Structural Characterization of Melanin Pigments from Commercial ...

Article pubs.acs.org/JAFC

Structural Characterization of Melanin Pigments from Commercial Preparations of the Edible Mushroom Auricularia auricula Rafael Prados-Rosales,*,† Stacy Toriola,† Antonio Nakouzi,† Subhasish Chatterjee,§ Ruth Stark,§ Gary Gerfen,‡ Paul Tumpowsky,∥ Ekaterina Dadachova,†,⊥ and Arturo Casadevall† †

Department of Microbiology and Immunology, ‡Department of Physiology and Biophysics, and ⊥Department of Radiology, Albert Einstein College of Medicine, Bronx, New York 10461, United States § Department of Chemistry, Graduate Center and Institute for Macromolecular Assemblies, City University of New York, New York, New York 10031-9101, United States ∥ Goodwin and Wells, New York, New York 10065, United States ABSTRACT: Many of the most widely consumed edible mushrooms are pigmented, and these have been associated with some beneficial health effects. Nevertheless, the majority of the reported compounds associated with these desirable properties are nonpigmented. We have previously reported that melanin pigment from the edible mushroom Auricularia auricula can protect mice against ionizing radiation, although no physicochemical characterization was reported. Consequently, in this study we have characterized commercial A. auricula mushroom preparations for melanin content and carried out structural characterization of isolated insoluble melanin materials using a panel of sophisticated spectroscopic and physical/imaging techniques. Our results show that approximately 10% of the dry mass of A. auricula is melanin and that the pigment has physicochemical properties consistent with those of eumelanins, including hosting a stable free radical population. Electron microscopy studies show that melanin is associated with the mushroom cell wall in a manner similar to that of melanin from the model fungus C. neoformans. Elemental analysis of melanin indicated C, H, and N ratios consistent with 5,6-dihydroxyindole-2-carboxylic acid/5,6dihydroxyindole and 1,8-dihydroxynaphthalene eumelanin. Validation of the identity of the isolated product as melanin was achieved by EPR analysis. A. auricula melanin manifested structural differences, relative to the C. neoformans melanin, with regard to the variable proportions of alkyl chains or oxygenated carbons. Given the necessity for new oral and inexpensive radioprotective materials coupled with the commercial availability of A. auricula mushrooms, this product may represent an excellent source of edible melanin. KEYWORDS: melanin, Auricularia auricula, mushroom, radiotherapy

■

INTRODUCTION Among the most consumed edible products with a high concentration of pigments are saffron, squid ink, and certain mushrooms. Besides their culinary interest, these biological molecules have other health-promoting beneficial properties. For instance, saffron is a spice obtained from the flower of Crocus sativus, which is rich in carotenoids like crocin and crocetin.1 Consumption of some carotenoids is associated with potent anti-tumor effects.2 Most commercially available ink is obtained from the cuttlefish, a thick-fleshed relative of the squid. Squid ink is mostly composed of melanin, polysaccharides, and proteins and has been shown to have antitumor effects. In addition, promotion of immunity or induction of some cytokines by squid ink was demonstrated by in vitro and in vivo studies.3 Several mushrooms, including Auricularia auricula, Grifola f rondosa, Lentinus tigrinus, Flammulina velutipes, Hericium erinaceus, Pleurotus ostreatus, and Tremella foliacea, are known for their edibility and their salubrious functional properties. In fact, 700 of the 10 000 known species of mushrooms are edible, and more than 200 are thought to have medicinal value.4 Many studies have characterized the composition of edible mushrooms and established the prevalence of bioactive molecules with diverse biological activity. The content and bioactivity of © 2015 American Chemical Society

these compounds depend on how the mushrooms are prepared and consumed.4 Studies of the medicinal properties of edible mushrooms revealed such beneficial effects as lowering of blood pressure, modulation of the immune system, reduction of blood lipid concentrations, inhibition of tumors, and prevention of inflammation or anti-microbial activity. A. auricula, commonly known as tree-ear, is an edible mushroom found worldwide. Although not widely consumed in Western countries, this mushroom has been used in Asian cuisine since ancient times. Several studies have reported diverse biological activities of this mushroom, including antitumor and anti-coagulant activity among others. Non-starch polysaccharide components are believed to be responsible for some of these effects. Other non-polysaccharide compounds from this mushroom, including melanins, can have biological effects such as the inhibition of bacterial quorum sensing.5 Physicochemical characterization of soluble melanin fractions from A. auricula fruit-bodies and assessment of their antioxidant capacity have already been reported.6−8 Received: Revised: Accepted: Published: 7326

March 31, 2015 July 8, 2015 August 5, 2015 August 5, 2015 DOI: 10.1021/acs.jafc.5b02713 J. Agric. Food Chem. 2015, 63, 7326−7332

Article

Journal of Agricultural and Food Chemistry

in a 1 M sorbitol/0.1 M pH 5.5 sodium citrate solution and incubated at 30 °C for 24 h with 10 mg/mL of lysing enzymes from Trichoderma harzianum (Sigma). The enzyme-digested materials were collected by centrifugation at 3000 rpm for 10 min and washed repeatedly with PBS until the supernatant was nearly clear. Proteinaceous materials were denatured by incubating the pellets with 4 M guanidine thiocyanate in a rocker for 12 h at room temperature. The recovered debris was collected by centrifugation, washed two or three times with ∼20 mL of PBS , and then incubated for 4 h at 65 °C in 10 mL of buffer (10 mM pH 8.0 Tris-HCl, 5 mM CaCl2, 5% SDS) containing 1 mg/mL of proteinase K (Boehringer, Mannheim, Germany). The debris was recovered and washed two or three times with ∼20 mL of PBS, after which lipids were extracted three successive times by the Folch method, maintaining chloroform, methanol, and saline solution in the final mixture at 8:4:3 (v/v/v). The product was suspended in 20 mL of 6 M HCl and boiled for 1 h to hydrolyze cellular contaminants associated with the melanin. The resulting acid-resistant insoluble material was dialyzed against distilled water for 14 d with daily water changes and lyophilized. Elemental analysis (carbon, nitrogen, and hydrogen) was performed by Intertek (Whitehouse, NJ). Scanning Electron Microscopy (SEM). Mushroom powders and melanin extracts were suspended in PBS, incubated on coverslips coated with 0.01% poly-L-lysine, and fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 24 h at 4 °C. Mushroom and melanin extracts were post-fixed with 1% OsO4, dehydrated progressively in 70%, 90%, 95%, and 100% ethanol, and critical-pointdried in CO2. The material was sputter-coated with gold using an EMitech K550 sputter coater and viewed in a Zeiss Supra 40 scanning electron microscope operating at a voltage of 10 kV. Transmission Electron Microscopy (TEM). Samples were fixed with 2% paraformaldehyde−2.5% glutaraldehyde−0.1 M sodium cacodylate buffer, followed by a post-fixation step with 1% osmium tetroxide and 1% uranyl acetate. Samples were then dehydrated by immersion in a graded series of ethanol concentrations and embedded in LX112 resin (LADD Research Industries, Burlington, VT). A Reichert Ultracut UCT instrument was used to cut ultrathin (80 nm thick) sections that were then stained with uranyl acetate followed by lead citrate. TEM was done using a JEOL 1200EX transmission electron microscope operating at 80 kV. Electron Paramagnetic Resonance (EPR) Spectroscopy. EPR spectra were obtained at 77 K using a Varian E112 X-Band model spectrometer equipped with a TE102 resonator. Approximately 5 mg samples of melanin preparations were loaded into 4 mm quartz EPR tubes (Wilmad LabGlass, Buena, NJ), which were held in place in the resonator using a quartz finger dewar. Typical parameters used to acquire the spectra were as follows: modulation amplitude, 1.6 G; microwave frequency, 9.11 GHz; and microwave power, 0.10 mW. The quartz EPR signal generated as a result of the irradiation was subtracted from the spectra of the irradiated samples. EPR spectra were also obtained after illumination of melanin suspensions with a 150 W fiber optic lamp for 30 min at 77 K or by suspending the melanins in 0.1 M ZnCl2. Solid-State Nuclear Magnetic Resonance (NMR) spectroscopy. Solid-state 13C NMR measurements were performed using a Varian (Agilent Technologies, Santa Clara, CA) DirectDrive1 (VNMRs) NMR spectrometer operating at a 1H frequency of 600 MHz (13C frequency of 150 MHz) and a temperature set to 25 °C. The 13C cross-polarization (CP) experiments were conducted at a magic-angle spinning (MAS) frequency of 20 kHz (±20 Hz) using a 1.6 mm HXY fastMAS probe filled with 4−6 mg of powdered sample. Ramped-amplitude 13C CPMAS (RAMP-CP)18 measurements were conducted to identify the carbon chemical moieties, and 13C multipleCP experiments19 were carried out to achieve high-throughput quantification of the carbon-containing pigment functional groups. Detailed experimental parameters for CPMAS measurements on fungal melanins have been reported elsewhere.20,21 The recycle delay for the ramp CP measurements and at the beginning of the multipleCP sequence was 3 s, and the duration of the re-polarization period in multiple-CP was 0.8 s for the pigment samples. The CP times were 1.0−1.5 ms for the RAMP-CP22 and 1.0 ms for the multiple-CP

There is an urgent need for counter-measures against ionizing radiation in fields that run the gamut from medical radiation therapeutics to the nuclear industry to space travel. Apart from external physical shielding, there is little that can be done currently to protect individuals who are exposed to large doses of radiation. However, we have observed,9 and others have confirmed,10,11 that the presence of melanin in the intestinal lumen during extra-corporeal irradiation translates into remarkable protection against the deleterious effects of extra-corporeal ionizing radiation in mice. The presumed mechanism of action for this melanin protective effect is shielding of the gut tissue, including the associated lymphatic tissue, which in turn provides cells to replenish radiationdepleted bone marrow. More specifically, melanin involves physical shielding of the cells via Compton scattering of the incoming photons, accompanied by the scavenging of Compton electrons and free radicals by the melanin macromolecules.9 Melanin is an insoluble and non-digestible pigment with a complex molecular structure that is generated by polymerization of indolic and phenolic compounds.12 Melanins are found in all biological kingdoms, but their structure and function remain poorly understood, as these heterogeneous pigments cannot be crystallized for high-resolution structural studies. There are five types of melanin commonly found in nature: eumelanin, pheomelanin, neuromelanin, pyomelanin, and allomelanin.13 Of the five, eumelanin is the most abundant and is a brown to black pigment.13−15 The eumelanin polymer is composed 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units.9 It can be highly concentrated in the human epidermal, retinal, and auditory systems.13,14 Structures for both oligomers for eumelanin and pheomelanin have been already proposed.9 Melanins are readily produced by many fungi, including edible fungi, where their functions could include energy transduction.16 Consequently, the edible fungi constitute a potential source of nutritional melanin that could find important use as a radiation countermeasure; e.g., the benefits from the A. auricula mushroom could be gained by ingesting the raw material. In addition to radiation protection, melanins have semiconductor properties that could be exploited in the design of edible electronic devices.17 Hence, there is a need for identifying good sources of melanin from edible biomaterials in such diverse areas as radioprotection and electronic design. As mushroom powders are now sold as food supplements, we conducted an essential study of the melanin content and associated molecular characteristics in commercial preparations of powdered A. auricula.

■

MATERIALS AND METHODS

Melanin Sources. Auricularia auricula mushroom preparations were obtained from Maypro (New York, NY, http://maypro.com/). As a control for L-DOPA melanin, we isolated melanin from Cryptococcus neoformans as described previously.16 Briefly, melanized cells were obtained by growing C. neoformans strain H99 (American Type Culture Collection, Rockville, MD) in defined minimal medium (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 μM vitamin B1 [Sigma Chemical Co., Cleveland, OH]) with 1 mM L-DOPA (Sigma) at 30 °C for 10 days with shaking at 150 rpm. Melanin Isolation. The same protocols were used to isolate melanin from melanized C. neoformans and powdered mushrooms. For C. neoformans, melanized cells were harvested by centrifugation at 2000 rpm and washed with phosphate-buffered saline (PBS). Similarly, 20 g samples of A. auricula mushroom powders were dissolved in PBS. Fungal cells and powdered mushroom material were each suspended 7327

DOI: 10.1021/acs.jafc.5b02713 J. Agric. Food Chem. 2015, 63, 7326−7332

Article

Journal of Agricultural and Food Chemistry measurement, each with 10 recursive cycles. The SPINAL method23 was used to apply high-power heteronuclear 1H decoupling of 170− 185 kHz, and 2048−7400 transients were acquired to generate each 13 C spectrum. 1D 13C spectral data sets were processed with 100−200 Hz of line broadening; chemical shifts were referenced externally to the ethylene (−CH2−) group of adamantane (Sigma) at δC = 38.48 ppm. Reproducibility of the spectroscopic measurements was assessed by obtaining replicate 13C spectra on each sample; biological replicates of these materials and previously reported C. neoformans melanins20 displayed minor variations in their 13C NMR spectra due to differences in cellular media, scale of synthesis, extent of polymerization, and pigment deposition within the cellular matrix.

10% of the dry weight of the fungal mass.9 To determine the melanin content of commercial mushroom preparations, we used a quantitative melanin isolation protocol adapted from the fungus C. neoformans. Twenty grams of powder was processed for melanin isolation, beginning with suspension in PBS (Figure 2A). Of note, we observed a remarkable expansion in

■

RESULTS Microscopic Analysis of Commercial Mushroom Preparations. Commercial edible mushroom preparations are usually available as powders. We studied the ultra-structural features of commercial powders of A. auricula edible mushrooms. A. auricula mushroom appears as a dark powder (Figure 1A). Analysis of these “raw” preparations by SEM showed that

Figure 1. Electron microscopy analysis of the A. auricula mushroom powders (A). A. auricula mushroom preparations were processed for scanning electron microscopy (SEM, B and C) and transmission electron microscopy (TEM, D). Scale bars in B and D are 10 μm, and scale bar in C is 1 μm.

Figure 2. Melanin isolation protocol from commercial mushroom preparations. (A) The protocol was adapted from that for the fungus C. neoformans and involved digestion of cell walls, de-proteination, delipidation, and acid hydrolysis. (B) Appearance of A. auricula commercial mushroom powders after dissolving in PBS and centrifugation (steps 1 and 2 in the melanin isolation protocol). Note that 20 g of A. auricula powder expanded when dissolved in PBS. (C) Appearance of the melanin extract from A. auricula mushrooms.

A. auricula powder was composed of a heterogeneous mixture of amorphous fragments with a size distribution ranging between 1 and 50 μm (Figure 1B). Higher magnifications of some of the mushroom pieces showed that the surface of these fragments appeared to have a rough morphology (Figure 1C). Melanin is associated with the cell wall in the human pathogen C. neoformans and often appears as an electron-dense layer in transmission electron micrographs.24 We performed TEM on mushroom extracts and found that both mushroom powders have very similar internal structure, including circular units with a dense cell wall made of several concentric layers embedded in a heterogeneous mass of material with different textures (Figure 1D). However, the A. auricula mushroom powder had more electron-dense material. The darker areas in this mushroom were associated with material randomly distributed across the section, although we could observe that the inner layers of the circular units were also highly electron dense (Figure 1D), suggesting that melanin might not have a regular distribution pattern in edible mushrooms. Melanin Content of Commercial Mushroom Preparations. A prior analysis of A. auricula mushrooms obtained at a local market revealed that melanin comprises approximately

the volume of the A. auricula powder upon hydration (Figure 2B). Subsequent steps in the melanin isolation from A. auricula involved digestion of cell-wall structural components and protein denaturation and digestion. Extraction of lipids by chloroform and methanol treatment revealed that 0.5% of the dry weight was due to lipids. Subsequent acid hydrolysis of the denatured, proteinase-digested and de-lipidated material reduced the wet weight by 50%. After hydrolysis, the product was dialyzed and lyophilized. The color of the extract was significantly darker than the original mushroom powder, which reflects enrichment in the melanin pigment (Figure 2C). Approximately 10% of the dry weight of the mushroom powder corresponded to melanin pigment that is resistant to acid hydrolysis. Ultrastructural characterization of the final (melanin) product by SEM revealed a very different morphology relative to the original mushroom powder, namely an amorphous material with little structural resemblance to the commercial 7328


Article


spectroscopy a particularly effective method for identifying and classifying them. A set of qualitative EPR criteria were established for melanin identification based on the increase in EPR signal upon (a) illumination with a bright white light or (b) addition of ZnCl2.9,24,25 As previously reported, irradiation of C. neoformans melanin with a bright light or supplementation with 0.1 M ZnCl2 increased the EPR by approximately 200% and 500%, respectively (Figure 4A).9,24 To assess whether the

powder (Figure 3). The removal of acid-digestible material was apparent as an increase in effective surface area. TEM analysis

Figure 3. Electron micrographs of isolated melanin from commercial A. auricula mushrooms. Note the hollowness of the isolated material. Scale bars are 100 μm (top panel), 20 μm (medium panel), and 10 μm (lower panel).

of the isolated melanin showed that the units observed by SEM were hollow and consisted mainly of a melanin ring with no associated internal material. High-magnification micrographs revealed that mushroom melanin is composed of small spherical units with an estimated diameter of 20−30 nm (Figure 3). Physicochemical Characterization of Mushroom Melanins. Melanins are complex, amorphous, and incompletely understood polymeric pigments.12 Demonstration that a compound is melanin by chemical means is difficult, and not all dark pigments in nature are melanins. Fungal melanins include DHI and DHICA monomer units with 6−9% nitrogen or 1,8-dihydroxynaphthalene (DHN) with no nitrogen in its structure.9 Elemental analysis of C. neoformans melanin showed 59% C, 7% H, and 6% N (C/N of 10.6, no S), indicating that C. neoformans melanin was primarily a DHI/DHICA eumelanin. In contrast, A. auricula melanin contained 41% C, 5% H, and 1.6% N (C/N of 24.8, no significant S), suggesting that it contained a mixture of DHICA/DHI and DHN eumelanin, as previously reported (Table 1).9 Of note, structural formulas for DHN melanins have been already reported.9 Melanin pigments are remarkable in that they contain a stable population of organic free radicals, making EPR

Figure 4. Electron paramagnetic resonance spectroscopy analysis of fungal melanins. (A) EPR of C. neoformans H99 melanin using standard conditions (red) or after exposure to either bright light stimulation (blue) or supplementation with ZnCl2 (black). (B) EPR of A. auricula commercial mushroom melanin using the same conditions as in A.

dark product resulting from the A. auricula commercial mushroom preparation was melanin, we performed EPR measurements before and after bright illumination and upon addition of ZnCl2. The EPR spectra of the material isolated from mushroom powder revealed a stable free radical signal like that described for melanin (Figure 4B). Illumination of this material for 30 min with white light from a 150 W fiber optic lamp and suspension in 0.1 M ZnCl2 increased the magnitude of the EPR signal by 150% and 900%, respectively (Figure 4B). Thus, the acid insolubility of the dark residue combined with the EPR results established the material derived from A. auricula as a melanin. NMR Spectroscopy of Commercial and C. neoformans Fungal Mushroom Preparations. The heterogeneous and amorphous character of melanin pigments combined with their insolubility in aqueous buffers requires specialized techniques such as solid-state NMR to elucidate the constituent chemical moieties present in the intact pigment. The 13C CPMAS spectrum of C. neoformans L-DOPA melanin revealed similar resonances to prior 13C NMR reports for fungal melanin ghosts, exhibiting major indole-based aromatic and/or olefinic

Table 1. Elemental Composition of Melanins

a

melanin

C

H

N

(O)a

C. neoformans A. auricula

58.99 41.18

7.11 5.56

5.56 1.66

28.34 51.60

Calculated from 100% − C − H − N. 7329


Article


NMR spectra supported the presence of chemically recalcitrant aliphatic frameworks associated with melanization in both pigmented samples that underwent exhaustive degradative treatments (Figure 2).

carbons (110−160 ppm) and additional long-chain methylene groups ((CH2)n, 20−40 ppm), oxygenated aliphatic carbons (CHnO, 50−105 ppm), and carboxyls or amides (COO or CONH, 170−173 ppm)20,21 (Figure 5). The resonances at 30,

■

DISCUSSION The aim of this study was to analyze the nature of the melanin pigment from A. auricula commercial mushroom preparations, in the context of exploring the feasibility of using those products as sources of edible protectants for ionizing radiation. SEM revealed significant ultrastructural differences in the morphology of the constituents of these preparations. The yield of the melanin obtained from the A. auricula commercial powder was in agreement with that reported previously from intact mushrooms.9 Scanning electron micrographs of isolated melanin showed a very different product compared to the initial mushroom preparations: isolated melanin consisted of a heterogeneous population of fragments with high specific surface area. This final material was resistant to acid hydrolysis, a property that it shares with C. neoformans fungal melanin. Isolated melanin from C. neoformans appears as empty melanin spheres that conserve the fungal cell’s shape and were thus named melanin “ghosts”. The morphology observed in the TEM images of A. auricula commercial mushroom preparations was more heterogeneous than fungal cells, and the dark material appeared to be associated with different areas of the respective materials. However, when the melanin recovered from the mushroom powder was analyzed by TEM, very discrete, closed, and empty dark structures were observed that retained the shape of the inner layer of cell walls. Hence, these A. auricula cell-wall ghosts could be viewed as comparable to the C. neoformans melanin ghosts that retained the shape of individual yeast cells.24,26 Electron micrographs at higher magnification showed that the commercial mushroom melanin is composed of small spherical units with an estimated diameter of 20−100 nm, with the smallest particle size estimated as 20− 30 nm. An in-depth microscopy analysis of C. neoformans melanin showed a similar size distribution,28 indicating that the ultrastructural morphology of melanin is similar in both microscopic and macroscopic fungi. Similar analysis of the surfaces of isolated melanin from hair melanosomes and Sepia of f icinalis showed substructures in the size range of a few to 30 nm.29 To confirm that the black residue recovered from commercial mushroom powder was melanin, we conducted a standardized test based on EPR analysis, which takes advantage of the fact that melanin pigments contain a stable free radical population.25 The dark material from commercial mushroom had a spectrum similar to that of C. neoformans melanin24 and responded to either illumination with a bright lamp or addition of ZnCl2 with a significant increase in the EPR signal.24 These results establish that the pigment conforms to spectroscopic criteria assigned to melanins. The molar C/N ratios of the C. neoformans and black mushroom melanin were 10.6 and 24.8, respectively, indicating differences between the melanin structures. No sulfur was found, so both materials could be classified as eumelanins. In agreement with previous studies on A. auricula melanin,9 the low percentage of N revealed by elemental analysis suggested that commercial A. auricula mushroom melanin contains some DHN melanin. Nonetheless, oxidative high-performance liquid chromatography of A. auricula melanin in the cited study detected the presence of pyrrole-2,3-dicarboxylic acid, an

Figure 5. 150 MHz 13C NMR spectra of C. neoformans melanin produced with natural abundance L-DOPA (top) and commercial mushroom melanin (bottom), measured at 20 kHz MAS using the quantitatively reliable multiple cross-polarization magic-angle spinning technique. Each spectrum is normalized by setting the largest peak to full scale.

24, and 15 ppm can be attributed to a typical pattern from −(CH2)n−CH2CH3 alkyl chains.20 The broad aromatic spectral envelope centered at ∼128 ppm is attributable to overlap of resonances from slightly dissimilar aromatic indole-based constituents, structural heterogeneity of the disordered pigment materials, and/or the presence of paramagnetic species, as demonstrated by the EPR results. The quantitatively reliable multiple-CPMAS 13C spectra of the purified melanin pigment obtained from commercial A. auricula mushroom materials had several features comparable to those of the C. neoformans melanin. However, the spectra also manifested several structural differences, including modest signal intensity from aromatic moieties (110−160 ppm), diminished signals from the alkyl chains (20−40 ppm), and significantly enhanced signals from oxygenated carbons (50− 105 ppm) (Figure 5). As deduced for the NMR spectra of C. neoformans melanin ghosts, key oxygenated aliphatic carbon moieties in the mushroom sample included signals from a range of functional groups, such as CH3O and CH2CH2O (40−60 ppm), CH2O (62 ppm), and CHO groups (69, 75, 86, and 105 ppm), that could be derived from attached cell-wall polysaccharides. A set of relatively sharper oxygenated carbon resonances for the mushroom-derived material suggests a comparatively ordered cell-wall matrix; the prominence of the oxygenated carbon NMR signals also conforms with elemental analysis results that imply a larger percentage of oxygen. Our 7330



■

oxidation product of DHI. Thus, it is possible that A. auricula contains a mixture of DHN and DHI/DHICA melanins. Of note, both C. neoformans and commercial mushroom melanins showed a higher C/N ratio compared to synthetic and human hair melanins, also suggesting a divergence in structural features and/or an association with denaturant- and proteinase-resistant materials for these latter eumelanins. Solid-state NMR analysis provided additional evidence for differences in carbon-containing structures between C. neoformans and commercial mushroom melanins. Analysis of the 13C CPMAS spectral features associated with carbon-containing moieties showed diminished contributions at chemical shifts corresponding to aromatic and alkyl chain moieties, but significantly enhanced signals from the oxygenated carbons. The larger proportion of oxygenated carbons was also reflected in our elemental analysis results, from which percentage O values can be calculated as 51.60 for A. auricula and 28.34 for C. neoformans, respectively. Given the comparable colors, EPR intensities, and EPR line widths for C. neoformans and A. auricula mushroom samples, the weaker mushroom aromatic spectral contributions could reflect broadening associated with closer proximity to the unpaired electrons. Similar to C. neoformans melanin, NMR signatures associated with cell-wall polysaccharides were detected in the commercial mushroom preparation.20,21,26,27 The sharper oxygenated carbon resonances displayed for commercial mushroom melanin suggested a more ordered cell-wall matrix as well as a different type of scaffolding on which the pigment can assemble. A previous study demonstrated that isolated melanin from A. auricula mushrooms protects mice from acute ionizing radiation when administered orally.9 Although the current study did not test the capacity of the fungal melanin isolated from mushroom powder to protect mice, the near identity of the melanins isolated from A. auricula whole mushrooms and commercially available powdered mushroom preparations suggests that the latter can offer a relatively cheap and plentiful source of melanin for future radioprotective studies. The location of melanin at the cell wall, according to our ultrastructural studies, suggests that cells will be protected from ionizing radiation going deeper into the cells. Given the ongoing necessity of new oral radioprotectors suitable for treatment before acute radiotherapy or to alleviate the effects of radiation during catastrophic events, ingestion of melanin from powdered mushrooms could represent a new inexpensive and safe protective protocol. In summary, we characterized the pigment derived from a commercial A. auricula mushroom as an edible source, established that it was a melanin by spectroscopic criteria, and characterized it using ultrastructural techniques and solidstate NMR as well as EPR. Melanin is insoluble and indigestible, yet it can provide medicinal effects by its presence in the gastrointestinal tract through its ability to mediate local shielding against external radiation. Melanin has the additional distinction of being an edible stable free radical, although the biological effects associated with this characteristic during its residence in the gastrointestinal tract remain to be determined. Melanins have electronic properties and metal-binding capacities that could be exploited for several purposes, making the availability of commercial non-toxic melanin sources promising for future studies to assess their clinical and technological usefulness.

Article

AUTHOR INFORMATION

Corresponding Author

*Tel.: (718) 430-2811. Fax: (718) 430-8771. E-mail: rafael. [email protected]. Notes

The authors declare the following competing financial interest(s): Three of the authors (E.D., P.T., and A.C.) are involved in an effort to develop melanin for radiation protection through the company Goodwin & Wells (http:// www.goodwinandwells.com/technology/). Furthermore, E.D. and A.C. hold patents describing the radioprotective properties of melanin. However, none of the authors have any connection with the supplier of these mushroom powders (Maypro).

■

ACKNOWLEDGMENTS A.C., A.N., S.T., and R.P.-R. are supported by grants from the National Institutes of Health. S.C. and R.S. are supported by NIH grant R01-AI052733. The 600 MHz NMR facilities used in this work are operated by The City College and the CUNY Institute for Macromolecular Assemblies, with additional infrastructural support provided by grant 8G12 MD007603 from the National Institute on Minority Health and Health Disparities, National Institutes of Health.

■

REFERENCES

(1) Bolhassani, A.; Khavari, A.; Bathaie, S. Z. Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. Biochim. Biophys. Acta, Rev. Cancer 2014, 1845, 20−30. (2) Khavari, A.; Bolhassani, A.; Alizadeh, F.; Bathaie, S. Z.; Balaram, P.; Agi, E.; Vahabpour, R. Chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine generates different anti-tumor effects against tumors expressing the E7 protein of human papillomavirus. Arch. Virol. 2015, 160, 499. (3) Lei, M.; Wang, J.; Wang, Y.; Pang, L.; Wang, Y.; Xu, W.; Xue, C. Study of the radio-protective effect of cuttlefish ink on hemopoietic injury. Asia Pac. J. Clin. Nutr. 2007, 16 (Suppl 1), 239−43. (4) Chang, R. Functional properties of edible mushrooms. Nutr. Rev. 1996, 54, S91−3. (5) Zhu, H.; He, C. C.; Chu, Q. H. Inhibition of quorum sensing in Chromobacterium violaceum by pigments extracted from Auricularia auricula. Lett. Appl. Microbiol. 2011, 52, 269. (6) Zou, Y.; Hu, W.; Ma, K.; Tian, M. Physicochemical properties and antioxidant activities of melanin and fractions from Auricularia auricula fruiting bodies. Food Sci. Biotechnol. 2015, 24, 15. (7) Zou, Y.; Yang, Y.; Zeng, B.; Gu, Z.; Han, Y. Comparison of Physicochemical Properties and Antioxidant Activities of Melanins from Fruit-Bodies and Fermentation Broths of Auricularia auricula. Int. J. Food Prop. 2013, 16, 803. (8) Zou, Y.; Xie, C.; Fan, G.; Gu, Z.; Han, Y. Optimization of ultrasound-assisted extraction of melanin from Auricularia auricula fruit bodies. Innovative Food Sci. Emerging Technol. 2010, 11, 611. (9) Revskaya, E.; Chu, P.; Howell, R. C.; Schweitzer, A. D.; Bryan, R. A.; Harris, M.; Gerfen, G.; Jiang, Z.; Jandl, T.; Kim, K.; Ting, L. M.; Sellers, R. S.; Dadachova, E.; Casadevall, A. Compton scattering by internal shields based on melanin-containing mushrooms provides protection of gastrointestinal tract from ionizing radiation. Cancer Biother. Radiopharm. 2012, 27, 570−6. (10) Kunwar, A.; Adhikary, B.; Jayakumar, S.; Barik, A.; Chattopadhyay, S.; Raghukumar, S.; Priyadarsini, K. I. Melanin, a promising radioprotector: mechanisms of actions in a mice model. Toxicol. Appl. Pharmacol. 2012, 264, 202−11. (11) Rageh, M. M.; El-Gebaly, R. H.; Abou-Shady, H.; Amin, D. G. Melanin Nanoparticles (MNPs) provide protection against wholebody-irradiation in mice via restoration of hematopoietic tissues. Mol. Cell. Biochem. 2015, 399, 59.

7331


Article

Journal of Agricultural and Food Chemistry (12) Chedekel, M. R.; Ahene, A. B.; Zeise, L. Melanin standard method: empirical formula 2. Pigm. Cell Res. 1992, 5, 240−6. (13) Solano, F. Melanins: Skin Pigments and Much MoreTypes, Structural Models, Biological Functions, and Formation Routes. New J. Sci. 2014, 2014, 498276. (14) Eisenman, H. C.; Casadevall, A. Synthesis and Assembly of Fungal Melanin. Appl. Microbiol. Biotechnol. 2012, 93, 931−940. (15) Plonka, P. M.; Grabacka, M. Melanin synthesis in microorganisms-biotechnology and medical aspects. Acta Biochim. Pol. 2006, 53 (3), 429−443. (16) Dadachova, E.; Bryan, R. A.; Huang, X.; Moadel, T.; Schweitzer, A. D.; Aisen, P.; Nosanchuk, J. D.; Casadevall, A. Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi. PLoS One 2007, 2, e457. (17) Kim, Y. J.; Wu, W.; Chun, S. E.; Whitacre, J. F.; Bettinger, C. J. Biologically derived melanin electrodes in aqueous sodium-ion energy storage devices. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 20912−7. (18) Metz, G.; Wu, X. L.; Smith, S. O. Ramped-Amplitude Cross Polarization in Magic-Angle-Spinning NMR. J. Magn. Reson., Ser. A 1994, 110, 219−227. (19) Johnson, R. L.; Schmidt-Rohr, K. Quantitative solid-state 13C NMR with signal enhancement by multiple cross polarization. J. Magn. Reson. 2014, 239, 44−9. (20) Chatterjee, S.; Prados-Rosales, R.; Frases, S.; Itin, B.; Casadevall, A.; Stark, R. E. Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors. Biochemistry 2012, 51, 6080−8. (21) Chatterjee, S.; Prados-Rosales, R.; Tan, S.; Itin, B.; Casadevall, A.; Stark, R. E. Demonstration of a common indole-based aromatic core in natural and synthetic eumelanins by solid-state NMR. Org. Biomol. Chem. 2014, 12, 6730−6. (22) Morcombe, C. R.; Zilm, K. W. Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 2003, 162, 479−86. (23) Fung, B. M.; Khitrin, A. K.; Ermolaev, K. An improved broadband decoupling sequence for liquid crystals and solids. J. Magn. Reson. 2000, 142, 97−101. (24) Wang, Y.; Aisen, P.; Casadevall, A. Melanin, melanin “ghosts,” and melanin composition in Cryptococcus neoformans. Infect. Immunity 1996, 64, 2420−2424. (25) Enochs, W. S.; Nilges, M. J.; Swartz, H. M. A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy. Pigm. Cell Res. 1993, 6, 91−9. (26) Tian, S.; Garcia-Rivera, J.; Yan, B.; Casadevall, A.; Stark, R. E. Unlocking the molecular structure of fungal melanin using 13C biosynthetic labeling and solid-state NMR. Biochemistry 2003, 42, 8105−9. (27) Zhong, J.; Frases, S.; Wang, H.; Casadevall, A.; Stark, R. E. Following fungal melanin biosynthesis with solid-state NMR: biopolymer molecular structures and possible connections to cellwall polysaccharides. Biochemistry 2008, 47, 4701−10. (28) Eisenman, H. C.; Nosanchuk, J. D.; Webber, J. B.; Emerson, R. J.; Camesano, T. A.; Casadevall, A. Microstructure of cell wallassociated melanin in the human pathogenic fungus Cryptococcus neoformans. Biochemistry 2005, 44, 3683−93. (29) Liu, Y.; Simon, J. D. Isolation and biophysical studies of natural eumelanins: applications of imaging technologies and ultrafast spectroscopy. Pigm. Cell Res. 2003, 16, 606−18.

7332
