food Chemistry, 49, 2343-2348. 47 48 Orhan, D. D., Özçelik, B., Özgen, S., & Ergun, F. (2010). Antibacterial, antifungal, and antiviral activities of 49 50 51 some flavonoids. Microbiological Research, 165, 496-504. 52 53 Ortuño, A., Reynaldo, I., Fuster, M. D., Botía, J., Puig, D. G., Sabater, F., Lidón, A. G., Porras, I., & Del Río, J. 54 55 (1997). Citrus cultivars with high flavonoid contents in the fruits. Scientia Horticulturae, 68, 231- 56 57 58 236. 59 60 61 62 63 20 64 65 Özyürek, M., Bener, M., Güçlü, K., & Apak, R. (2014). Antioxidant/antiradical properties of microwave-

1 assisted extracts of three wild edible mushrooms. Food Chemistry, 157, 323-331. 2 3 Palacios, I., Lozano, M., Moro, C., D’Arrigo, M., Rostagno, M. A., Martínez, J. A., García-Lafuente, A., 4 5 6 Guillamón, E., & Villares, A. (2011). Antioxidant properties of phenolic compounds occurring in 7 8 edible mushrooms. Food Chemistry, 128, 674-678. 9 10 Park, Y. K., Koo, M. H., Ikegaki, M., & Contado, J. l. (1997). Comparison of the flavonoid aglycone contents of 11 12 13 Apis mellifera propolis from various regions of Brazil. Arquivos de biologia e tecnologia, 40, 97-106. 14 15 Pękal, A., & Pyrzynska, K. (2014). Evaluation of Aluminium Complexation Reaction for Flavonoid Content 16 17 Assay. Food Analytical Methods, 7, 1776-1782. 18 19 20 Puttaraju, N. G., Venkateshaiah, S. U., Dharmesh, S. M., Urs, S. M. N., & Somasundaram, R. (2006). 21 22 Antioxidant Activity of Indigenous Edible Mushrooms. Journal of Agricultural and Food Chemistry, 23 24 25 54, 9764-9772. 26 27 Ramírez-Anguiano, A. C., Santoyo, S., Reglero, G., & Soler-Rivas, C. (2007). Radical scavenging activities, 28 29 endogenous oxidative enzymes and total phenols in edible mushrooms commonly consumed in 30 31 32 Europe. Journal of the Science of Food and Agriculture, 87, 2272-2278. 33 34 Rausher, M. D. (2006). The evolution of flavonoids and their genes. In E. Goteworld (Ed.), The science of 35 36 flavonoids (pp. 71-95). New York: Springer. 37 38 39 Robards, K., & Antolovich, M. (1997). Analytical Chemistry of Fruit BioflavonoidsA Review. Analyst, 122, 40 41 11R-34R. 42 43 44 Ruiz-Rodríguez, A., Polonia, I., Soler-Rivas, C., & Wichers, H. J. (2011). Ligninolytic enzymes activities of 45 46 Oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and 47 48 substrates. International Biodeterioration & Biodegradation, 65, 285-293. 49 50 51 Ruiz-Rodriguez, A., Soler-Rivas, C., Polonia, I., & Wichers, H. J. (2010). Effect of olive mill waste (OMW) 52 53 supplementation to Oyster mushrooms substrates on the cultivation parameters and fruiting 54 55 bodies quality. International Biodeterioration & Biodegradation, 64, 638-645. 56 57 58 59 60 61 62 63 21 64 65 Sapozhnikova, Y., Byrdwell, W. C., Lobato, A., & Romig, B. (2014). Effects of UV-B Radiation Levels on

1 Concentrations of Phytosterols, Ergothioneine, and Polyphenolic Compounds in Mushroom 2 3 Powders Used As Dietary Supplements. Journal of Agricultural and Food Chemistry, 62, 3034-3042. 4 5 6 Sharma, S. K., & Gautam, N. (2015). Chemical, Bioactive, and Antioxidant Potential of Twenty Wild Culinary 7 8 Mushroom Species. BioMed Research International, 2015, 12. 9 10 Soler-Rivas, C., Jolivet, S., Arpin, N., Olivier, J. M., & Wichers, H. J. (1999). Biochemical and physiological 11 12 13 aspects of brown blotch disease of Agaricus bisporus. FEMS Microbiology Reviews, 23, 591-614. 14 15 Tevfik, O., Cihan, D., Oznur, A., & Ibrahim, T. (2011). Screening of Antioxidant, Antimicrobial Activities and 16 17 Chemical Contents of Edible Mushrooms Wildly Grown in the Black Sea Region of Turkey. 18 19 20 Combinatorial Chemistry & High Throughput Screening, 14, 72-84. 21 22 Velísek, J., & Cejpek, K. (2011). Pigments of higher fungi: A review. Czech Journal of Food Sciences, 29, 87- 23 24 25 102. 26 27 Winkel, B. S. J. (2006). The biosynthesis of flavonoids. In E. Grotewold (Ed.), The science of flavonoids (pp. 28 29 274). New York: Springer-verlag New York. 30 31 32 Yongcai, Q., Xin, Z., & Yaung-Iee, L. (2013). Antioxidant and anticancer effects of edible and medicinal 33 34 mushrooms. Journal of the Korean Society of Food Science and Nutrition, 42, 655-662. 35 36 Zhang, N., Chen, H., Zhang, Y., Xing, L., Li, S., Wang, X., & Sun, Z. (2015). Chemical composition and 37 38 39 antioxidant properties of five edible Hymenomycetes mushrooms. International Journal of Food 40 41 Science & Technology, 50, 465-471. 42 43 44 Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of flavonoid contents in mulberry and 45 46 their scavenging effects on superoxide radicals. Food Chemistry, 64, 555-559. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 22 64 65 1 2 3 4 Table 1. Taxonomic distribution of sequences related to the three key enzymes involved in the biosynthesis of flavonoids extracted from Pfam (particularly those 5 6 7 present in the fungal kingdom). 8 9 10 Enzyme Function PFamily Kingdom * Phyllum Class Order Family Genus Specie Seq. 11 No Kingdom NS NS NS NS NS 20 37 12 Viridiplantae NS NS NS NS NS 312 1007 13 Metazoa NS NS NS NS NS 91 142 14 15 Agaricaceae Agaricus bisporus 1 Transform Tricholomataceae Tricholoma matsutake 3 16 Agaricomycetes Agaricales 17 L-phenylalanine Amanitaceae Amanita muscaria 1 18 to ammonia and Pleurotaceae Pleurotus eryngii 1 19 trans-cinnamic Lyase Ustilaginomycetes Ustilaginales Ustilaginaceae Pseudozyma brasiliensis 1 20 Basidiomycota PAL acid (first aromatic Rhodosporidium toruloides 1 21 phenolic (PF 00221) Fungi 22 mucilaginosa 1 compound in the No class Sporidiobolales No Family Rhodotorula 23 glutinis 3 24 phenyl propanoid 25 pathway) Sporidiobolus salmonicolor 3 26 Eurotiomycetes Eurotiales Aspergillaceae Penicillum roqueforti 1 27 Ascomycota Sordariomycetes Hypocreales Nectriaceae Fusarium graminearum 1 28 Lecanoromycetes Lecanorales Parmeliaceae Letharia vulpina 1 29 Uncategorized NS NS NS NS NS 28 102 30 31 Catalyzes the Chalcone Viridiplantae NS NS NS NS NS 751 2687 32 conversion of and 33 4-coumaroyl-CoA stilbene 34 CHS and malonyl-CoA synthases, Uncategorized NS NS NS NS NS 61 199 35 to naringenin C-terminal 36 chalcone (first domain 37 flavonoid) (PF02797) 38 39 Catalyzes the che Viridiplantae NS NS NS NS NS 146 398 40 mical reaction: a Chalcone CHI Uncategorized NS NS NS NS NS 15 55 41 chalcone <-> a (PF02431) 42 flavanone No Kingdom NS NS NS NS NS 2 2 43 44 *Only inside Eukariota domain. PAL, phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI, chalcone isomerase. NS, non-specified data. 45 46 47 48 23 49 Table 2. Results of the alignments of the Chalcone synthase (CHS) sequence from Vitis vinifera with other organisms using Blastp. Data included are those showing higher percentage of homology. 1 2 % alignment 3 Organism Term searched Genus Specie Homology with (query 4 cover) 5 6 Plant Vitis vinifera Vitis vinifera Chalcone synthase 100 Arabidopsis 7 Plant Arabidopsis thaliana Chalcone synthase 97 8 thaliana 9 Morus alba var. multicaulis Chalcone synthase 98 10 11 Plant Mulberry Morus notabilis Chalcone synthase 98 12 Morus alba Chalcone synthase 65 13 Plant Strawberry Fragaria ananassa Chalcone synthase 98 14 15 Solanum lycopersicum Chalcone synthase 1&2 98 16 Plant Solanum Solanum tuberosum Chalcone synthase 98 17 Solanum pinnatisectum Chalcone synthase 98 18 19 Citrus reticulata Chalcone synthase 98 20 Citrus ichangensis Chalcone synthase 98 21 Plant Citrus Citrus maxima Chalcone synthase 98 22 Hypothetical protein CICLE 23 Citrus clementina 98 24 v 10015535mg 25 Arabidopsis Plant Arabidopsis thaliana Chalcone synthase 97 26 thaliana 27 No significant similarity Animal Human Homo sapiens 0 28 found 29 No significant similarity Animal Mouse Mus musculus 0 30 found 31 Caenorhabditis No significant similarity 32 Animal Caenorhabditis elegans 0 33 elegans found Saccharomyces No significant similarity 34 Fungi Saccharomyces cerevisiae 0 35 cerevisiae found 36 bisporus var. burnettii Hypothetical protein Agaricus 7 37 JB137-S8 AGABI2DRAFT 11251 38 bisporus var. bisporus Hypothetical protein Agaricus 7 39 H97 AGABI2DRAFT 193398 Fungi Agaricus 40 bisporus var. burnettii Hypothetical protein Agaricus 3 41 JB137-S8 AGABI2DRAFT 74201 42 bisporus var. bisporus Hypothetical protein 43 Agaricus 3 44 H97 AGABI2DRAFT 207020 Pleurotus Hypothetical protein 45 Fungi Pleurotus ostreatus PC15 17 46 ostreatus PLEOSDRAFT 1059391 47 Homeodomain 1 mating 48 Fungi Pleurotus eryngii* Pleurotus eryngii var. tuoliensis type protein (genome not 3 49 completely sequenced) 50 No significant similarity Fungi Lentinula edodes* Lentinula edodes 0 51 found 52 Ganoderma No significant similarity 53 Fungi Ganoderma lucidum 0 lucidum* found 54 Lactarius No significant similarity 55 Fungi Lactarius deliciosus 0 56 deliciosus* found Hypothetical protein 57 Pseudomonas pseudoalcaligenes 55 58 Others Microbes N619_00405 59 Gordonia rhizosphera Stilbene synthase 34 60 61 *Genomes from mushrooms not completely sequenced (in 2015). 62 63 24 64 65 Figure captions

1 Figure 1. Number of scientific works published up to October 2015 determining flavonoids in mushrooms 2 3 (according to Web of Science). References were shorted by the impact factor of the journal where they 4 5 were published the year that they were published (except for 2015). 6 7 8 9 10 Figure 2. Total phenols and the colorimetric results obtained after using the method to determine ‘total 11 12 13 flavonoids’ in several mushrooms species (at 510 nm). 14 15 16 17 Figure 3. VIS spectra of quercetin, L-DOPA and ergosterol standard compounds when they were treated 18 19 20 with Al(Cl3)3 and NaNO2 solutions as indicated in the test used to measure ‘total flavonoids’ in mushrooms. 21 22 23 24 Figure 4. HPLC chromatogram at 340 nm of a) onion waste b) substrate supplemented with onion waste 25 26 27 after cultivation of P. ostreatus and c) P. ostreatus fruiting bodies grown on substrate supplemented with 28 29 onion waste (Rt= 8.76 min. quercetin 3,7- or 3,4’-diglucoside; Rt= 10.02 min. quercetin 3,7- or 3,4’- 30 31 32 diglucoside; Rt= 10.81 min. isorhamnetin 3-diglucoside (or 3-sophoroside); Rt=16.02 min. quercetin 3- 33 34 glucoside and Rt= 17.39 min. isorhamnetin 3-glucoside). 35 36 37 38 39 Figure 5. Mycelial growth of P. ostreatus grown on MEA media supplemented with different concentrations 40 41 of a) luteolin dissolved in ethanol and b) caffeic acid. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 25 64 65 Table 1 Click here to download high resolution image Table 2 Click here to download high resolution image Figure 1 (in color) Click here to download high resolution image Figure 1 (bw) Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Supplementary Material Click here to download Figure: Appendix-supplementary Table 1.docx Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Antioxidant activities and Ganoderma total phenolics of ethanol Food Science and Choi, YM. et 14, 5, Total lucidum and 1 2005 0.645 extracts from several edible Colorimetric NF Ethanol Biotechnology al. 700-703 flavonoids Pleurotus mushrooms produced in eryngii Korea. Influence of heat treatment on the antioxidant activities NaNO + 99, 2, Total 2 80% Lentinus 2 2006 Food Chemistry 2.433 Choi, Y. et al. and polyphenolic compounds 10% AlCl · VIS 510 nm 381–387 flavonoids 3 Ethanol edodes of Shiitake (Lentinus edodes) H2O mushroom. Antioxidant activities and Journal of total phenolics of pickles Ganguli, A. 5, 2-3, Total UV/VIS 415 Agaricus 3 2006 Culinary Science NF produced from the edible 10% Al(NO ) Ethanol et al. 131-142 flavonoids 3 3 nm bisporus and Technology mushroom, Agaricus bisporus. Antioxidant and antimicrobial Annals of Mercan, N. et properties of ethanolic extract 56, 4, Total UV/VIS 415 4 2006 0.427 10% Al(NO ) Ethanol Lepista nuda Microbiology al. from Lepista nuda (Bull.) 339-344 flavonoids 3 3 nm Cooke. Free-radical scavenging 5, 20, African Journal of Gezer, K. et capacity and antimicrobial Total UV/VIS 415 Wild edible 5 2006 NF 1924- 10% Al(NO ) Ethanol Biotechnology al. activity of wild edible flavonoids 3 3 nm mushrooms 1928 mushroom from Turkey. Antioxidant and antimicrobial 5, 11, African Journal of Turkoglu, A. Total UV/VIS 415 Morchella 6 2006 NF activities of Morchella conica 1146- 10% Al(NO ) Ethanol Biotechnology et al. flavonoids 3 3 nm conica Pers. 1150 Antioxidant and antimicrobial Turkoglu, A. 101, 1, Total UV/VIS 415 Laetiporus 7 2007 Food Chemistry 3.052 activities of Laetiporus 10% Al(NO ) Ethanol et al. 267–273 flavonoids 3 3 nm sulphureus sulphureus (Bull.) Murrill Food and Effect of Lactarius piperatus 45, 9, NaNO + Barros, L. et Total 2 Lactarius 8 2007 Chemical 2.186 fruiting body maturity stage 1731– 10% AlCl · VIS 510 nm Methanol al. flavonoids 3 piperatus Toxicology on antioxidant activity 1737 H2O

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used measured by several biochemical assays. Bioactive properties of the medicinal mushroom NaNO + Barros, L. et Leucopaxillus giganteus 105, 1, Total 2 Leucopaxillus 9 2007 Food Chemistry 3.052 10% AlCl · VIS 510 nm Methanol al. mycelium obtained in the 179–186 flavonoids 3 giganteus H O presence of different nitrogen 2 sources. Free radical-scavenging and inhibition of nitric oxide 103, 4, NaNO + Ethyl Lim, HW. et Total 2 Tricholoma 10 2007 Food Chemistry 3.052 production by four grades of 1337– 10% AlCl · VIS 510 nm acetate or al. flavonoids 3 matsutake pine mushroom (Tricholoma 1342 H2O butanol matsutake Sing.) Effect of fruiting body Lactarius Journal of maturity stage, on chemical 55, 21, NaNO + Barros, L. et Total 2 deliciosus 11 2007 Agricultural and 2.532 composition and 8766- 10% AlCl · VIS 510 nm Methanol al. flavonoids 3 and Food Chemistry antimicrobial activity of 8771 H O 2 L. piperatus Lactarius sp mushrooms. Antibacterial metabolites Gowdaman, 7, 2, 763- Total Phellinus 12 2007 Plant Archives NF from wood rot fungus Colorimetric NF Ethanol V. et al. 765 flavonoids hohnelli Phellinus hohnelli. Effect of Lactarius piperatus Food and fruiting body maturity stage 45, 9, NaNO + Barros, L. et Total 2 13 2007 Chemical 2.186 on antioxidant activity 1731- 10% AlCl · VIS 510 nm Methanol L. piperatus al. flavonoids 3 Toxicology measured by several 1737 H2O biochemical assays. L. deliciosus, Antimicrobial activity and Sarcodon European Food bioactive compounds of 225, 2, NaNO + Barros, L. et Total 2 imbricatus 14 2007 Research and 1.159 Portuguese wild edible 151 EP 10% AlCl · VIS 510 nm Methanol al. flavonoids 3 and Technology mushrooms methanolic 156 H O 2 Tricholoma extracts portentosum

Appendix- Supplementary Table 1

Lactarius Evaluation of the antioxidant deterrimus, activity of four edible Suillus mushrooms from the Central 99, 14, Bioresource Sarikurkcu, Total UV/VIS 415 collitinus, 15 2008 4.453 Anatolia, Eskisehir – Turkey: 6651– 2% AlCl Methanol Technology C. et al. flavonoids 3 nm Boletus edulis Lactarius deterrimus, Suillus 6655 and collitinus, Boletus edulis, Xerocomus Xerocomus chrysenteron. chrysenteron

Agaricus bisporus, Agaricus Antioxidant activity of arvensis, NaNO + Barros, L. et Agaricus sp. mushrooms by 111, 1, Total 2 Agaricus 16 2008 Food Chemistry 2.696 10% AlCl · VIS 510 nm Methanol al. chemical, biochemical and 61–66 flavonoids 3 romagnesii, H O electrochemical assays. 2 Agaricus sivaticus and Agaricus silvicola

B. edulis, Calocybe gambosa, Cantharellus cibarius, Food and Wild and commercial 46, 8, NaNO + Craterellus Barros, L. et Total 2 17 2008 Chemical 2.321 mushrooms as source of 2742– 10% AlCl · VIS 510 nm Methanol cornucopioide al. flavonoids 3 Toxicology nutrients and nutraceuticals. 2747 H2O s, Marasmius oreades, A. bisporus, A. sylvaticus and A. silvicola

Journal of Phenolic compound 56, 16, P. eryngii, G. Kim, MY. et Total 80% 18 2008 Agricultural and 2.562 concentration and antioxidant 7265- HPLC-DAD UV 280 nm lucidum and al. flavonoids Methanol Food Chemistry activities of edible and 7270 A. bisporus 3

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used medicinal mushrooms from Korea. Phenolics and antioxidant Food Science and activity of mushroom NaNO + Barros, L. et 14, 1, Total 2 19 2008 Techonology 0.702 Leucopaxillus giganteus 10% AlCl · VIS 510 nm Water L. giganteus al. 47- 55 flavonoids 3 International mycelium at different carbon H2O sources. Innovative Food In-vitro antioxidant activities Science and Jayakumar, of an Ethanolic extract of the 10, 2, Rutin and 95% Pleurotus 20 2009 2.174 HPLC UV 300 nm Emerging T. et al. oyster mushroom Pleurotus 228–234 chrysin Ethanol ostreatus Technologies ostreatus. Morchella rotunda, Morchella crassipes, Morchella Antioxidant activities, metal esculenta var. Food and 47, 9, Gursoy, N. contents, total phenolics and Total UV/VIS 415 umbrina, 21 2009 Chemical 2.114 2381– AlCl Methanol et al. flavonoids of seven flavonoids 3 nm Morchella Toxicology 2388 Morchella species. deliciosa, Morchella elata, M.conica and Morchella angusticeps. Acetone, water, ethanol, Micologia Antioxidant properties of NaNO + Bruijn, J. et 21, 1, 11- Total 2 ethyl 22 2009 Aplicada NF extracts obtained from Grifola 10% AlCl · VIS 510 nm Grifola gargal al. 18 flavonoids 3 acetate, n- International gargal mushrooms. H O 2 hexane or ethanol:wat er mixtures

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Mixtures of Pleurotus sp., Phenolic profiles of selected methanol, Hygrocybe Asian Journal of edible wild mushrooms as NaNO + ethanol, conica, 2, 3, 392- Total 2 23 2009 Food and NF Hip, SY. et al. affected by extraction 10% AlCl · 510 nm acetone, Schizophyllu 401 flavonoids 3 AgroIndustry solvent, time and H2O water or m commune temperature. absolute and Lentinus hexane ciliatus Comparitive study on the antioxidant, anticancer and African Journal of Jagadish, antimicrobial property of 8, 4, 654- Total 24 2009 0.565 AlCl UV 367 nm Ethanol A. bisporus Biotechnology L.K., et al. Agaricus bisporus (J. E. 661 flavonoids 3 Lange) Imbach before and after boiling. Accumulation of antioxidant 100, 3, 70% Bioresource Zheng, W. et phenolic constituents in Total UV 280-310 Inonotus 25 2009 4.253 1327- HPLC-DAD Aqueous Technology al. submerged cultures of flavonoids nm obliquus 1335 acetone Inonotus obliquus. International Ethyl Antioxidant activities of Journal of Food Liang, L. et 60, 2, Total acetate, n- 26 2009 1.313 extracts and subfractions Colorimetric NF I. obliquus Sciences and al. 175-184 flavonoids butanol or from Inonotus obliquus. Nutrition water International Antioxidant activity of edible Journal of Percario, S. blushing wood mushroom, 11, 2,133- Total UV/VIS 415 27 2009 0.661 AlCl Water A. sylvaticus Medicinal et al. Agaricus sylvaticus Schaeff. 139 flavonoids 3 nm Mushrooms (Agaricomycetideae) in vitro. Phenolics and antioxidant activity of a ready-to-eat NaNO + Nutrition and Singla, R. et 39, 3, Total 2 28 2009 NF snack food prepared from the 10% AlCl · VIS 510 nm NF A. bisporus Food Science al. 227-234 flavonoids 3 edible mushroom ( Agaricus H2O bisporous).

Appendix- Supplementary Table 1

A QCAR model for predicting Environmental Froufe, 20, 5-6, Total Wild 29 2009 3.237 antioxidant activity of wild Colorimetric NF NF Research H.J.C. et al. 579-590 flavonoids mushrooms mushrooms.

Effect of integrated application of gamma irradiation and modified NaNO + Jiang, T. et atmosphere packaging on 122, 3, Total 2 Ethanol or 30 2010 Food Chemistry 3.458 10% AlCl · VIS 510 nm L. edodes al. physicochemical and 761–767 flavonoids 3 water H O microbiological properties of 2 shiitake mushroom (Lentinus edodes).

Influence of UV-C treatment on antioxidant capacity, Postharvest NaNO + Jiang, T. et antioxidant enzyme activity 56, 3, Total 2 31 2010 Biology and 2.256 10% AlCl · VIS 510 nm Ethanol L.edodes al. and texture of postharvest 209–215 flavonoids 3 Technology H O shiitake (Lentinus edodes) 2 mushrooms during storage.

Chemical composition and Acetonitrile Journal of 58, 11, Russula Chen, XH. et antioxidant activities of and 32 2010 Agricultural and 2.816 6966– Quercetine HPLC-DAD UV 273 nm griseocarnos al. Russula griseocarnosa sp. hydrocloric Food Chemistry 6971 a nov. acid

L. edodes, Journal of Central Antioxidant properties of Hericium 11, 4, Total 50% 33 2010 European NF Mujić, I. et al. selected edible mushroom AlCl NF erinaceus 387-392 flavonoids 3 Ethanol Agriculture species. and Agrocybe aegerita

Appendix- Supplementary Table 1

Evaluation of antioxidant Ramaria activities of 3 edible flava, mushrooms: Ramaria flava Food Science and Gursoy, N. 19, 3, Total UV/VIS 415 Rhizopogon 34 2010 0.505 (Schaef.: Fr.) Quél., AlCl Methanol Biotechnology et al. 691-696 flavonoids 3 nm roseolus and Rhizopogon roseolus (Corda) Russula T.M. Fries., and Russula delica delica Fr.

Lycoperdon perlatum, Ca ntharellus Antimicrobial properties, cibarius, Clav antioxidant activity and aria Pharmacognosy Ramesh, CH. bioactive compounds from 2, 2, 107– Total UV/VIS 415 vermiculris, R 35 2010 NF 10% Al(NO ) Methanol Research et al. six wild edible mushrooms of 112 flavonoids 3 3 nm amaria western ghats of Karnataka, Formosa, Mar India. asmius oreades and Pleurotus pulmonarius In vitro free radical Indian Journal of 48, 12, Cold/hot scavenging activity of wild Total UV/VIS 415 Pleurotus 36 2010 Experimental 0.702 Pal, J. et al. 1210- 10% Al(NO ) water and edible mushroom, Pleurotus flavonoids 3 3 nm squarrosulus Biology 1218 methanol squarrosulus (Mont.) Singer Chemical composition and Russula 22, 9, Asian Journal of Chen, XH. et antioxidant activity of two Total virescens and 37 2010 0.247 6867- Colorimetric NF NF Chemistry al. edible mycorrhizal fungi from flavonoids Cantharellus 6878 South China. cibarius Antioxidant activities of Pharmacognosy Ozen, T. et Sarcodon imbricatum wildly 6 ,22, 89- Total UV/VIS 415 38 2010 0.432 10% Al(NO ) Methanol S. imbricatus Magazine al. grown in the black sea region 97 flavonoids 3 3 nm of Turkey.

Appendix- Supplementary Table 1

Antioxidant properties of cap 15, 3, NaNO + Total 2 Ethanol or Coprinus 39 2010 Molecules 1.988 Li, B. et al. and stipe from Coprinus 1473- 10% Al(NO ) VIS 510 nm flavonoids 3 3 hot water comatus comatus. 1486 · H2O

Comparison Antioxidant components and of fee Folin– Journal of Food antioxidant/antiradical Al-Laith, 23, 1, 15- Total phenolic Ciocalteu 80% Tirmania 40 2010 Composition and 1.948 activities of desert truffle AAA. 22 flavonoids content after method (765 Ethanol nivea Analysis (Tirmania nivea) from various formaldehyde mn) Middle Eastern origins. precipation.

Antioxidant activities and polyphenolic properties of International 3, 6, NaNO + Singla, R. et raw and osmotically Total 2 99% 41 2010 Journal of Food 0.947 1290- 10% AlCl · VIS 510 nm A. bisporus al. dehydrated dried mushroom ( flavonoids 3 Ethanol Properties 1299 H O Agaricus bisporous) snack 2 food. Antioxidant and free radical scavenging activities of International culinary-medicinal Ethyl C. cibarius Journal of Ebrahimzade 12, 3, 265 Total 42 2010 0.639 mushrooms, Golden Colorimetric NF acetate or and Pleurotus Medicinal h, MA. et al. -272 flavonoids Chanterelle Cantharellus methanol porrigens Mushrooms cibarius and Angel's Wings Pleurotus porrigens.

Anti-Human Papillomavirus (HPV) 16 E6 activity of Ling International Zhi or Reishi medicinal Journal of mushroom, Ganoderma 12, 3, Total Dichlorome 43 2010 0.639 Lai, LK. et al. Colorimetric NF G. lucidum Medicinal lucidum (W. Curt.: Fr.) P. 279-286 flavonoids thane Mushrooms Karst. (Aphyllophoromycetideae) extracts.

Appendix- Supplementary Table 1

Antioxidant activities of 127,4, NaNO + extract and fractions from Total 2 Tuber 46 2011 Food Chemistry 3.655 Guo, T. et al. 1634– 10% Al(NO ) VIS 510nm Ethanol Tuber indicum Cooke & flavonoids 3 3 indicum 1640 · H O Massee. 2

Protective effect of two edible P. eryngii and mushrooms against oxidative 128, 3, Methanol or Auricularia 47 2011 Food Chemistry 3.655 Oke, F. et al. Luteolin HPLC-DAD UV 278 nm cell damage and their 613–619 water auricula- phenolic composition. judae

In vitro antioxidant, anticholinesterase and antimicrobial activity studies Hexane, Agaricus Food and on three Agaricus species 49, 6, ethyl essettei, Öztürk, M. et Total UV/VIS 415 48 2011 Chemical 2.999 with fatty acid compositions 1353– 10% Al(NO ) acetate or Agaricus al. flavonoids 3 3 nm Toxicology and iron contents: A 1360 aqueous bitorquis and comparative study on the methanol A. bisporus. three most edible mushrooms.

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used A. bisporus, Total B. edulis, C. AlCl NF flavonoids 3 gambosa, C. Antioxidant properties of cibarius, C. Palacios, I. et phenolic compounds 128, 3, cornucopioid, 49 2011 Food Chemistry 3.655 Methanol al. occurring in edible 674–678 Hygrophorus mushrooms. Catechin marzuolus, L. 280 y and HPLC-DAD deliciosus 320nm myricetin and P. ostreatus. Determination of total phenol, Pleurotus International flavonoid and antioxidant Imran, MM. 18, 2, Total UV/VIS 415 florida and 50 2011 Food Research 0.282 activity of edible mushrooms AlCl Methanol et al. 579-582 flavonoids 3 nm Pleurotus Journal Pleurotus florida and eous Pleurotus eous. Detection: Analysis of antioxidant amonia Presence of Journal of activity, total phenol, total solution + yellow color P. eous and Chemical and Sathyaprabh flavonoid and screening of Total sulphuric acid 51 2011 NF 3, 6, 1-6 Water Pleurotus Pharmaceutical a, G. et al. phytocomponents in flavonoids Cuantification platypus Research Pleurotus platypus and : NaNO2 + Pleurotus eous. VIS 510 nm 10% AlCl3 · H2O International Antioxidant properties of the 12, 10, NaNO + Armillaria Journal of Lung, MY. et edible Basidiomycete Total 2 UV/VIS 415 Hot water 52 2011 2.6 6367- 10% AlCl · mellea Molecular al. Armillaria mellea in flavonoids 3 nm or methanol 6384 H O (micellium) Sciences submerged cultures. 2

Antioxidant properties and antioxidant compounds of 16, NaNO + Cold/hot Yeh, JY. et Total 2 UV/VIS 415 Grifola 53 2011 Molecules 2.386 various extracts from the 4, 3197- 10% AlCl · water or al. flavonoids 3 nm frondosa edible Basidiomycete Grifola 3211 H2O methanol Frondosa (Maitake).

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Evaluation of the antioxidant 60% and antityrosinase activities 10, acetone, African Journal of Alam, N. et naringenin, Pleurotus 54 2011 NI of three extracts from 11, 2978- HPLC-DAD UV 280 nm 80% Biotechnology al. hesperetin nebrodensis Pleurotus nebrodensis 2986 methanol or fruiting bodies. hot water Screening of antioxidant, Total Twelve Combinatorial antimicrobial activities and flavonoids commonly Chemistry & High Ozen, T. et chemical contents of edible 14, 2, 72- 55 2011 1.785 and Colorimetric NF NF consumed Throughput al. mushrooms wildly grown in 84 anthocyanin wild edible Screening the Black Sea region of s mushrooms Turkey. Effect of spermine on International bioactive components and NaNO + Journal of Jahangir, antioxidant properties of 13, 5, Total 2 56 2011 0.94 10% AlCl · VIS 510 nm Methanol A. bisporus Agriculture and MM. et al. sliced 771-775 flavonoids 3 H O Biology buttonmushroom (Agaricus 2 bisporus) during storage.

Effect of high argon pressure and modified atmosphere Journal of Food packaging on the NaNO + Lagnika, C. 50, 3, Total 2 57 2011 Nutrition and 0.679 white mushroom(Agaricus 10% AlCl · VIS 510 nm Water A. bisporus et al. 167-176 flavonoids 3 Research bisporus) physico-chemical H2O and microbiological properties.

Antioxidant properties L. edodes Journal of Food Da Silva, AC. of Lentinus 34, 6, Total 58 2011 0.54 Colorimetric NF NF and Agaricus Quality et al. edodes and Agaricus 386-394 flavonoids blazei blazei extracts.

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Nitric oxide fumigation stimulates flavonoid and 135, 3, NaNO + R. Dong, J. et Total 2 59 2012 Food Chemistry 3.334 phenolic accumulation and 1220– 10% Al(NO ) VIS 510nm Methanol griseocarnos al. flavonoids 3 3 enhances antioxidant activity 1225 · H2O a of mushroom. Towards chemical and Twelve nutritional inventory of NaNO + Pereira, E. et 130, 2, Total 2 Portuguese 60 2012 Food Chemistry 3.334 Portuguese wild edible 10% AlCl · VIS 510 nm Methanol al. 394–403 flavonoids 3 wild edible mushrooms in different H O 2 mushrooms habitats. Asian Pacific Identification and 2, 1, Journal of Shirmila, JG. determination of antioxidant suppleme Total UV/VIS 420 Omphalotus 61 2012 0.502 AlCl Methanol Tropical et al. constituents of nt, 386- flavonoids 3 nm nidiformis Biomedicine bioluminescent mushroom. 391 Clitocybe maxima, Catathelasma Chemical composition of five ventricosum, wild edible mushrooms Total Food and 50, 5, Stropharia collected from Southwest flavonoids, 62 2012 Chemical 3.01 Liu, YT. et al. 1238– HPLC-DAD UV 273 nm Ethanol rugoso- China and their quercetin Toxicology 1244 annulata, C. antihyperglycemic and and catechin cornucopioide antioxidant activity. s and Laccaria amethystea

Boletus Iranian Journal of Mushrooms as possible 11, 4, Acetone aestivalis, B. Kosanić, M. Total UV/VIS 415 63 2012 Pharmaceutical 0.54 antioxidant and antimicrobial 1095- AlCl and edulis and et al. flavonoids 3 nm Research agents. 1102 methanol Leccinum carpini

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Targeted phenolic analysis in Food Science and 21, 3, Total UV/VIS 415 64 2012 0.695 Li, H et al. Hericium erinaceum and its 10% Al(NO ) Methanol H. erinaceum Biotechnology 881-888 flavonoids 3 3 nm antioxidant activities. Antioxidant and Tricholoma cholinesterase inhibition fracticum, activities of three Tricholoma Tricholoma Food Analytical 5, 3, 495- Total UV/VIS 415 65 2012 1.969 Tel, G.et al. species with total phenolic 10% Al(NO ) Methanol imbricatum Methods 504 flavonoids 3 3 nm and flavonoid contents: The and edible mushrooms from Tricholoma Anatolia. terreum Phellinus Antioxidant and antifungal gilvus, Revista Ayala- NaNO + potential of methanol extracts 29, 3, Total 2 UV/VIS 415 Phellinus 66 2012 Iberoamericana 1.312 Zavala, FJ. et 10% AlCl · Methanol of Phellinus spp. from 132-138 flavonoids 3 nm rimosus and de Micología al. H O Sonora, Mexico. 2 Phellinus badius Antioxidant activity of various Ethyl 21, NaNO + Food Science and Sudha, G. et extracts from an Total 2 acetate, 67 2012 0.695 3, 661- 10% AlCl · VIS 510 nm P. eous Biotechnology al. edible mushroom Pleurotus flavonoids 3 methanol or 668 H O eous. 2 hot water A. bisporus, Rutin, P. eryngii var. Flavonoids contents and 2,2- myricetin, eryngii, P. diphenyl-1-picrylhydrazyl morin, eryngii var. Current topics in Akyuz, M. et radical scavenging activity of 10, 2, quercetin, UV 254-280 80 % ferulae, 68 2012 Nutraceutical 0.262 HPLC-DAD al. some edible mushrooms 133-136 kaempferol, nm Methanol Pleurotus Research from Turkey: A. bisporus and catechin, sajor-caju, P. Pleurotus spp. naringenin ostreatus and and naringin Pleurotus florida

Appendix- Supplementary Table 1

Physicochemical and Food and nutritional characteristics of NaNO + Singla, R. et 5, 2, 808- Total 2 69 2012 Bioprocess 4.115 organic acid-treated button 10% AlCl · VIS 510 nm NF A. bisporus al. 815 flavonoids 3 Technology mushrooms (Agaricus H2O bisporous).

Study on antioxidation Journal of Food 31, 10, Huang, XD. effect in vitro and in vivo of Total 70 2012 Science and 0.695 1062- NF NF NF I. obliquus et al. total flavonoids from Inonotus flavonoids Biotechnology 1068 obliquus. Immunomodulatory activities International of different solvent extracts Water or n- Journal of from Tricholoma matsutake 14, 6, Total 71 2012 0.838 Yin, X. et al. Colorimetric NF butyl T. matsutake Medicinal (S. Ito et S. Imai) Singer 549-556 flavonoids alcohol. Mushrooms (higher Basidiomycetes) on normal mice. Phellinus Polar fractionation affects the fastuosus, International antioxidant properties of Phellinus Water or Journal of Ayala-Zavala, methanolic extracts from 14, 6, Total grenadensis, 72 2012 0.838 Colorimetric NF ethyl Medicinal FJ. et al. species of genus Phellinus 563-573 flavonoids Phellinus acetate Mushrooms Quel. (higher merrillii, and Basidiomycetes). Phellinus badius Pharmacological activity of Journal of the chaga mushroom on 41, 10, Hot water Korean Society of Baek, GH. et Total 73 2012 NF extraction conditions and 1378- Colorimetric NF or hot I. obliquus Food Science and al. flavonoids immunostimulating 1387 ethanol Nutrition polysaccharide. Journal of the Antioxidant properties and 41, 9, 14 Korean Society of Hong, MH. et ubiquinone contents in Total 74 2012 NF 1235- Colorimetric NF Methanol commercial Food Science and al. different parts of several flavonoids 1241 mushrooms Nutrition commerical mushrooms.

Appendix- Supplementary Table 1

Contents of some metabolites in the peel and International flesh of the Journal of Wang, Y. et 14, 1, 79- Total Wolfiporia 75 2012 0.838 medicinal mushroom Wolfipo Colorimetric NF NF Medicinal al. 83 flavonoids cocos ria cocos (F.A. Wolf) Mushrooms Ryvarden et Gilb. (Higher Basidiomycetes).

Amanita crocea, Amanita mairei, UV 280-370; Boletus Portuguese wild mushrooms comparison porosporus, at the “pharma–nutrition” of mass Boletus Food Research Leal, AR. et Chrysin HPLC- 80% 76 2013 3.05 interface: nutritional 50, 1, 1–9 spectras regius, International al. derivatives DAD/MS Methanol characterization and and Gyromitra antioxidant properties. retention esculenta, times Helvella lacunosa, Russula aurea and R. virescens Comparison In vitro and in vivo of mass Food and antioxidant activity of 51, -,310– Myricetin HPLC-ESI- spectras 80% 77 2013 Chemical 2.61 Liu, J. et al. ethanolic extract of white A. bisporus 316 and catechin MS/MS and Ethanol Toxicology button mushroom (Agaricus retention bisporus). times A. bisporus, Extraction of total phenolic Trametes Journal of Natural and flavonoids from edible NaNO + versicolor, Abugria, DA. 3, 3, 37- Total 2 Mehtnaol or 78 2013 Products and NF wild and cultivated medicinal 10% AlCl · VIS 510 nm Ganoderma et al. 42 flavonoids 3 water Plant Resources mushrooms as affected by H2O applanatum different solvents. and Fomes fomentarius. 15

Appendix- Supplementary Table 1

Antioxidant analysis of different types of edible 20, 3, NaNO + 60% A. bisporus International Food Total 2 79 2013 NF Gan, C. et al. mushrooms (Agaricus 1095- 10% AlCl · VIS 510 nm Ethanol or and Agaricus Research Journal flavonoids 3 bisporous and Agaricus 1102 H2O water brasiliensis brasiliensis).

Hypsizygus te ssulatus, P. Evaluation of bioactivities eryngii, P. Tropical Journal 12, 6, NaNO + Wong, FC. and phenolic content of Total 2 90 % florida, 80 2013 of Pharmaceutical 0.495 1011- 10% AlCl · VIS 510 nm et al. selected edible mushrooms flavonoids 3 Ethanol Auricularia Research 1016 H O in Malaysia. 2 polytricha and Flammulina velutipes International In vitro antioxidant activities, Journal of total phenolics and flavonoid Shirmila, J, et 5, 2, 161- Total UV/VIS 415 Macrolepiota 81 2013 Pharmacy and NF of wild edible mushroom AlCl Methanol al. 166 flavonoids 3 nm mastoidea Pharmaceutical Macrolepiota Mastoidea (Fr.) Sciences Singer. Nutritive value, flavonoid Myricetin, Journal of Soil content and radical 13, 1, kaempferol, UV 254-280 80% Terfezia 82 2013 Science and Plant 0.582 Akyüz, M. scavenging activity of the HPLC-DAD 143-151 naringin and nm Methanol boudieri Nutrition truffle (Terfezia boudieri naringenin Chatin). Antioxidant properties in the Pleurotus oyster mushrooms (Pleurotus NaNO + djamor var. Arbaayah, 4, 4, 661– Total 2 83 2013 Mycosphere NF spp.) and split gill mushroom 10% AlCl · VIS 510 nm Ethanol djamor and HH. et al. 673 flavonoids 3 (Schizophyllum commune) H2O Schizophyllu ethanolic extracts. m commne Phytochemicals in edible wild NaNO + Journal of Food Wandati, TW. 2, 3, 137- Total 2 UV/VIS 415 P. florida and 84 2013 1.791 mushrooms from selected 10% AlCl · Ethanol Research et al. 144 flavonoids 3 nm A. biporus areas in Kenya. H2O

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Phytochemical screening and Food and effects on cell-mediated Llaurado, G. 24, 3, Total 85 2013 Agricultural 0.984 immune response of Colorimetric NF NF Pleurotus et al. 295-304 flavonoids Immunology Pleurotus fruiting bodies powder. NaNO + Total 2 10% AlCl · VIS 517 nm flavonoids 3 H2O Acetylcholinesterase UV 280 nm; inhibition and in vitro and in 18, Quercetin, Hasnat, A. et comparison 86 2013 Molecules 2.095 vivo antioxidant activities of 6, 6663- kaempferol, Water G. lucidum al. of mass Ganoderma lucidum grown 6678 cateuin, HPLC- spectras on germinated brown rice. myricetin, DAD/MS and rutin, retention epicatechin times L. deliciosus, Comparison Lactarius Chrysin, Bioactive microconstituents of mass sanguifluus, Food and genistein, Kalogeropoul and antioxidant properties of 55, -, 378- spectras Lactarius 87 2013 Chemical 2.61 kaempferol GC-MS Methanol os, N. et al. wild edible mushrooms from 385 and semisanguiflu toxicology and the island of Lesvos, Greece. retention us, R. delica quercetin times and Suillus bellinii A. bisporus, Antioxidant properties and Journal of Food 50, Hypsizygus Babu, DR. et electrochemical behavior of Total UV/VIS 415 95% 88 2013 Science and 2.024 2, 301- AlCl ulmarius and al. cultivated commercial Indian flavonoids 3 nm Etahnol Technology 308 Calocybe edible mushrooms. indica Nutritional and bioactive potential of two wild edible Lentinus NaNO + Ethanol, Food Science and Singdevsach mushrooms (Lentinus sajor- 22, 1, Total 2 sajor-caju 89 2013 0.656 10% AlCl · VIS 510 nm methanol or Biotechnology an, SK. et al. caju and Lentinus torulosus) 137-145 flavonoids 3 and Lentinus H O water from Similipal Biosphere 2 torulosus Reserve, India. 17

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used L. edodes, G. Cytotoxicity of blended lucidum, A. International versus single bisporus, G. Journal of Durgo, K. et medicinal mushroom extracts 15, 5, Total 90 2013 1.123 Colorimetric NF NF frondosa, T. Medicinal al. on human cancer cell lines: 435-448 flavonoids massutake Mushrooms contribution of polyphenol and P. and polysaccharide content. ostreatus Antioxidant capacity and the correlation with major Ethanol, phenolic compounds, 2013, -, NaNO + BioMed Research Vamanu, E. Total 2 UV/VIS 410 methanol or 91 2013 2.706 anthocyanin, and tocopherol 11 (ID 10% AlCl · B. edulis International et al. flavonoids 3 nm cold/hot content in various extracts 313905) H O 2 water from the wild edible Boletus edulis mushroom. Antioxidant and antiedema properties of solid-state International cultured honey mushroom, Journal of Lai, MN. et 15, 1, 1- Total Ethanol or 92 2013 1.123 Armillaria mellea (Higher Colorimetric NF A. mellea Medicinal al. 8 flavonoids water Basidiomycetes), extracts Mushrooms and their polysaccharide and polyphenol contents.

Termitomyce s microcarpus, Termitomyce Evaluation of nutraceutical s radicatus, International components and antioxidant Termitomyce Journal of Kumari, B. et 15,2, 191- Total 93 2013 1.123 potential of North Indian wild Colorimetric NF NF s badius, Medicinal al. 197 flavonoids culinary-medicinal Termitomyce Mushrooms termitophilous mushrooms. s medius, Termitomyce s heimii andTermitom yces striatus

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Antioxidant and immunomodulating activities International Cryptosporus of exo- and Journal of Jeong, SC. et 15, 3, Total volvatus and 94 2013 1.123 endopolysaccharide fractions Colorimetric NF NF Medicinal al. 251-266 flavonoids Cordyceps from submerged mycelia Mushrooms militaris cultures of culinary-medicinal mushrooms. L. edodes, A. blazei, C. Journal of the Antioxidant and anticancer militaris, G. Korean Society of 42, 5, Total 95 2013 NI Qi, Y. et al. effects of edible and Colorimetric NF Methanol lucidum, I. Food Science and 655-662 flavonoids medicinal mushrooms. obliquus and Nutrition Phellinus linteus Antioxidant activity NaNO + Ethanol, Journal of Food Debnath, T. of Inonotus obliquus grown 37, 4, Total 2 96 2013 0.853 10% AlCl · VIS 517 nm methanol or I. obliquus Biochemistry et al. on germinated brown rice 456-464 flavonoids 3 H O water extracts. 2 P. ostreatus, L. edodes, L. sulphureus, Agaricus Antioxidant property of edible NaNO + campestris, Woldegiorgis, 157, 15, Total 2 97 2014 Food Chemistry 3.391 mushrooms collected from 10% AlCl · VIS 510 nm Methanol Termocyes AZ. et al. 30–36 flavonoids 3 Ethiopia. H2O clypeatus, T. microcarpus and Termocytes letestui Rutin, Comparison T. boudieri Antioxidant/antiradical quercetin, of mass Chatin, B. Özyürek, M. properties of microwave- 157, 15, hesperidin, UPLC–ESI- spectras 80% 98 2014 Food Chemistry 3.391 edulis and et al. assisted extracts of three wild 323–331 naringenin, MS/M and Methanol Lactarius edible mushrooms. kaempferol retention volemus and apigenin times 19

Appendix- Supplementary Table 1

Antioxidant and P. florida, P. Journal of Food electrochemical properties of 51, 11, sajor-caju, Babu, DR. et Total UV/VIS 415 95% 99 2014 Science and 2.203 cultivated Pleurotus spp. and 3317- AlCl Pleurotus al. flavonoids 3 nm Ethanol Technology their sporeless/low sporing 3324 cystidiosus mutants. and P. djamor

Scientific validation of the Chinese caterpillar International medicinal mushroom, Journal of Rathor, R. et 16, 6, Total Ophiocordyce 100 2014 0.927 Ophiocordyceps sinensis Colorimetric NF Water Medicinal al. 541-553 flavonoids ps sinensis (Ascomycetes) from India: Mushrooms immunomodulatory and antioxidant activity. A comparative study of the antimicrobial, antioxidant, International and cytotoxic activities of Journal of Dong, CH. et methanol extracts from fruit 16, 5, Total 101 2014 0.927 Colorimetric NF Methanol C. militaris Medicinal al. bodies and fermented 485-495. flavonoids Mushrooms mycelia of caterpillar medicinal mushroom Cordyc eps militaris (Ascomycetes). Wound healing activity of an International aqueous extract of the Journal of Gupta, A. et Lingzhi or Reishi 6, 4, 345- Total 102 2014 0.927 Colorimetric NF Water G. lucidum Medicinal al. medicinal mushroom Ganode 354. flavonoids Mushrooms rma lucidum (higher Basidiomycetes).

Vitamins, phenolics and LWT-Food NaNO + Jaworska, G. antioxidant activity of culinary 59, 2 (1), Total 2 103 2014 Sciences and 2.416 10% AlCl · VIS 510 nm Methanol Suillus luteus et al. prepared Suillus luteus (L.) 701-706 flavonoids 3 Technology H O Roussel mushroom. 2

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Important nutritional Journal of Food constituents, flavour 51, 8, Gogavekar, Total UV/VIS 415 104 2014 Science and 2.203 components, antioxidant and 1483- AlCl Methanol P. sajor-caju SS. et al flavonoids 3 nm Technology antibacterial properties of 1491 Pleurotus sajor-caju.

Evaluation of nutritional and Russula Journal of nutraceutical potentials of NaNO + Ethanol, vesca, R. Consumer Singdevsach 9, 2, 111- Total 2 105 2014 0.719 three wild edible mushrooms 10% AlCl · VIS 510 nm methanol or delica and Protection and an, SK. et al. 120 flavonoids 3 from Similipal Biosphere H O water Termitomyce Food Safety 2 Reserve, Odisha, India. s eurrhizus

Meripilus Wild-growing International giganteus, A. lignicolous mushroom specie Journal of Food Karaman, M. 65, 3, Total aegerita, F. 106 2014 1.206 s as sources of novel agents Colorimetric NF NF Sciences and et al. 311-319 flavonoids fomentarius with antioxidative and Nutrition and Xylaria antibacterial potentials. polymorpha Effects of UV-B radiation Comparison levels on concentrations of of mass A. bisporus, Journal of 62, 14, Sapozhnikov phytosterols, ergothioneine, Total LC–ESI- spectras 80% P. ostreatus 107 2014 Agricultural and 2.912 3034- a, Y. et al. and polyphenolic compounds flavonoids MS/M and Methanol and L. Food Chemistry 3042 in mushroom powders used retention edodes as dietary supplements. times Antioxidant and anti- inflammatory activities of 79, 4, Journla of Food Total 80% Tremella 108 2014 1.696 Li, H. et al. methanol extracts of C460- AlCl VIS 640 nm Science flavonoids 3 Methanol fuciformis Tremella fuciformis and its C468 major phenolic acids.

DNA protection, total NaNO + 75% Journal of Food Hasnat, MA. phenolics and antioxidant 38, 1, 6- Total 2 109 2014 0.741 10% AlCl · VIS 517 nm Ethanol or R. virescens Biochemistry et al. potential of the mushroom 17 flavonoids 3 H O water Russula virescens. 2

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Application of GC, GC-MSD, ICP-MS and Ethaol, spectrophotometric methods hexane, Food Analytical Cayan, F. et for the determination of 7, 2, 449- Total UV/VIS 415 Chroogomph 110 2014 1.956 10% AlNO ethyl Methods al. chemical composition and in 458 flavonoids 3 nm us rutilus acetate or vitro bioactivities of water Chroogomphus rutilus: the edible mushroom species.

Effect of different drying methods and 24-month Drying Jaworska, G. storage on water activity, 32, 3, Total 111 2014 1.518 Colorimetric NF NF B. edulis Technologies et al. rehydration capacity, and 291-300 flavonoids antioxidants in Boletus edulis mushrooms.

Qualitative phytochemical screening, total phenolic Journal of Pure content, in vitro antioxidant 8, 6, Thakur, M. et Total 112 2014 and Applied 0.073 activity and antimicrobial 4735- Colorimetric NF Methanol L. sanguifluus al. flavonoids Microbiology activities in methanolic 4741 extracts of Lactarius sanguifluus (Paulet) Fr. Complex chemical evaluation Journal of Applied Tuber Kruzselyi, D. of the summer truffle (Tuber 87, -, Total UV/VIS 415 113 2014 Botany and Food 0.758 AlCl Methanol aestivum et al. aestivum Vittadini) fruit 291-295 flavonoids 3 nm Quality Vittadini bodies.

Antioxidant, antigungal and P. eryngii, P. Archives of 66, 4, Milovanovic, anticancer activities of Se- Total UV/VIS 415 96% ostreatus and 114 2014 Biological 0.718 1379- 10% Al(NO ) I. et al. enriched Pleurotus spp. flavonoids 3 3 nm Ethanol P. Sciences 1388 mycelium extracts. pulmonarius

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Intrastrain comparison of the chemical composition and 2014, -, Scientific World Phan, CW. et antioxidant activity of an 111 Total Pleurotus 115 2014 NF AlCl VIS 435 nm Ethanol Journal al. edible mushroom, Pleurotus (ID 37865 flavonoids 3 giganteus giganteus, and its potent 1) neuritogenic properties. P. ostreatus, A. bisporus, M. oreades, Total UV/VIS 415 C. AlCl flavonoids 3 nm cornucopioide s, and Tuber Biological activity of fluidized 23, 5, melanosporu Food Science and Vamanu, E. 70% 116 2014 0.653 bed ethanol extracts from 1483- m Biotechnology et al. Ethanol several edible mushrooms. 1490

T. Comparison Rutin and melanosporu HPLC-DAD of retention catechin m and P. times ostreatus

Pleurotus citrinopileatus , Pleurotus Food Science and Miao, QJ. et Research on total flavonoids 7 , 39, Total 117 2014 0.393 Colorimetric NF NF geesteranus, Technology al. bioactivity of edible fungus. 206-209 flavonoids A. auricula and P. ostreatus Chemical composition, antioxidant and anti- UV 220-450 LWT-Food inflammatory properties for nm and 55, 1, Total 118 2014 Sciences and 2.416 Lin, JT. et al. Ethanolic extracts from HPLC-DAD comparison Ethanol P. eryngii 374-382 flavonoids Technology Pleurotus eryngii fruiting of retention bodies harvested at different times time. 23

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Phytochemical investigation, Industrial Crops Tel-Cayan, antioxidant and 76, -, Total UV/VIS 415 Ganoderma 119 2015* 2.837 10% Al(NO ) Methanol and Products G. et al. anticholinesterase activities 749–754 flavonoids 3 3 nm adspersum of Ganoderma adspersum. Fumigation with essential oils improves sensory quality and NaNO + Jiang, T. et 172, -, Total 2 80% 120 2015* Food Chemistry 3.391 enhanced antioxidant ability 10% AlCl · VIS 510 nm L. edodes al. 692–698 flavonoids 3 Ethanol of shiitake mushroom H2O (Lentinus edodes). Antioxidant activity of Metanol, different fractions of hexane, International methanolic extract of the choroform, Journal of Ebrahimzade 17, 6, Total 121 2015* 0.927 golden chanterelle Colorimetric NF ethyl C. cibarius Medicinal h, MA. et al. 557-565 flavonoids mushroom Cantharellus acetate, n- Mushrooms cibarius (Higher butanol or Basidiomycetes) from Iran. water NaNO + Total 2 Chemical, bioactive, and 10% AlCl3 · VIS 510 nm Water 2015, -, flavonoids 20 culinary BioMed Research Sharma, SK. antioxidant potential of H2O 122 2015* 1.579 12 (ID mushroom International et al. twenty wild Ethyl 346508) Anthocyanidi species culinary mushroom species. Colorimetric VIS 546 nm alcohol and ns ClH Nutraceutical properties of the methanolic extract of NaNO + Food and Kozarski, M. 6, 6, Total 2 123 2015* 2.791 edible mushroom Cantharellu 10% AlCl · VIS 510 nm Methanol C. cibarius Function et al. 1875-86 flavonoids 3 s cibarius (Fries): primary H2O mechanisms. Functional nutraceutical International profiling of wild edible and 10 wild and Journal of Khaund, P. et 17, 2, Total 124 2015* 0.927 medicinal mushrooms Colorimetric NF Ethanol edible Medicinal al. 187-197 flavonoids consumed by ethnic tribes in mushrooms Mushrooms India.

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used International Antidiabetic and antioxidant G. lucidum Journal of activities of eight 17, 2, Total 125 2015* 0.927 Wu, T. et al. Colorimetric NF NF and T. Medicinal medicinal mushroom species 129-40 flavonoids fuciformis Mushrooms from China. Antioxidant activities and bioactive compound International determination from caps and Journal of Mishra ,KK. stipes of specialty medicinal 16, 6, Methanol or C. indica and 126 2015* 0.927 Tannins Colorimetric NF Medicinal et al. mushrooms Calocybe indica 555-67. water P. sajor-caju Mushrooms and Pleurotus sajor-caju (higher Basidiomycetes) from India. NaNO + Total 2 Antioxidant capacity of 10% AlCl3 · VIS 510 nm Brazilian Journal Tajalli, F. et several Iranian, wild and 46, 3, flavonoids 127 2015* 0.592 H2O Methanol A. bisporus of Microbiology al. cultivated strains of the 769-776 Anthocyanidi VIS 510-700 button mushroom. NaCH Co ns 3 2 nm

Production of Agaricus Internatinal brasiliensis mycelium from 50, 9, Journal of Food Mokochinski, Total 128 2015* 1.384 food industry residues as a 2052- AlCl VIS 425 nm Methanol A. brasiliensis Science and JB. et al. flavonoids 3 source of antioxidants and 2058 Technology essential fatty acids.

The effect of selenium on Pholiota LWT-Food NaNO + Gasecka, M. phenolics and flavonoids in 63, 1, Total 2 nameko, P. 129 2015* Sciences and 2.416 10% AlCl · VIS 510 nm Methanol et al. selected edible white rot 726-731 flavonoids 3 eryngii and P. Technology H O fungi. 2 ostreatus Antioxidant and anticholinesterase activities Methanol or G. lucidum Pharmaceutil 53, 6, Total 130 2015* 1.241 Tel, G. et al. of five Colorimetric NF ethyl and Funalia Biology 824-830 flavonoids wild mushroom species with acetate trogii total bioactive contents.

Appendix- Supplementary Table 1

Method Detection Volumen, Compounds Solvents Mushroom Impact utilized to device or Number Year Journal First author Title Issue, found in used for specie factor measure wavelength pages mushrooms extraction studied flavonoids used Polyporus sulphureus, Metal concentration and Macrolepiota Sarikurkcu, 175, -, Total UV/VIS 415 Methanol or 131 2015* Food Chemistry 3.391 antioxidant activity of edible AlCl procera, L. C. et al. 549-555 flavonoids 3 nm water mushrooms from Turkey. perlatum and Gomphus clavatus Cytotoxicity of some edible C. comatus, mushrooms extracts over Methanol, T. fracticum, liver hepatocellular 11, 4, acetone, n- Pharmacognosy Total Rhizopogon 132 2015* 1.256 Sadi, G. et al. carcinoma cells in suppleme Colorimetric NF hexane, Magazine flavonoids luteolus and conjunction with their nt, 6-18 cloroform Lentinus antioxidant and antibacterial or water tigrinus properties. L. edodes, T. Internatinal Chemical composition and NaNO + fuciformis, A. Journal of Food Zhang, N. et antioxidant properties of five 50, 2, Total 2 133 2015* 1.384 10% AlCl · VIS 510 nm Ethanol polytricha, G. Science and al. edible Hymenomycetes 465-471 flavonoids 3 H O lucidum and I. Technology mushrooms. 2 obliquus Antioxidant potential of white International oyster culinary- Journal of Ganeshpurka 17, 5, Total 50% 134 2015* 0.927 medicinal mushroom, Colorimetric NF P. florida Medicinal r, A. et al. 491-498 flavonoids Ethanol Pleurotus florida (Higher Mushrooms Basidiomycetes). Effects of Selenium presence International G. in mycelia of Ganoderma Journal of Milovanovic, 17, 1, 11- Total UV/VIS 415 96% applanatum 135 2015* 0.927 species (Higher 10% Al(NO ) Medicinal I. et al. 20 flavonoids 3 3 nm Ethanol and G. Basidiomycetes) on their Mushrooms lucidum medicinal properties. Screening of antimicrobial, P. ostreatus, Annals of Clinical antioxidant properties and Chowdhury, Total UV/VIS 415 L. edodes 136 2015* Microbiology and 2.189 bioactive compounds of 14, 1, 8 10% Al(NO ) Methanol MMH. et al. flavonoids 3 3 nm and H. Antimicrobials some edible mushrooms tessulatus cultivated in Bangladesh. 26

Appendix- Supplementary Table 1