International Journal of Medicinal Mushrooms, 18(5): 413–424 (2016) Quantification of Water-Soluble Metabolites in Medicinal Mushrooms Using Proton NMR Spectroscopy Yu-Chang Lo,1,2,3 Shih-Chang Chien,4 Darya O. Mishchuk,5 Carolyn M. Slupsky,5,6,* & Jeng-Leun Mau1,2,3,* 1Department of Food Science and Biotechnology, National Chung Hsing University (NCHU), Taichung, Taiwan, R.O.C.; 2NCHU-UCD Plant and Food Biotechnology Center, NCHU, Taichung, Taiwan, R.O.C.; 3Agricultural Biotechnology Center, NCHU, Taichung, Taiwan, R.O.C.; 4The Experimental Forest Management Office, NCHU, Taichung, Taiwan, R.O.C.; 5Department of Food Science and Technology, University of California, Davis, California; 6Department of Nutrition, University of California, Davis, California *Address all correspondence to: Jeng-Leun Mau, Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, R.O.C.; Tel.: +886-4-2285-4313; [email protected]; and Carolyn M. Slupsky, Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616; Tel.: +1-530-752-6804, +1-530-219-575; cslupsky@ ucdavis.edu ABSTRACT: The water-soluble metabolites in 5 mushrooms were identified and quantified using proton nuclear magnetic resonance (NMR) spectroscopy and software for targeted metabolite detection and quantification. In total, 35 compounds were found in Agaricus brasiliensis, 25 in Taiwanofungus camphoratus, 23 in Ganoderma lucidum (Taiwan) and Lentinus edodes, and 16 in G. lucidum (China). Total amounts of all identified metabolites inA. brasilien- sis, T. camphoratus, G. lucidum, G. lucidum (China), and L. edodes were 149,950.51, 12,834.18, 9,549.09, 2,788.41, and 111,726.51 mg/kg dry weight, respectively. These metabolites were categorized into 4 groups: free amino acids and derivatives, carbohydrates, carboxylic acids, and nucleosides. Carbohydrates were the most abundant metabolites among all 4 groups, with mannitol having the highest concentration among all analyzed metabolites (848–94,104 mg/ kg dry weight). Principal components analysis (PCA) showed obvious distinction among the metabolites of the 5 dif- ferent kinds of mushrooms analyzed in this study. Thus PCA could provide an optional analytical way of identifying and recognizing the compositions of flavor products. Furthermore, the results of this study demonstrate that NMR- based metabolomics is a powerful tool for differentiating between various medicinal mushrooms. KEY WORDS: medicinal mushrooms, NMR, principal component analysis, water-soluble metabolite ABBREVIATIONS: DW, dry weight; GABA, 4-aminobutyric acid (γ-aminobutyric acid); HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; PCA, principal component analysis I. INTRODUCTION treat disease are species such as Ganoderma lucidum, Agaricus brasiliensis, Taiwanofungus camphoratus, Mushrooms have been consumed as food and food and Lentinus edodes. flavorings because of their unique and subtle flavors. The lingzhi or reishi medicinal mushroom, G. Mushroom flavor derives primarily from nonvolatile lucidum (Curtis: Fr.) P. Karst. (Ganodermataceae, components such as amino acids and nucleotides.1 Agaricomycetes), has long been used in traditional Mushrooms have been reported to be low in calories Chinese medicine as an anti-inflammatory, antitumor, and fat but rich in proteins, polysaccharides, and vita- antiviral, antibacterial, and antiparasitic agent, as well mins, and they exhibit abilities to lower the level of as in blood pressure regulation, cardiovascular dis- cholesterol in humans.2,3 Moreover, some mushroom orders, kidney toxicity, hepatotoxicity, and chronic species have also shown certain medicinal effects and bronchitis. It has also been suggested for use as a are widely used to alleviate the symptoms of cer- protective nerve tonic.5 tain diseases such as diabetes, hypercholesterolemia, A. brasiliensis S. Wasser et al. (=A. blazei and cancer.4 Among the mushrooms typically used to Murrill s. Henem., Agaricaceae, Agaricomycetes) is 1521-9437/16/$35.00 © 2016 Begell House, Inc. www.begellhouse.com 413 414 Lo et al. reportedly used as a healthy food for the prevention accomplished by combining NMR spectroscopy of cancer, diabetes, hyperlipidemia, arteriosclerosis, with metabolite identification and quantification and chronic hepatitis.6 It has also been traditionally in NMR spectra using a technique called “targeted used in food because of its unique flavor. Research profiling.”16 in vitro and in vivo showed that A. brasiliensis has The Chenomx NMR Suite is a commercial the ability to stimulate the body’s immune system metabolomics software package that uses targeted and show positive clinical results in colorectal and profiling and contains standard spectral libraries/ gynecological cancers.7,8 databases that include hundreds of metabolites.16 T. camphoratus (M. Zang & G.H. Su) Sheng Targeted profiling is based on a combination of H. Wu et al. (=Antrodia cinnamomea, Polyporales, mathematical modeling and a database of NMR Basidiomycetes) is a traditional medicinal mushroom spectral signatures of individual metabolites.17 NMR in Taiwan and is found only in the high mountainous spectroscopy is an efficient technology for metabo- regions of Taiwan, with Cinnamomum kanehirai as lomics because it is not destructive, does not rely on its single host. This fungus grows inside the hol- separating mixtures before analysis, and can concur- low trunk of the tree, where it is dark and humid. rently identify hundreds of metabolites in samples.16 The fruiting body is difficult to culture successfully, Once metabolites in biological samples are even in a well-controlled environment. Such unique identified and quantified, the information can be growth conditions not only enable this medicinal analyzed further using different methods of mul- mushroom to generate important compounds that tivariate analysis, including principal component are difficult to replicate in the laboratory, but they analysis (PCA). PCA is an unsupervised statisti- also make it one of the most expensive medicinal cal analysis technique that is used to find sets of fungi in the world. Extracts from this fungus have correlated variables that can describe differences been found to exhibit various anticancer activities, between data sets.17 In this study a combination of and its active components, such as triterpenoids and 1H NMR spectroscopy and PCA was used to iden- polysaccharides, have shown potent cytotoxicity tify the metabolites that differentiate A. brasiliensis, against cancer cells.9 In addition, T. camphoratus L. edodes, T. camphoratus, and G. lucidum from 2 has been used as a remedy for drug intoxication, different locations (one grown in Taiwan and other diarrhea, abdominal pain, hypertension, itchy skin, obtained in China). and liver cancer.10 The shiitake mushroom, L. edodes (Berk.) II. MATERIALS AND METHODS Singer (=Lentinula edodes, Marasmiaceae, Agarico- mycetes), is a traditional delicacy in Asia and can A. Samples and Preparation be ground to powder form and used as a flavoring to enhance the taste of foods and increase a product’s Four different species of mushrooms, includ- appeal.11 ing A. brasiliensis, T. camphoratus, G. lucidum, Nuclear magnetic resonance (NMR)–based and G. lucidum (China), were obtained from the metabolomics is a modern and effective analytical Biotechnology Center, Grape King Inc., Chungli technique used to identify and quantify components City, Taiwan; L. edodes was purchased from Q-Yo in different biological fluids and in extracts from Bio-Technology Farm, Pusin, Chunghua, Taiwan. tissues, including plants and mushrooms. NMR was All 5 mushrooms were obtained in the dried form discovered in 1938 by Isidor Rabi. Since then, NMR and randomly divided into 3 samples (~50 g each), spectroscopy has become a widely used analytical ground to powder, and stored in desiccators before technique.12 One important application of NMR- use. The moisture content of the freeze-dried based metabolomics is in identifying biomarkers samples was measured using an MF-50 Infrared of disease in human body fluids such as urine and Moisture Analyzer (A&D Co., Tokyo, Japan). serum.13–15 This complicated task can be effectively Each sample powder (1 g) was mixed with 10 mL International Journal of Medicinal Mushrooms Quantification of Metabolites in Medicinal Mushrooms Using Proton NMR Spectroscopy 415 ultrapure (type I) water obtained from a Synergy C. Statistical Analysis UV system (Millipore, Billerica, MA); they were preheated to 80°C, vortexed for 15 minutes, The metabolite concentrations were expressed as and centrifuged using a model 5810 centrifuge the mean ± standard error of 3 triplicate extraction/ (Eppendorf, Hauppauge, NY) at 2000 × g for 15 analyses and subjected to an analysis of variance for minutes. The collected supernatant was lyophi- a completely random design using a SAS version lized using a FreeZone 4.5-L Benchtop Freeze Dry 9.4 (SAS Institute, Inc., Cary, NC). To determine System (Labconco, Kansas City, MO), sealed, and the significance of differences between means, stored at −80°C until further analysis. Duncan multiple range tests at the level of α = 0.05 were used. PCA was carried out using Xlstat 2014 B. NMR Spectroscopy (Addinsoft, Brooklyn, NY), in which the computa- tions were performed using a Pearson correlation Each lyophilized extract was redissolved in 1 mL of matrix. PCA was elucidated to show the main dif-