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Critical Reviews in Biotechnology

ISSN: 0738-8551 (Print) 1549-7801 (Online) Journal homepage: http://www.tandfonline.com/loi/ibty20

A review on the constituents in mushrooms: , and

Chia-Wei Phan, Joon-Keong Wang, Shiau-Chuen Cheah, Murali Naidu, Pamela David & Vikineswary Sabaratnam

To cite this article: Chia-Wei Phan, Joon-Keong Wang, Shiau-Chuen Cheah, Murali Naidu, Pamela David & Vikineswary Sabaratnam (2017): A review on the nucleic acid constituents in mushrooms: nucleobases, nucleosides and nucleotides, Critical Reviews in Biotechnology, DOI: 10.1080/07388551.2017.1399102 To link to this article: http://dx.doi.org/10.1080/07388551.2017.1399102

Published online: 10 Nov 2017.

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Download by: [University of Florida] Date: 11 November 2017, At: 02:55 CRITICAL REVIEWS IN BIOTECHNOLOGY, 2017 https://doi.org/10.1080/07388551.2017.1399102

REVIEW ARTICLE A review on the nucleic acid constituents in mushrooms: nucleobases, nucleosides and nucleotides

Chia-Wei Phana,b , Joon-Keong Wangc, Shiau-Chuen Cheahc, Murali Naidua,b, Pamela Davida,b and Vikineswary Sabaratnama,d aMushroom Research Centre, University of Malaya, Kuala Lumpur, Malaysia; bDepartment of Anatomy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; cFaculty of Medicine and Health Sciences, UCSI University, Kuala Lumpur, Malaysia; dInstitute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

ABSTRACT ARTICLE HISTORY Mushrooms have become increasingly important as a reliable food source. They have also been Received 19 March 2017 recognized as an important source of bioactive compounds of high nutritional and medicinal val- Revised 14 October 2017 ues. The nucleobases, nucleosides and nucleotides found in mushrooms play important roles in Accepted 24 October 2017 the regulation of various physiological processes in the human body via the purinergic and/or 0 receptors. Cordycepin, a 3 - found in Cordyceps sinensis has received KEYWORDS much attention as it possesses many medicinal values including anticancer properties. In this Mushroom; nucleic acid; review, we provide a broad overview of the distribution of nucleobases ( and ; ; ); pyrimidine nucleobases (, , and ); nucleosides (, , cordycepin; 50-nucleotides; and ); as well as novel nucleosides/tides in edible and nonedible mushrooms. flavor This review also discusses the latest research focusing on the successes, challenges, and future perspectives of the analytical methods used to determine nucleic acid constituents in mush- rooms. Besides, the exotic taste and flavor of edible mushrooms are attributed to several non- volatile and water-soluble substances, including the 50-nucleotides. Therefore, we also discuss the total flavor 50-nucleotides: 50- (50-GMP), 50- monophosphate (50-IMP), and 50- monophosphate (50-XMP) in edible mushrooms.

Introduction nucleosides present in mushrooms. In fact, nucleosides are thought to be the main bioactive compounds in the Mushrooms are macrofungi with large, easily observed mushroom Cordyceps (Sordariomycetes), a famous trad- and collectable fruiting bodies. There are more than itional Chinese medicine with high medicinal and com- 14,000 species of mushrooms and at least 2000 of them mercial value [10]. Nucleosides consist of a nucleobase are edible [1]. Out of these 2000 edible mushrooms, approximately 200 are wild species [1]. Mushrooms (also termed as a nitrogenous base) and a 5-carbon have been appreciated for their flavor and texture and (either or ). On the other hand, Downloaded by [University of Florida] at 02:55 11 November 2017 are recognized as important sources of nutrients [2–4]. a is composed of a nucleoside (a nucleobase The nutritional value of edible mushrooms is due to their and a 5-carbon sugar), in addition with one or more high , fiber, , mineral contents and low fat group (Figure 1). levels [5]. Culinary mushrooms are also very useful for To date, nucleosides, such as cytidine, uridine, vegetarian diets because they provide many of the essen- adenosine, guanosine, and inosine, were tial amino acids for adult requirements. More importantly, reported to be involved in the regulation and modula- edible mushrooms contain many different bioactive com- tion of various physiological processes in the human pounds including , steroids, , ter- body via the purinergic and/or pyrimidine receptors penoids, and nucleosides, all of which have been shown to [11]. These nucleosides serve as precursor molecules for exert various human health benefits [6]. making DNA and RNA which are vital in almost all cellu- While the health effects and underlying mechanisms lar functions. Furthermore, nucleosides and/or nucleoti- of mushroom polysaccharides have been more exten- des were reported to enhance brain function, affect sively evaluated [7–9], little attention was paid to the immune modulation, influence fatty acids ,

CONTACT Chia-Wei Phan [email protected] Department of Anatomy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Vikineswary Sabaratnam [email protected] Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia ß 2017 Informa UK Limited, trading as Taylor & Francis Group 2 C.-W. PHAN ET AL.

chromatography (RPLC), hydrophilic interaction chroma- tography (HILIC), ion-exchange chromatography (IEC) and denaturing HPLC (DHPLC). Electrophoretic methods encompass CE and capillary electrochromatography (CEC). Capillary electrophoresis with (UV) and/or mass spectrometry (MS) detection has been widely used in the food industry for analysis of nucleosides and nucleotides. In fact, due to the high polarity of nucleotides, an ion-pairing agent or Figure 1. The composition of a nucleotide which comprised an ion-exchange column is required for analysis by of a nucleoside (a nucleobase and 5-carbon sugar) and one or HPLC. more phosphate group.

contribute to absorption in the gut, and improve Nucleobases, nucleosides and nucleotides in gastrointestinal tract repair after damage [12,13]. Ganoderma spp It is noteworthy to highlight that even though Ganoderma spp belong to the family Polyporaceae and healthy people can produce and recycle nucleosides in are one of the most important medicinal fungi of their bodies, dietary nucleosides sourced from nucleo- Basidiomycetes. Ganoderma spp, in particular tide-rich food can be useful to meet the demand for Ganoderma lucidum (Curtis) P. Karst, has a long history nucleosides in situations like illness recovery or immune of use for promoting health and longevity [20]. system malfunction. Due to the remarkable reservoir of Ganoderma lucidum is called Reishi in the Japanese lan- nucleosides in mushrooms, scientists have recently guage and Lingzhi in Chinese. The first isolation of found the urge to qualitatively and quantitatively deter- adenine, adenosine, uracil and uridine from Ganoderma mine the distribution of nucleosides in mushrooms [14]. species was carried out by Yu and Zhai in 1979 on Figure 2 shows the chemical structures of different Ganoderma capense (Lloyd) Teng [21]. Ganoderma nucleobases and nucleosides reported in mushrooms. capense is a Ganoderma species that morphologically This paper mainly summarizes the types of nucleo- sides and nucleotides found in mushrooms and the resembles G. lucidum to a certain extent [22,23]. analytical methods employed to identify and quantify According to Yu and Zhai [21], mycelia from G. lucidum – them. In addition, newly discovered mushroom nucleo- were extracted with 92 95% followed by sus- sides and their total flavor nucleotides are also pension in water and further extracted with ethyl ether. reviewed. The resulting was subjected to ion- exchange chromatography to give a neutral and basic fractions. Upon subfractioning, the neutral fraction Mode of detection of nucleobases, nucleosides yielded uracil and uridine via adsorption chromatog- and nucleotides raphy on macroporous resin. The basic fraction, on the Novel analytical and detection methods play an essen- other hand, yielded adenine and adenosine by further

Downloaded by [University of Florida] at 02:55 11 November 2017 tial role in biotechnology. Further progress in biotech- extraction with chloroform and butanol. nology very much depends on the introduction of A study of Ganoderma nucleotides was subsequently 0 increasingly sophisticated analytical tools for the separ- carried out by Kim et al. [24]. According to the study, 5 - 0 0 ation, characterization and quantitation of natural prod- guanosine monophosphate (5 -GMP) and 5 -xanthosine ucts. Consequently, the concepts, methods and monophosphate (5’-XMP) were found in both young strategies of “analytical chemistry” will inevitably be and mature basidiocarps. However, the levels of both adopted by biotechnology. the nucleotides in the young basidiocarp were higher Separations of nucleobases, nucleosides and nucleo- than those in the matured basidiocarp. Adenosine iso- tides have been accomplished by two general methods: lated from G. lucidum water extract was found to inhibit chromatography and electrophoresis [15–17]. In gen- platelet aggregation [25]. The water extract of G. luci- eral, capillary electrophoresis (CE) yields better reso- dum was separated by a Sephadex G-25 column. Then, lution and higher separation efficiency when compared polyacrylamide gel chromatography with mobile phase to chromatography [18]. Capillaries that are narrow of 10 mM potassium phosphate (pH 5.1) containing also reduce the “band-broadening”, which are often 20% methanol or 15% methanol, or 10 mM potassium encountered during chromatography [19]. The chroma- phosphate (pH 8.5) containing 20% methanol or 15% tographic methods include reversed-phase liquid methanol, was used to obtain an active fraction [26]. CRITICAL REVIEWS IN BIOTECHNOLOGY 3

Nucleobases

NH2 O O

N N N N NH NH

N N N N N H H N NH2 H adenine guanine

O NH2 O H3C NH N NH

N O N O N O H H H thymine cytosine uracil

Nucleosides

NH2 O NH2 O N N N N NH NH

N N N O N O N N NH2 HO HO HO HO O O O O

OH OH OH OH OH OH OH OH adenosine guanosine cytidine uridine OH O O

H3C N N N N NH NH N N N N N O N O N N HO HO HO H HO CH2 CH2 O O O O

OH OH OH OH OH OH OH inosine thymidine xanthosine nebularine O NH2 NH2 O N N N NH N N NH

N N N N O N N N NH2 HO HO HO HO O O O O Downloaded by [University of Florida] at 02:55 11 November 2017 OH OH OH OH cordycepin 2'-deoxyadenosine 2'- 2'-

NH 2 OH NH2

N N N N N N O N N N N Cl HO HO HO CH O 2 O O

OH OH OH OH 2'- 2'-deoxyinosine 2'-chloroadenosine Figure 2. Nucleobases and nucleosides reported in mushrooms. 4 C.-W. PHAN ET AL.

Table 1. List of nucleobases and nucleosides isolated from Ganoderma. Nucleic acid constituents Pharmacological Mode of detection Ganoderma species Nucleobases Nucleosides functions References Ion-exchange G. capense Adenine, uracil Adenosine, uridine – [21] chromatography Size-exclusion G. lucidum – Adenosine Suppression of [25] chromatography platelet aggregation Micellar electrokinetic G. lucidum and Uracil Adenosine, uridine – [27] chromatographic G. japonicum HPLC-DAD G. lucidum and Uracil, hypoxanthine, Cytidine, uridine, inosine, [28] G. sinense thymine, adenosine, guanosine, thymidine guanine, adenine HPLC-DAD G. lucidum, Uracil, hypoxanthine, Cytosine, cytidine, uridine, 2’- – [29] G. duroppora, and thymine, adenine, deoxyuridine, inosine, G. sinense guanosine, thymidine, adenosine, 2’- deoxyadenosine Zwitterionic hydrophilic G. lucidum, G. sinense, Adenine, uracil, guanine, Adenosine, cytosine, cytidine, – [30] interaction chromatog- and G. atrum thymine, uridine, guanosine, inosine, raphy (ZIC-HILIC) thymidine, 20-deoxyuridine, 20-deoxyadenosine, 20-deoxy- cytidine, 20-deoxyguanosine, 20-deoxyinosine HPLC G. lucidum Adenine Adenosine – [31] Proton NMR spectroscopy G. lucidum – Adenosine – [33]

With this procedure, adenosine was well separated from the major nucleosides, which accounted for 60% of the cytidine, guanosine and uridine. total nucleosides and nucleobases in Ganoderma Furthermore, a micellar electrokinetic chromato- species. graphic (MEKC) method was developed to determine Nucleosides in Ganoderma atrum Zhao, Hsu & Zhang the nucleosides (adenosine and uridine); as well as the were elucidated [30]. The content of uridine, guanine, nucleobase (uracil) in aqueous extracts of G. lucidum 20-deoxyinosine, and 20-deoxyguanosine were abundant and G. japonicum (Fr.) Lloyd [27]. The separations were in G. atrum, while the content of thymidine, 20-deoxyuri- conducted in a Beckman P/ACE 5000 CE system with dine, and adenine was significantly lower when com- peak detection by direct UV at 254 nm. The nucleobases pared to G. lucidum and G. sinense [27]. and nucleosides were separated within 10 min using a Besides, a chromatographic experiment was con- borate buffer (80 mM; pH 9) containing sodium dodecyl ducted on G. lucidum by using a YL9100 HPLC system sulfate (25 mM) at 22 kV, 20 C and an injection time of [31]. This reverse phase chromatography was performed 5 s. Furthermore, Gao et al. [28] used a HPLC-diode array using a C18 column with methanol and dihydrogen detector-mass spectrometry (HPLC–DAD-MS) method to phosphate as the mobile phase in isocratic elution detect nucleosides and nucleobases in two species of method at a flow rate of 1.0 ml/min. Detection was car- Ganoderma: G. lucidum and G. sinense Zhao, Hsu and ried out at 254 nm, which gave a sharp peak of adenine Downloaded by [University of Florida] at 02:55 11 November 2017 Zhang. The results showed that the quantity of nucleo- and adenosine at a retention time of 6.53 ± 0.02 min bases and nucleosides (guanine, adenine, uracil, cyti- and 12.41 ± 0.02, respectively. dine, hypoxanthine, uridine, thymine, inosine, Overall, the separation technique for nucleic acid con- guanosine, thymidine and adenosine) in G. lucidum stituents has progressively improved. Chromatography were higher than those in G. sinense, in particular the system was developed with addition of multiple col- cytidine. umns and detectors. For example, the size exclusion A total of 42 samples of G. lucidum and its related chromatography column, hydrophilic interaction chro- species including G. duroppora Lloyd and G. sinense matography column, and reversed phase chromatog- were collected from 11 regions in China [29]. A HPLC- raphy column; as well as detectors such as diode array DAD method was applied to simultaneously quantify 13 detector, evaporative light scattering detector and mass nucleosides and nucleobases (cytosine, uracil, cytidine, spectrometry detector [32]. Most recently, proton nuclear hypoxanthine, uridine, thymine, 20-deoxyuridine, aden- magnetic resonance (NMR) spectroscopy was also used to ine, inosine, guanosine, thymidine, adenosine and identify and quantify nucleosides in the water extracts of 2’-deoxyadenosine) in the Ganoderma samples. The G. lucidum [33]. Table 1 shows the list of nucleobases, nucleoside and nucleobase profiles showed variation. nucleosides, and nucleotides detected in Ganoderma and Inosine, guanosine and adenosine were determined as their mode of detection. CRITICAL REVIEWS IN BIOTECHNOLOGY 5

Nucleobases, nucleosides and nucleotides in was 10 Mpa. On the other hand, a HPLC coupled with Cordyceps spp DAD and light scattering detection (ELSD) method was developed by Wang et al. [44]. Most recently, Yang For centuries, Cordyceps, an endoparasitic fungus, has et al. [45] had developed an ion-pairing reversed-phase been used in traditional Chinese medicine. The pharma- liquid chromatography–mass spectrometry (IP-RP- cological uses of Cordyceps sinensis (Berk.) Sacc. and C. LC–MS) to determine nucleobases and nucleosides in militaris (Fr.) L. have been extensively reviewed [34,35]. wild natural C. sinensis stroma and cultured stroma of C. The full genome of C. militaris has been sequenced and militaris. A perfluorinated carboxylic acid, namely penta- the advances in the production of bioactive composi- decafluorooctanoic acid (PDFOA, 0.25 mM) was used as tions of C. militaris has been recently reviewed by Cui a volatile ion-pairing agent and all separations were [36]. To date, adenosine is regarded as the most signifi- performed by employing a reversed-phase column cant bioactive constituent in Cordyceps [37]. Adenosine (Agilent ZORBAX SB-Aq column). As a result, three serves as a quality control marker in preparing standar- 0 0 nucleotides: 5 - (UMP), 5 - dized Cordyceps extract for commercialization purposes. 0 (AMP) and 5 -guanosine Interestingly, this mushroom was given close attention monophosphate (GMP) were detected along with seven in the athletic world, as it was reported to boost ATP nucleosides (adenosine, guanosine, uridine, inosine, level and improve utilization [38]. cytidine, thymidine and cordycepin), and six nucleo- A simple capillary electrophoresis (CE) method was bases (adenine, guanine, uracil, hypoxanthine, cytosine developed to simultaneously determine six main and thymine). nucleosides and nucleobases in Cordyceps, including A more rapid ultra-performance liquid chromatog- adenine, uracil, adenosine, guanosine, uridine and raphy (UPLC) method was also developed for the inosine [39]. The CE method was subsequently opti- determination of 14 nucleosides and nucleobases in mized into a capillary electrochromatography (CEC) Cordyceps [46]. The separation was performed on a method whereby a simultaneous determination of 11 Waters Acquity UPLC system with Acquity UPLC BEH nucleosides and nucleobases could be achieved [40]. The C18 column and gradient elution of 0.5 mM acetic acid additional five nucleosides/bases included cytosine, hypo- and acetonitrile in 5 min. Most recently, Qian et al. [47] 0 , 2 -deoxyuridine, thymidine and cordycepin. analyzed Cordyceps with a multicolumn liquid chroma- Besides electrophoresis, HPLC was also used to deter- tography (MC-LC) that encompasses a size exclusion mine the nucleosides in Cordyceps. An RP-HPLC method pre-column, a size exclusion column (SEC) and a was developed for a simultaneous determination of 11 reversed phase column (RP). nucleosides and nucleobases, including adenosine, cor- Despite the common uses of CE, HPLC, UPLC and LC- dycepin, cytidine, guanosine, inosine, thymidine, uri- MS, many have highlighted their drawbacks, for example dine, cytosine, guanine, thymine and uracil in Cordyceps low sensitivity and low separation efficiency for polar [41]. This technique employed a Zorbax 300SB C18 ana- compounds like nucleosides and nucleobases. For this lytical column (4.6 250 mm i.d, 5 mm) with gradient reason, a new technique that combined a hydrophilic elution coupled with diode-array detection (DAD) sys- interaction chromatography (HILIC) with an electrospray tem. Subsequently, a group of researchers attempted to tandem mass spectrometry (ESI–MS/MS) was Downloaded by [University of Florida] at 02:55 11 November 2017 perform an acid on Cordyceps before subject- developed [48]. This HILIC–ESI–MS/MS system was found ing it to HPLC analysis using a Zorbax SB-AQ analytical to be more sensitive compared to routine HPLC while column (4.6 mm 250 mm i.d., 5 mm) [42]. According to showing good linearity, repeatability and recovery. the study, after the screening of perchloric acid, sulfuric To summarize the data collected from past experi- acid, formic acid, and hydrochloric acid; ments, the adenosine level was found to be higher in perchloric acid was chosen and was able to hydrolyze cultured stroma of C. sinensis than in the natural wild nucleic acids into nucleobases without degrading the stroma. Moreover, the cultured C. sinensis had higher end products. Therefore, the optimized hydrolysis condi- adenosine than those in cultured C. militaris. The list of tions for Cordyceps sample is as such: eight rounds of nucleic acid constituents found in Cordyceps was hydrolysis with 100% perchloric acid for 1 h at 95–100 C. depicted in Table 2. Subsequently, researchers have developed several modifications on HPLC to achieve precise measurement The production of cordycepin in Cordyceps of nucleosides and their nucleobases in stroma of militaris Cordyceps. A HPLC coupled with pressurized organic solvent extraction was developed by Li et al. [43]. The Cordycepin is an analog of adenosine and it carries a solvent used was methanol and the pressure applied structural formula of 30-deoxyadenosine. Cordycepin 6 C.-W. PHAN ET AL.

Table 2. List of nucleobases, nucleosides and nucleotides isolated from Cordyceps. Nucleic acid constituents Mode of detection Mushroom Nucleobases Nucleosides Nucleotides References Capillary C. sinensis and Adenine, uracil Adenosine, guanosine, uridine, – [39] electrophoresis C. militaris inosine Capillary electro- C. sinensis and Cytosine, uracil, hypoxan- Uridine, 20-deoxyuridine, – [40] chromatography C. militaris thine, adenine, inosine, guanosine, thymi- dine, adenosine, cordycepin RP-HPLC C. sinensis and Cytosine, guanine, thy- Adenosine, cordycepin, cytidine, – [41] C. militaris mine, uracil guanosine, inosine, thymi- dine, uridine HPLC C. sinensis and Cytosine, uracil, hypoxan- ––[42] C. militaris thine, guanine, thy- mine and adenine HPLC coupled with C. sinensis and Adenine, cytosine, guan- Adenosine, cordycepin, cytidine, – [43] pressurized solvent C. militaris ine, thymine, uracil guanosine, thymidine, uri- extraction dine, 20-deoxyuridine HPLC-DAD-ELSD C. sinensis and Uracil, adenine, Cordycepin, adenosine, uridine, – [44] C. militaris hypoxanthine inosine, guanosine Ion pairing-RP-LCMS C. sinensis and Adenine, guanine, uracil, Adenosine, guanosine, uridine, 50-uridine monophosphate [45] C. militaris hypoxanthine, cyto- inosine, cytidine, thymidine, (UMP), 50- adenosine sine, thymine cordycepin, monophosphate (AMP), 50-guanosine monophos- phate (GMP), UPLC C. militaris Adenine, cytosine, uracil, Adenosine, cytidine, uridine, – [46] guanine, hypoxan- guanosine, inosine, thymi- thine, thymine dine, 20-deoxyuridine, cordycepin Multi-column LC Cordyceps sp. Hypoxanthine, guanine Uridine, inosine, guanosine [47] HILIC-ESI-MS/MS C. sinensis Thymine, uracil, hypoxan- Thymidine, 20-deoxyuridine, cor- [48] thine, adenine, guan- dycepin, uridine, adenosine, ine, cytosine, xanthine inosine, cytidine, guanosine and 2-chloroadenosine

was first isolated from C. militaris. It is noteworthy to C18 column by isocratic elution with acetonitrile–water mention that cordycepin is found only in trace amounts (5:95; v/v), flow rate of 1.0 ml/min and UV detection at in natural C. sinensis and that it cannot be detected in 260 nm [61]. the cultured C. sinensis [34,49]. Therefore, cordycepin Since the demand of cordycepin has peaked, large- serves as a useful marker for authentication and quality scale production of cordycepin by the cultivation of C. control of C. militaris. militaris has become a significant issue recently. The Since the discovery of cordycepin, C. militaris has nitrogen source plays a vital role in cordycepin biosyn- begun to gain much attention and it is often used as a thesis. Peptone has been identified as the best nitrogen substitute for C. sinensis. Cordyceps militaris is favored source for cordycepin [62]. The carbon/ because the cultivation process is simple compared to nitrogen ratio is very important for the accumulation of that of C. sinensis; the latter needs the larvae of the cordycepin. In the case of submerged culture, additional þ Downloaded by [University of Florida] at 02:55 11 November 2017 “ghost moth” (Hepialus humuli)[50] as a host. On the feeding of NH4 to the medium enhanced cordycepin other hand, C. militaris can be mass-produced by using production [63]. Furthermore, adenosine could be rice substrate and buried the larvae and pupae of other added into the liquid culture to boost cordycepin pro- insects. duction as shown by Masuda et al. [64]. In submerged The therapeutic potential of cordycepin is enormous culture, ferrous sulfate could also be added to enhance and its functions are extensively researched and cordycepin biosynthesis as iron ion was shown to upre- reviewed [51–52]. Cordycepin is implicated in regulating gulate the synthetase (purA) various physiological functions, including anti-tumor which is important during the purine nucleotide biosyn- [53], anticancer [49], anti-inflammation [54,55], antipla- thetic pathway [65]. telet [56], antidepression [57] and antiviral effects [58]. The method of employing the mutant strain of C. This compound was also found to demonstrate militaris was proposed to obtain a maximal yield of cor- protection [59] and even neuroprotective activities [60]. dycepin. A mutant strain G81–3 was obtained by ion Cordycepin can be separated and isolated by using beam irradiation [66]. The irradiated mutant showed a CE, HPLC-MS and LCMS methods. It has a maximum 179% higher cordycepin production as compared to the absorption wavelength of 259–260 nm. A simple HPLC wild strain. Besides, different culture conditions also method was developed to separate cordycepin on a affect mycelial growth and cordycepin production CRITICAL REVIEWS IN BIOTECHNOLOGY 7

during submerged culture of C. militaris. A separate extract, prevented serum deprivation-induced rat pheo- study showed that apart from surface liquid culture chromocytoma (PC12) cell apoptosis by acting on the

techniques [67], the combination of submerged and A2a [76] and mitogen-activated protein static cultures has been shown to be favorable for the (MAPK) family pathway [77]. higher production of cordycepin by C. militaris [68]. Interestingly, cordycepin was also detected in the Much spent media have been discarded as a result of ethanolic extract of A. camphorata. Chen et al. [78] iden- massive industrial production of this mushroom tified three main constituents of the mushroom, includ- through solid fermentation. Therefore, it would be ing adenosine, cordycepin, and a sterol named zhankuic environmental friendly and economical to extract cordy- acid A, by HPLC analysis. It is suggested that combin- cepin from the spent substrate of C. militaris [69]. Ni ation of the two nucleosides: adenosine and cordyce- et al. [70] reported that cordycepin could be extracted pin, has made A. camphorata an effective anticancer from these spent mushroom composts by column chro- agent [79]. matographic methodology. It is noteworthy to empha- size that the chemical synthesis of cordycepin is Nucleobases, nucleosides and nucleotides in other feasible; however, it is not favorable as the process is edible mushrooms complicated and it would involve a large amounts of organic solvents. A study of the distribution of nucleic acid constituents in other edible mushrooms continues. The study by Yang et al. [80] had indicated that Poria cocos F.A. Wolf, Nucleobases, nucleosides and nucleotides in Tuber spp Tremella fuciformis Berk., and Polyporus umbellatus (Pers.) Fr., had no detectable nucleosides and nucleoti- Truffles of the genus Tuber are very popular for their des when tested with a capillary zone electrophoresis aroma and taste which make them very precious and (CZE) system. expensive. The two most widely found truffles, that is, Meanwhile, four nucleosides (adenosine, guanosine, Tuber indicum Cooke & Massee (the Chinese truffle) and uridine and inosine) and 50-guanosine monophosphate T. melanosporum Vittad (the black truffle) are morpho- (GMP) were detected in Lasiosphaeria sp., Agrocybe logically alike but their aromas are very different [71]. aegerita (V. Brig.) Singer, Boletus nigricans (Fr.) Spreng., For the first time, a total of seven nucleosides and Boletus fulvus Fodere Voy, Tricholoma matsutake (S. Ito nucleobases (adenine, hypoxanthine, uridine, adeno- & S. Imai) Singer, and Auricularia auricula-judae (Bull.) J. sine, guanine, guanosine, and inosine) were identified Schrot€ [80]. Of all the mushrooms tested, the puffball in Tuber fruiting bodies and submerged fermentation mushroom, Lasiosphaeria sp. had the highest amount of mycelia [72]. The species that were analyzed included: adenosine and uridine. T. indicum, T. aestivum, T. borchii var., T. himalayense, T. Ten wild edible mushrooms were collected in the sinense and T. melanosporum. A dispersive solid phase Croatian regions of Istria and Slavonia [81]. The mush- extraction (DSPE) combined with LC–MS was developed rooms, that is, Morchella deliciosa Fr., Craterellus cornu- and it was reported to be inexpensive and rapid. Also, a copoides (L.) Pers, Cantharellus cibarius Fr., Agaricus D3520 macroporous resin was chosen as the DSPE sor- bisporus (Lange) Imbach, Panus tigrinus (Bull.) Singer, Downloaded by [University of Florida] at 02:55 11 November 2017 bent to remove any protein interferences. Interestingly, Entoloma clypeatum (L.) P. Kumm., Morchella crassipes the fermentation mycelia were found to contain higher (Vent.) Pers., Calocybe gambosa (Fr.) Donk, Entoloma nucleosides and nucleobases content than those of the saundersii (Fr.) Sacc., and Agrocybe aegerita were ana- fruiting bodies by 25 times. lyzed by using a reverse-phase HPLC. Uridine, adeno- sine, guanosine and xanthosine were detected in all the Nucleobases, nucleosides and nucleotides in mushrooms studied. Uridine and xanthosine were found to be the highest in E. saudersi. On the other Antrodia camphorata hand, the white button mushroom, A. bisporus was Antrodia camphorata (M. Zang et C. H. Su) Sh. H. Wu found to contain the highest amount of guanosine. et al. is a rare fungus native to the forests of Taiwan For the first time, adenosine, ADP and cytidine were [73]. It is famous among the indigenous people in reported to be present in the ethanolic extract of the Taiwan as a liver tonic [74]. Three nucleosides, namely giant elm bracket mushroom, Rigidoporus ulmarius cytidine, adenosine and thymidine were identified in (Sowerby) Imazeki [82]. The adenosine and ADP were the ethanolic extract of A. camphorata by Huang et al. proposed to be responsible for the anti-angiogenic and [75]. Subsequently, it was confirmed that adenosine, anti-inflammation properties of the mushroom. Another which was found in abundance in A. camphorata study on the giant oyster mushroom, Pleurotus 8 C.-W. PHAN ET AL.

Table 3. List of nucleobases and nucleosides isolated from some edible and medicinal mushrooms. Nucleic acid constituents Parts of Mushroom Mode of detection mushroom Nucleobases Nucleosides Nucleotides References Tuber indicum, T. aestivum, Dispersive solid phase Fruiting body and Adenine, hypoxan- Uridine, adenosine, – [72] T. borchii var., T. hima- extraction (DSPE) mycelium thine, guanine guanosine, inosine layense, T. sinense, and combined with T. melanosporum. LC–MS Antrodia camphorata HPLC Fruiting body – Cytidine, adenosine, thy- – [75,78] midine, cordycepin Lasiosphaeria sp. Capillary zone elec- Fruiting body – Adenosine, guanosine, 50-guanosine [80] trophoresis (CZE) inosine monophosphate Agrocybe aegerita Capillary zone elec- Fruiting body – Adenosine, guanosine, – [80] trophoresis (CZE) uridine Boletus nigricans Capillary zone elec- Fruiting body – Adenosine, guanosine, – [80] trophoresis (CZE) uridine Boletus fulvus Capillary zone elec- Fruiting body – Adenosine, guanosine, 50-guanosine [80] trophoresis (CZE) uridine, inosine monophosphate Tricholoma matsutake Capillary zone elec- Fruiting body – Adenosine, uridine, – [80] trophoresis (CZE) inosine Auricularia auricular Capillary zone elec- Fruiting body – Adenosine, guanosine, – [80] trophoresis (CZE) uridine, inosine Morchella deliciosa RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine Craterellus cornucopoides RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine, cytidine Cantharellus cibarius RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine, cytidine Agaricus bisporus RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine, cytidine Panus tigrinus RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine, cytidine Entoloma clypeatum RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine Morchella crassipes RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine Calocybe gambosa RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine, cytidine Entoloma saundersii RP-HPLC Fruiting body – Adenosine, guanosine, – [81] xanthosine, uridine Rigidoporus ulmarius HPLC Mycelium – Adenosine, cytidine Adenosine [82] diphosphate Pleurotus giganteus LCMS Fruiting body – Uridine [83] Termitomyces albuminosus HPLC Fruiting body Uracil, hypoxan- Cytidine, uridine, inosine, – [84] thine, adenine guanosine, thymidine, aadenosine Lentinus edodes HPLC Fruiting body Uracil, hypoxan- Cytidine, uridine, inosine, – [84] thine, adenine guanosine, adenosine Downloaded by [University of Florida] at 02:55 11 November 2017

giganteus (Berk) Karunarathna and Hyde had reported summary of nucleobases and nucleosides found in dif- the presence of uridine in the aqueous and ethanolic ferent mushroom species. extracts of the mushroom by using LC-MS method [83]. – The high uridine content (1.66 1.80 g/100 g mushroom New nucleosides in mushrooms extract) of the P. giganteus extracts is correlated to its high neurite outgrowth stimulatory activity of the N6–(2-hydroxyethyl)-adenosine (Figure 3(a)) is a major mushroom. Yuan et al. [84] made a comparison of active constituent of Cordyceps cicadae. Unlike C. milita- nucleobase and nucleoside content between two edible ris and C. sinensis, C. cicadae is an entomopathogenic mushrooms: Termitomyces albuminosus (Berk.) R. Heim mushroom that parasitizes the host named Cicada flam- and Lentinus edodes (Berk.) Singer. Both the mushrooms mata [85,86]. When the host dies, the fruiting body or contain: uracil, cytidine, hypoxanthine, uridine, adenine, stroma of C. cicadae emerges through the exoskeleton inosine, guanosine and adenosine. However, thymidine of the insect. The adenosine analog, N6–(2-hydrox- was not detected in L. edodes. Table 3 provides a yethyl)-adenosine, detected in the fruiting body was CRITICAL REVIEWS IN BIOTECHNOLOGY 9

Figure 3. New nucleosides isolated from mushroom: (a) N6-(2-hydroxyethyl)-adenosine, (b) N2–(1-methoxycarbonylethyl) guano- sine, (c) clitidine, (d) clitidine 50-mononucleotide, (e) 9-b-D-ribopyranosylpurine and (f) clitocine. reported to suppresse lipopolysaccharide (LPS)-stimu- Clitocybe inversa (renamed as Lepista flaccida)[91]. lated release of pro-inflammatory cytokines by RAW Clitocine was initially found to possess insecticidal activ- 264.7 macrophages [87]. ity against the pink bollworm [92]. The apoptotic ability Detection of toxic nucleosides from poisonous mush- of the compound was found to be useful in several in rooms is common. A new purine nucleoside coupled vitro and in vivo models, for example human with an a- derivative, N2–(1-methoxycarbo- colon cancer cell lines [93,94], human cervical cell lines nylethyl)guanosine (Figure 3(b)) was isolated from the [95], multidrug-resistant human hepatocellular carcin- fruiting body of Amanita exitialis Zhu L. Yang and T.H. oma HepG2 cells [96,97], and L1210 leukemic tumor- Li, a poisonous mushroom [88]. The guanosine analog bearing mice [98]. compound was found to be toxic in a brine shrimp lethality test. The flavor compounds, 50-nucleotides in Clitocybe acromelalga Ichimura is a poisonous mush- mushrooms room found in Japan and it is always mistaken as the edible Lepista inversa (Scop.) Pat. A new nucleoside The characteristic flavor substances of mushrooms can called clitidine (Figure 3c) was isolated from the fruiting be classified into nonvolatile and volatile components. Downloaded by [University of Florida] at 02:55 11 November 2017 body of C. acromelalga [89]. Subsequently, its mononu- The nonvolatile and water-soluble substances, including cleotide, clitidine 50-mononucleotide (Figure 3(d)) was the 50-nucleotides, free amino acids and soluble carbo- also detected and isolated from the fruiting body of C. hydrates are the attributes for the taste of an edible acromelalga. All the mentioned compounds were mushroom. Non-protein nitrogenous components (e.g. deduced from spectral data and confirmed by chemical purine base) in mushrooms could also contribute to conversions. overall mushroom flavor characteristics. In fact, the typ- More recently, a novel nucleoside, 9-b-D-ribopyrano- ical flavor of mushrooms are due to the mono- sylpurine (Figure 3(e)) was isolated from the fruiting sodium glutamate (MSG)-like amino acids such as body of Tricholoma japonicum A. Kawam along with and , as well as the flavors 50- other nucleosides: adenosine, uridine, and nebularine nucleotides [99]. [90]. This is the first report regarding the isolation of 9- Mushroom’s nucleotides encompass the 50-adeno- b-D-ribopyranosylpurine from an edible mushroom. The sine monophosphate (50-AMP), 50-cytosine monophos- ribopyranosylpurine was found to inhibit human umbil- phate (50-CMP), 50-guanosine monophosphate (50-GMP), ical vein endothelial cell proliferation. 50-inosine monophosphate (50-IMP), 50-uridine mono- Clitocine (Figure 3(f)) is an exocyclic amino nucleo- phosphate (50-UMP), and 50- side isolated both from the wild and edible mushroom, (50-XMP) (Figure 4). It is important to highlight that the 10 C.-W. PHAN ET AL.

Figure 4. Six 50-nucleotides detected in mushrooms.

mushroom’s total flavor 50-nucleotides that give the nucleotides in the descending order for the four mush- mushroom the umami taste, are the 50-GMP, 50-IMP and rooms were (mg/g): P. ostreatus (14.81), A. bisporus 50-XMP. 50-GMP. These nucleotides that act as a flavor (7.54), F. velutipes (4.94) and L. edodes (2.52). The dis- enhancer like the (MSG), that crepancy of the findings could be due to strain differen- give the mushroom a “meaty” flavor. The synergistic ces of the mushrooms. effects between the flavor 50-nucleotides and MSG-like Apart from that the total flavor 50-nucleotides found

Downloaded by [University of Florida] at 02:55 11 November 2017 component (glutamic acid), greatly increase the overall in some other important edible mushrooms are as fol- taste of mushrooms [100]. lows (mg/g): Hericium erinaceus, the monkey head In the recent years, many investigations were done mushroom (10.31); Hypsizygus marmoreus, the shimeji to explore the 50-nucleotides in various mushroom spe- mushroom (4.76); Grifola frondosa, the maitake mush- cies. The most cultivated edible mushrooms are the A. room (1.46); Tremella fuciformis, the silver ear (0.12); bisporus (button mushroom), Lentinula edodes (shiitake), Auricularia auricula-judae, the wood ear mushroom Pleurotus ostreatus (Jacq.) P. Kumm (oyster mushroom), (0.10); and Sparassis crispa, the cauliflower mushroom and Flammulina velutipes (Curtis) Singer (winter mush- (0.09). Among the highest 50-nucleosides-containing room) [101,102]. mushrooms were the Tricholoma giganteum Massee Yang et al. [103] studied the total flavor 50-nucleotide (13.6 mg/g) and Dictyophora indusiata (Vent.) Desv (50-IMP þ50-GMP þ50-XMP; mg/g) of the four mush- (9.04 mg/g) [105]. rooms using a HPLC method. The flavor 50-nucleotides Beluhan et al. [108] studied the 50-nucleotides in dif- in descending order for the mushrooms were: L. edodes ferent Croatian mushroom species. The mushrooms (11.60), white F. velutipes (8.60), yellow F. velutipes studied were Agaricus campestris Schwein., Boletus (6.32), P. ostreatus (6.09), and A. bisporus (2.96). Most edulis Bull., Calocybe gambosa (Fr.) Donk, Cantharellus recently, according to Phat et al. [104], the flavor 5’- cibarius Fr., Craterellus cornucopioides (L.) Pers., Entoloma CRITICAL REVIEWS IN BIOTECHNOLOGY 11

clypeatum (L.) P. Kumm, Macrolepiota procera (Scop.) Table 4. 50-Nucleotides contents in different edible mushroom Singer, Morchella elata Fr. and Pleurotus ostreatus. The species (mg/g dry weight). mushroom was mixed with deionized water (10:1) and Flavor nucleotide Mushroom species (50-GMPþ 50-IMPþ 50-XMP) References heated in water bath for one min. The mushroom sus- Agaricus bisporus 2.96 [103] pension was filtered using HPLC injection through a Agaricus campestris 1.29 [106] 0.2-mm nylon filter. Nucleotide determination assay was Agrocybe aegerita 1.82 [81] Amanita virgineoides 36.93 [104] performed on a Synergy Hydro column with mobile Auricularia auricula-judae 0.40 [104] phase 50 mM phosphate buffer (pH 5.8) and methanol Boletus edulis 1.63 [106] Calocybe gambosa 1.01 [106] at a flow rate of 0.4 ml/min and detection at 254 nm. Cantharellus cibarius 0.38 [106] The chromatographic peak and retention time were Craterellus cornucopioides 13.88 [106] then compared with the authentic standards. The indi- Dictyophora indusiata 9.04 [105] Entoloma clypeatum 1.24 [106] vidual 5’-nucleotides were in the range of 0.1–7.0 mg/g Entoloma saundersii 0.14 [81] dry weight. However, C. cornucopioides (the black chan- Flammulina velutipes (white) 8.60 [103] 0 Flammulina velutipes (yellow) 6.32 [103] terelle) contained the highest total flavor 5 -nucleotides, Grifola frondosa 1.46 [104] that is, 13.88 mg/g dry weight. The lowest total 50- Hericium erinaceus 10.31 [104] Hypsizigus marmoreus 4.76 [104] nucleotides (0.38 mg/g) was found in C. cibarius, the Lentinus edodes 11.60 [103] chanterelle mushroom. Macrolepiota procera 0.52 [106] Morchella crassipes 0.91 [81] Besides the nature of the mushroom species, the fla- Morchella deliciosa 0.05 [81] 0 vor 5 -nucleotides of mushrooms were also closely Morchella elata 5.32 [106] related to: (i) the parts of the mushroom, (ii) the matur- Panus tigrinus 0.36 [81] Pleurotus cornucopiae 1.49 [104] ity stages of mushrooms, (iii) the storage time and (iv) var. citrinopileatus the processing of the mushrooms [107]. A study by Cho Pleurotus eryngii 4.77 [104] 0 0 Pleurotus ferulae 0.74 [104] et al. [108] showed that the contents of 5 -AMP and 5 - Pleurotus ostreatus 6.09 [103] GMP were higher in the pileus (cap-like structure of Pleurotus salmoneo-stramineus 0.14 [104] Polyozellus multiplex 1.39 [104] basidiocarps) than in the stipe (stem or stalk-like struc- Ramaria botrytis 1.08 [104] ture) of Tricholoma matsutake Sing. Furthermore, in the Sparassis crispa 0.09 [104] 0 Tremella fuciformis 0.12 [104] case of L. edodes, the flavor 5 -nucleotides were higher Tricholoma giganteum 13.6 [105] (1.34 2.81 mg/g) in the pileus when compared to that Tricholoma matsutake 0.80 [91] in the stipe (0.86 1.41 mg/g) [109]. These results were Tuber melanosporum 3.41 [93] Volvariella volvacea 9.00 [111] able to justify the reason why the pilei of T. matsutake and L. edodes were more popular for culinary purposes when compared to the stipes. On the other hand, the 50-nucleotides content is gen- storage time. This was exemplified by Tseng and Mau erally higher in the mushroom mycelium when com- [113] who showed that the total 50-nucleotide in the pared to their fruiting bodies. Liu et al. [110] reported white button mushroom peaked at day 3 of fruiting that a total of five 50-nucleotides (i.e. 50-AMP, 50-CMP, 50- body formation. GMP, 50-UMP and 50-IMP) were identified from Tuber Most recently, measurement of the amount of 50- Downloaded by [University of Florida] at 02:55 11 November 2017 fruiting bodies and fermentation mycelia. The total con- nucleotides in fresh, frozen, canned, and salted A. bispo- tent of 50-nucleotides in the fruiting bodies ranged from rus was carried out by Liu et al. [114]. The results 0.61 to 1.15 mg/g, whereas it ranged from 1.06 to showed that the amount of flavor 50-nucleotides (mg/g) 1.98 mg/g in the fermentation mycelia. According to the in ascending order were: salted (1.71), canned (2.83), study, the flour, added in the fermentation fresh (4.17), and frozen A. bisporus (6.22). In addition, medium, was able to increase the yield of 5’-nucleotides drying also affects the 50nucleotides in A. bisporus. in Tuber mycelium. According to Pei et al. [115], the content of 50-GMP and The total flavor 50-nucleotides content of A. bisporus 50-AMP, as well as the total 50-nucleotides content, sig- and Volvariella volvacea (Bull.) Singer (paddy straw nificantly (p < 0.05) increased during freeze drying and mushroom) was studied at different stages of maturity. also free drying coupled with microwave vacuum tech- At all maturity stages, the total flavor 50-nucleotides of nique. Overall, the quantities of the total flavor 5’- the paddy straw mushroom was almost five times nucleoties vary with the mushrooms species and their higher than that of the white button mushroom. means of handling, and there is no unambiguous rule Overall, the total flavor content peaked at the mid-stage to grade the mushrooms based on the 50-nucleotide for both the mushrooms [111,112]. In addition, the fla- content. Table 4 shows the total flavor 5’-nucleotides in vor of mushrooms tends to increase with post-harvest different mushroom species. 12 C.-W. PHAN ET AL.

Conclusions and its potent neuritogenic properties. Sci World J. 2014;378651. Nucleosides like adenosine and cordycepin serve as the [6] Valverde ME, Hernandez-perez T, Paredes-lopez O. fingerprint biomarkers to authenticate the mushroom Edible mushrooms: improving human health and species, as well as for quality control of the mushroom promoting quality . Int J Microbiol. 2015;376387. extracts. Nucleobases and nucleosides are beyond the [7] Zhang S, He B, Ge J, et al. Characterization of chem- ical composition of Agaricus brasiliensis polysacchar- genetic makeup of the mushrooms. They are reported ides and its effect on myocardial SOD activity, MDA to exhibit medicinal prospects in conjunction with the and caspase-3 level in ischemia-reperfusion rats. Int J special purinergic signaling mediated extracellularly by Biol Macromol. 2010;46:363–366. purine nucleosides and nucleotides such as adenosine [8] Sun C, Rosendahl a. H, Wang XD, et al. and ATP. It is also obvious that there is need for further -K (PSK) in cancer-old story, new possi- – studies on the novel nucleosides of mushrooms, as well bilities? Curr Med Chem. 2012;19:757 762. [9] Cheng PG, Phan CW, Sabaratnam V, et al. as to elucidate their structure-function relationships. Polysaccharides-rich extract of Ganoderma lucidum Investigation of more mushroom species is required to (M.A. Curtis:Fr.) P. Karst accelerates wound healing in obtain a better understanding of their flavor nucleoti- streptozotocin-induced diabetic rats. Evid Based des. More information on the nonvolatile flavors of Complementary Altern Med. 2013;2013:1–9. edible mushrooms would be helpful to market the [10] Shashidhar MG, Giridhar P, Udaya Sankar K, et al. mushrooms. Hence, many food-producing industries Bioactive principles from Cordyceps sinensis: a potent food supplement – a review. J Funct Foods. 2013;5: and mushroom growers are working towards enhancing 1013–1030. and preserving the flavor nucleotides of some popular [11] Jacobson KA, Constanzi S, Ohno M, et al. Molecular mushrooms, for instance, the Agaricus spp. (button recognition at purine and pyrimidine nucleotide (P2) mushrooms). receptors. CTMC. 2004;4:805–819. [12] Schlimme E, Martin D, Meisel H. Nucleosides and nucleotides: natural bioactive substances in milk and Disclosure statement colostrum. Brit J Nutr. 2000;84(Suppl 1):S59–S68. [13] Yamamoto S, Wang MF, Adjei AA, et al. Role of No potential conflict of interest was reported by the authors. nucleosides and nucleotides in the immune system, gut reparation after injury, and brain function. – Funding Nutrition. 1997;13:372 374. [14] Chirivı J, et al. Metabolomic profile and nucleoside We acknowledge the support of this work by the UCSI composition of Cordyceps nidus sp. nov. Pioneer Scientist Incentive Fund (PSIF) (Proj-in-CELT-001) and (Cordycipitaceae): a new source of active com- University of Malaya BKP grant (BK011-2017). This work was pounds. PLoS One. 2017;e0179428. also supported by the University of Malaya High Impact [15] Brown PR, Robb CS, Geldart SE. Perspectives on anal- Research MoE Grants, namely UM.C/625/1/HIR/MoE/SC/02 yses of nucleic acid constituents: the basis of genom- and UM.C/625/1/HIR/MOHE/ASH/01(H-23001-G000008). ics. J Chromatogr A. 2002;965:163–173. [16] Ishimaru M, Haraoka M, Hatate H, et al. Simultaneous analysis of purine and pyrimidine compounds associ- ORCID ated with the freshness and taste of marine foods. Food Anal Methods. 2015;9:1606–1615. Chia-Wei Phan http://orcid.org/0000-0003-1373-1374 Downloaded by [University of Florida] at 02:55 11 November 2017 [17] Garcıa-Gomez D, Rodrıguez-Gonzalo E, Carabias- Martınez R. 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