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1H-NMR-Based Metabolic Profiling of Cordyceps militaris to Correlate the Development Process and Anti-Cancer Effect
1Translational Research Division, Biomedical Institute of Mycological Resource, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong University, Incheon 22000, Republic of Korea , 2College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea, 3Kainos Medicine, Inc., Seongnam 13488, Republic of Korea, 4Department of Oriental Medicine, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong University, Incheon 22000, Republic of Korea, 5Mushtech Cordyceps Institute, Hoengseonggun, Republic of Korea, 6Department of Microbiology, College of Medicine, Catholic Kwandong University, Gangneung 25601, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2019; 29(8): 1212-1220
Published August 28, 2019 https://doi.org/10.4014/jmb.1904.04004
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Medicinal mushrooms have long been used in Asian culture and recently received considerable attention through discovery of useful natural products with biological activities [1].
The effective biological activities of
Metabolomics is focused on the quantitative and qualitative high-throughput chemical profiling analysis of biological resources, which is merged and based on multivariate statistics data from nuclear magnetic resonance spectrometry (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and transform infrared spectroscopy (FTIR) [21, 22]. For identifying metabolic profiling of
The development periods of
-
Fig. 1.
Characteristics of the development periods of (C. militaris fruit body.A ) Growth period displays the developing perithecia on the fruit body at 6 weeks’ incubation. (B ) In the matured period at 8 weeks’ incubation, the majority of ascospores inside the perithecia were matured on the fruit bodies. (C ) In the aging period, after culturing for 10 weeks, fewer ascospores were observed inside the perithecia on the fruit bodies since most of the ascospores were released. Scale bar = 2 cm
In the present study, we performed metabolic profiling of
Materials and Methods
Solvents and Chemicals
We obtained cordycepin, dimethyl sulfoxide (DMSO), methanol-
Fruit Body Production of C. militaris and Sample Preparation for 1H-NMR Spectroscopy
1H-NMR Metabolomic Profiling
To extract intracellular metabolites for 1H-NMR spectroscopy, 100 mg of each powdered sample of
Data Preprocessing and Multivariate Statistical Analysis of 1H-NMR Data
1H-NMR data were processed and the peak assignment was conducted using MestReNOVA 6 version 6.0.4 (Mestrelab Research SL, Spain) and Chenomx NMR Suite software (version 8.2, Chenomx Inc.). Multivariate statistical analyses were performed by one-way ANOVA followed by a Tukey’s significant difference test using PASW Statistics 22 software (IBM, USA). Significance was determined with a
Quantitative Analysis of Cordycepin and β-Glucan
To quantify cordycepin in each of the developmental periods, 10 mg of cordycepin standard was dissolved in DMSO to prepare stock solution. It was serially diluted with DMSO and the concentration of the diluted solutions was determined using AZURA HPLC with UV system at 260 nm (KNAUER, Germany). For HPLC analysis, 10 mg of 50% ethanol extract of
MTT Cell Proliferation Assay
HepG2 is a hepatocellular carcinoma cell line and was purchased from American Type Culture Collection (ATCC, USA). HepG2 cells were cultured in DMEM medium supplemented with 10% FBS. The cells were incubated at 37ºC in 5% CO2 (SCI-165D, Water-Jacket System, Astec Co. Ltd., Japan). Before the treatment of
Results and Discussion
1H-NMR Metabolic Profiling and Multivariate Data Analysis of Development Periods of C. militaris
Fig. 2 and Table 1 showed that 44 metabolites were identified in 50% ethanol extracts from different development periods (
-
Table 1 . Metabolite assignments and chemical shifts of
C. militaris .No. Metabolite compound Chemical shift (ppm) 1 Leucine 0.95 (t, J = 6.5 Hz), 1.69 (m), 3.76 (m)2 Valine 0.98 (d, J = 6.87 Hz), 1.05 (d,J = 6.0 Hz)3 Ethanol 1.22 (t, J = 7.07 Hz), 3.62 (dd,J 1 = 9.88 Hz,J 2 = 3.80 Hz)4 Lactate 1.3 (d, J = 6.91 Hz)5 Threonine 1.34 (d, J = 6.58 Hz), 4.22 (m)6 Lysine 1.42 (m), 1.74 (m), 3.00 (t, J = 7.5 Hz)7 Alanine 1.46 (d, J = 8.31 Hz)8 Putrescine 1.78 (m) 9 Arginine 1.94 (m), 3.24 (t, J = 6.93 Hz), 3.76 (t,J = 6.11 Hz)10 Glutamate 2.02 (m), 2.14 (m), 2.38 (m) 11 Proline 2.02 (m), 2.06 (m), 4.06 (dd, J 1 = 8.63 Hz,J 2 = 6.42 Hz)12 Methionine 2.14 (s), 2.62 (t, J = 7.58 Hz)13 Acetoacetate 2.3 (s), 3.42 (s) 14 Pyruvate 2.34 (s) 15 Succinate 2.38 (s) 16 2-Oxoglutarate 2.43 (t, J = 6.95 Hz), 2.98 (t,J = 6.84 Hz)17 Glutamine 2.46 (m) 18 Citrate 2.5 (d, J = 15.92 Hz), 2.7 (d,J = 15.59 Hz)19 Methylamine 2.58 (s) 20 Aspartate 2.64 (m), 2.84 (m) 21 Asparagine 2.94 (m), 3.98 (m) 22 Tyrosine 3.02 (dd, J 1 = 4.76 Hz,J 2 = 12.48 Hz), 7.18 (d,J = 8.41 Hz)23 Malonate 3.1 (s) 24 Glucose 3.17 (d, J = 5.57 Hz), 3.19 (d,J = 6.50 Hz), 3.38 (m), 3.46 (m), 3.82 (m), 5.2 (d,J = 4.07 Hz)25 TMAO 3.22 (s) 26 Choline 3.22 (s), 4.02 (m) 27 Betaine 3.26 (s), 3.9 (s) 28 Glycine 3.54 (s) 29 Glycerol 3.54 (m), 3.62 (m) 30 Phenylacetate 3.54 (s), 7.64 (m), 7.38 (m) 31 Xylitol 3.62 (m), 3.72 (m) 32 Xylose 3.66 (m), 3.94 (m), 5.18 (d, J = 3.66 Hz)33 Mannitol 3.66 (dd, J 1 = 11.76 Hz,J 2 = 6.2 Hz), 3.84 (dd,J 1 = 11.87 Hz,J 2 = 2.8 Hz)34 Trehalose 3.85 (m), 5.18 (d, J = 3.8 Hz)35 Guanidoacetate 3.78 (s) 36 Trigonelline 4.46 (s), 8.1 (m), 8.86 (m), 9.14 (s) 37 Cordycepin 6.06 (d, J = 2.50 Hz), 8.27 (s), 8.41 (s)38 Maleate 6.02 (s) 39 Adenosine 6.06 (d, J = 2.50 Hz), 8.27 (s), 8.41 (s)40 Fumarate 6.5 (s) 41 4-Hydroxyphenylacetate 6.86 (d, J = 8.6 Hz), 7.18 (d,J = 8.65 Hz)42 Phenylalanine 7.32 (m), 7.36 (m), 7.42 (m) 43 Xanthine 7.94 (s) 44 Formate 8.44 (s)
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Fig. 2.
Regions of the 600 MHz 1H-NMR spectra identification of C. militaris fruit body.C. militaris metabolites list was described in Table 1. The chemical shift of 44 metabolites was indicated on the NMR spectrum.
-
Fig. 3.
PCA and PLS–DA plots of multivariate statistical analyses to differentiate the three development periods of (C. militaris fruit body.A ) PCA score plot based on two principal components (PC1 64.6% and PC2 24.1%). (B ) PLS-DA score plot based on two PLS components: PLS1 component 1 64.6% and PLS2 component 2 24.1% (R2Y = 0.995, Q2Y = 0.990, R2Y intercept = 0.279, Q2Y intercept = -0.314).
Quantification of Cordycepin and β-Glucan in C. militaris Extracts
We have previously reported that cordycepin was enriched in the senescence process of
-
Table 2 . Cordycepin content in
C. militaris extraction.Samples Cordycepin content, mg/g Growth period 13.356 ± 0.541 Mature period 29.014 ± 3.214 Aging period 39.674 ± 4.057 Values (means ± standard deviation, SD;
n = 3).
In addition to cordycepin, we conducted β-glucan assay because it is one of the most potent mushroom-derived substances and is also known to exhibit favorable biological properties including anti-cancer and immunoenhancement effects [28]. The results of the total glucan and β-glucan contents are presented in Table 3. The total glucan content in the growth, mature, and aging periods of
-
Table 3 . β
- glucan content inC. militaris extraction.Samples Glucan content (dry weight basis) % (w/w) Total-glucan β-glucan Growth period 66.91 ± 5.0 43.41 ± 2.44 Mature period 62.70 ± 4.2 39.84 ± 2.17 Aging period 70.53 ± 3.81 47.86 ± 2.42 Values (means ± standard deviation, SD;
n = 3).
Metabolic Changes and Cancer Cell Growth Inhibitory Activity of Development Periods of C. militaris Fruiting Bodies
To understand the biochemical pathway of
-
Fig. 4.
Schematic diagram of the metabolic pathway and relative levels of the compounds in This diagram was modified from pathways in (KEGG) database. ANOVA was performed to assess the statistical significance of differences between groups (*C. militaris of different three development periods.p < 0.05, **p < 0.01, ***p < 0.001, and ns; no significance). Results are presented as means ± standard deviation (SD). Red squares indicate the metabolites which were significanly different in their metabolites in aging period.
Similar to our research, Park
-
Fig. 5.
Growth inhibition of human hepatic carcinoma HepG2 cells by ethanol extraction of Values are expressed as means ± standard error (SE) of three experiments. *C. militaris . HepG2 cells were treated with various concentration (0, 5, 10, 20, 50, and 100 μg/ml) of theC. militaris extraction according to each of the development periods.p < 0.05, **p < 0.01 and ***p < 0.001 compared with control.
In the present study, we analyzed metabolic profiling and showed the anti-cancer effect of
Supplemental Materials
Acknowledgments
This research was supported by the Bio-industry Technology Development Program (316025-05) of IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries), the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (No. 2019R1A2C2005157), and a research fund from Catholic Kwandong University (CKURF 201805580001).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Elsayed EA, El Enshasy H, Wadaan MA, Aziz R. 2014. Mushrooms: a potential natural source of anti-inflammatory compounds for medical applications.
Mediators Inflamm. 2014 : 805841. - Sung GH, Hywel-Jones NL, Sung JM, Luangsa-Ard JJ, Shrestha B, Spatafora JW. 2007. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi.
Stud. Mycol. 57 : 5-59. - Shrestha B, Zhang W, Zhang Y, Liu X. 2012. The medicinal fungus Cordyceps militaris: research and development.
Mycological progress. 11 : 599-614. - Das SK, Masuda M, Sakurai A, Sakakibara M. 2010. Medicinal uses of the mushroom Cordyceps militaris: current state and prospects.
Fitoterapia 81 : 961-968. - Hur H. 2008. Chemical Ingredients of Cordyceps militaris.
Mycobiology 36 : 233-235. - Smiderle FR, Baggio CH, Borato DG, Santana-Filho AP, Sassaki GL, Iacomini M,
et al . 2014. Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1-->3)-beta-D-glucan.PLoS One. 9 : e110266. - Reis FS, Barros L, Calhelha RC, Ciric A, van Griensven LJ, Sokovic M,
et al . 2013. The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties.Food Chem. Toxicol. 62 : 91-98. - Jeong JW, Jin CY, Park C, Han MH, Kim GY, Moon SK,
et al . 2012. Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt.Int. J. Oncol. 40 : 1697-1704. - Jeong MH, Lee CM, Lee SW, Seo SY, Seo MJ, Kang BW,
et al . 2013. Cordycepin-enriched Cordyceps militaris induces immunomodulation and tumor growth delay in mouse-derived breast cancer.Oncol. Rep. 30 : 1996-2002. - Lee S, Lee HH, Kim J, Jung J, Moon A, Jeong CS,
et al . 2015. Anti-tumor effect of Cordyceps militaris in HCV-infected human hepatocarcinoma 7.5 cells.J. Microbiol. 53 : 468-474. - Zhu ZY, Pang W, Li YY, Ge XR, Chen LJ, Liu XC,
et al . 2014. Effect of ultrasonic treatment on structure and antitumor activity of mycelial polysaccharides from Cordyceps gunnii.Carbohydr. Polym. 114 : 12-20. - Lee EJ, Kim WJ, Moon SK. 2010. Cordycepin suppresses TNF-alpha-induced invasion, migration and matrix metalloproteinase-9 expression in human bladder cancer cells.
Phytother. Res. 24 : 1755-1761. - Cunningham KG, Manson W, Spring FS, Hutchinson SA. 1950. Cordycepin, a metabolic product isolated from cultures of Cordyceps militaris (Linn.) Link.
Nature 166 : 949. - Yoshikawa N, Yamada S, Takeuchi C, Kagota S, Shinozuka K, Kunitomo M,
et al . 2008. Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.Naunyn Schmiedebergs Arch. Pharmacol. 377 : 591-595. - Song J, Wang Y, Teng M, Cai G, Xu H, Guo H,
et al . 2015. Studies on the antifatigue activities of cordyceps militaris fruit body extract in mouse model.Evid Based Complement Alternat. Med. 2015 : 174616. - Shao LW, Huang LH, Yan S, Jin JD, Ren SY. 2016. Cordycepin induces apoptosis in human liver cancer HepG2 cells through extrinsic and intrinsic signaling pathways.
Oncol Lett. 12 : 995-1000. - Tuli HS, Sandhu SS, Sharma AK. 2014. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin.
3 Biotech. 4 : 1-12. - Zheng P, Xia Y, Xiao G, Xiong C, Hu X, Zhang S,
et al . 2011. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine.Genome Biol. 12 : R116. - Xia Y, Luo F, Shang Y, Chen P, Lu Y, Wang C. 2017. Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin.
Cell Chem Biol. 24 : 1479-1489. - Hasko G, Cronstein BN. 2004. Adenosine: an endogenous regulator of innate immunity.
Trends Immunol. 25 : 33-39. - Wang JH, Byun J, Pennathur S. 2010. Analytical approaches to metabolomics and applications to systems biology.
Semin Nephrol. 30 : 500-511. - Corte L, Tiecco M, Roscini L, De Vincenzi S, Colabella C, Germani R,
et al . 2015. FTIR metabolomic fingerprint reveals different modes of action exerted by structural variants of N-alkyltropinium bromide surfactants onEscherichia coli and Listeria innocua cells.PLoS One 10 : e0115275. - Park SJ, Hyun S-H, Suh HW, Lee S-Y, Sung G-H, Kim SH,
et al . 2013. Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1 H nuclear magnetic resonance spectroscopy.Metabolomics 9 : 236-246. - Hyun SH, Lee SY, Sung GH, Kim SH, Choi HK. 2013. Metabolic profiles and free radical scavenging activity of Cordyceps bassiana fruiting bodies according to developmental stage.
PLoS One 8 : e73065. - Oh J, Yoon DH, Shrestha B, Choi HK, Sung GH. 2019. Metabolomic profiling reveals enrichment of cordycepin in senescence process of Cordyceps militaris fruit bodies.
J. Microbiol. 57 : 54-63. - Shrestha B, Han SK, Sung JM, Sung GH. 2012. Fruiting body formation of cordyceps militaris from multi-ascospore isolates and their single ascospore progeny strains.
Mycobiology 40 : 100-106. - Lee SG, Hyun S-H, Sung G-H, Choi H-K. 2014. Simple and rapid determination of cordycepin in cordyceps militaris fruiting bodies by quantitative nuclear magnetic resonance spectroscopy.
Analytical Lett. 47 : 1031-1042. - Chan GC-F, Chan WK, Sze DM-Y. 2009. The effects of β-glucan on human immune and cancer cells.
J. Hematol. Oncol. 2 : 25. - Cho J-H, Lee J-Y, Lee M-J, Oh H-N, Kang D-H, Jhune C-S. 2013. Comparative analysis of useful β-glucan and polyphenol in the fruiting bodies of Ganoderma spp.
J. Mushrooms 11 : 164-170. - Kang C, Wen T-C, Kang J-C, Meng Z-B, Li G-R, Hyde KD. 2014. Optimization of large-scale culture conditions for the production of cordycepin with Cordyceps militaris by liquid static culture.
ScientificWorldJournal. 2014 : 510627. - Papaspyridi L-M, Zerva A, Topakas E. 2018. Biocatalytic synthesis of fungal β-glucans.
Catalysts 8 : 274. - Huang W, Haferkamp I, Lepetit B, Molchanova M, Hou S, Jeblick W,
et al . 2018. Reduced vacuolar beta-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.Proc. Natl. Acad. Sci. USA 115 : 4791-4796. - Park SE, Kim J, Lee YW, Yoo HS, Cho CK. 2009. Antitumor activity of water extracts from Cordyceps militaris in NCI-H460 cell xenografted nude mice.
J. Acupunct. Meridian Stud. 2 : 294-300. - Lee HH, Lee S, Lee K, Shin YS, Kang H, Cho H. 2015. Anti-cancer effect of Cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis.
Daru 23 : 35. - Jin Y, Meng X, Qiu Z, Su Y, Yu P, Qu P. 2018. Anti-tumor and anti-metastatic roles of cordycepin, one bioactive compound of Cordyceps militaris.
Saudi J. Biol. Sci. 25 : 991-995. - Karaduman D, Eren B, Keles ON. 2010. The protective effect of beta-1,3-D-glucan on taxol-induced hepatotoxicity: a histopathological and stereological study.
Drug Chem. Toxicol. 33 : 8-16. - Yoon TJ, Koppula S, Lee KH. 2013. The effects of beta-glucans on cancer metastasis.
Anticancer Agents Med. Chem. 13 : 699-708. - Cui ZY, Park SJ, Jo E, Hwang IH, Lee KB, Kim SW,
et al . 2018. Cordycepin induces apoptosis of human ovarian cancer cells by inhibiting CCL5-mediated Akt/NF-kappaB signaling pathway.Cell Death Discov. 4 : 62. - Jafaar ZM, Litchfield LM, Ivanova MM, Radde BN, Al-Rayyan N, Klinge CM. 2014. beta-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression.
Int. J. Oncol. 44 : 1365-1375. - Shomori K, Yamamoto M, Arifuku I, Teramachi K, Ito H. 2009. Antitumor effects of a water-soluble extract from Maitake (Grifola frondosa) on human gastric cancer cell lines.
Oncol. Rep. 22 : 615-620.
Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2019; 29(8): 1212-1220
Published online August 28, 2019 https://doi.org/10.4014/jmb.1904.04004
Copyright © The Korean Society for Microbiology and Biotechnology.
1H-NMR-Based Metabolic Profiling of Cordyceps militaris to Correlate the Development Process and Anti-Cancer Effect
Junsang Oh 1, 2, Eunhyun Choi 3, Deok-Hyo Yoon 1, Tae-Yong Park 1, 4, Bhushan Shrestha 5, Hyung-Kyoon Choi 2 and Gi-Ho Sung 1, 6*
1Translational Research Division, Biomedical Institute of Mycological Resource, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong University, Incheon 22000, Republic of Korea , 2College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea, 3Kainos Medicine, Inc., Seongnam 13488, Republic of Korea, 4Department of Oriental Medicine, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong University, Incheon 22000, Republic of Korea, 5Mushtech Cordyceps Institute, Hoengseonggun, Republic of Korea, 6Department of Microbiology, College of Medicine, Catholic Kwandong University, Gangneung 25601, Republic of Korea
Correspondence to:Gi-Ho Sung
sung97330@gmail.com
Abstract
The study of metabolomics in natural products using the diverse analytical instruments including GC-MS, LC-MS, and NMR is useful for the exploration of physiological and biological effects and the investigation of drug discovery and health functional foods. Cordyceps militaris has been very attractive to natural medicine as a traditional Chinese medicine, due to its various bioactive properties including anti-cancer and anti-oxidant effects. In this study, we analyzed the metabolite profile in 50% ethanol extracts of C. militaris fruit bodies from three development periods (growth period, matured period, and aging period) using 1H-NMR, and identified 44 metabolites, which are classified as 16 amino acids, 10 organic acids, 5 carbohydrates, 3 nucleotide derivatives, and 10 other compounds. Among the three development periods of the C. militaris fruit body, the aging period showed significantly higher levels of metabolites including cordycepin, mannitol (cordycepic acid), and β-glucan. Interestingly, these bioactive metabolites are positively correlated with antitumor growth effect; the extract of the aging period showed significant inhibition of HepG2 hepatic cancer cell proliferation. These results showed that the aging period during the development of C. militaris fruit bodies was more highly enriched with bioactive metabolites that are associated with cancer cell growth inhibition.
Keywords: Cordyceps militaris, metabolomics, cordycepin, &beta,-glucan, nuclear magnetic resonance, anti-cancer effect
Introduction
Medicinal mushrooms have long been used in Asian culture and recently received considerable attention through discovery of useful natural products with biological activities [1].
The effective biological activities of
Metabolomics is focused on the quantitative and qualitative high-throughput chemical profiling analysis of biological resources, which is merged and based on multivariate statistics data from nuclear magnetic resonance spectrometry (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and transform infrared spectroscopy (FTIR) [21, 22]. For identifying metabolic profiling of
The development periods of
-
Figure 1.
Characteristics of the development periods of (C. militaris fruit body.A ) Growth period displays the developing perithecia on the fruit body at 6 weeks’ incubation. (B ) In the matured period at 8 weeks’ incubation, the majority of ascospores inside the perithecia were matured on the fruit bodies. (C ) In the aging period, after culturing for 10 weeks, fewer ascospores were observed inside the perithecia on the fruit bodies since most of the ascospores were released. Scale bar = 2 cm
In the present study, we performed metabolic profiling of
Materials and Methods
Solvents and Chemicals
We obtained cordycepin, dimethyl sulfoxide (DMSO), methanol-
Fruit Body Production of C. militaris and Sample Preparation for 1H-NMR Spectroscopy
1H-NMR Metabolomic Profiling
To extract intracellular metabolites for 1H-NMR spectroscopy, 100 mg of each powdered sample of
Data Preprocessing and Multivariate Statistical Analysis of 1H-NMR Data
1H-NMR data were processed and the peak assignment was conducted using MestReNOVA 6 version 6.0.4 (Mestrelab Research SL, Spain) and Chenomx NMR Suite software (version 8.2, Chenomx Inc.). Multivariate statistical analyses were performed by one-way ANOVA followed by a Tukey’s significant difference test using PASW Statistics 22 software (IBM, USA). Significance was determined with a
Quantitative Analysis of Cordycepin and β-Glucan
To quantify cordycepin in each of the developmental periods, 10 mg of cordycepin standard was dissolved in DMSO to prepare stock solution. It was serially diluted with DMSO and the concentration of the diluted solutions was determined using AZURA HPLC with UV system at 260 nm (KNAUER, Germany). For HPLC analysis, 10 mg of 50% ethanol extract of
MTT Cell Proliferation Assay
HepG2 is a hepatocellular carcinoma cell line and was purchased from American Type Culture Collection (ATCC, USA). HepG2 cells were cultured in DMEM medium supplemented with 10% FBS. The cells were incubated at 37ºC in 5% CO2 (SCI-165D, Water-Jacket System, Astec Co. Ltd., Japan). Before the treatment of
Results and Discussion
1H-NMR Metabolic Profiling and Multivariate Data Analysis of Development Periods of C. militaris
Fig. 2 and Table 1 showed that 44 metabolites were identified in 50% ethanol extracts from different development periods (
-
Table 1 . Metabolite assignments and chemical shifts of
C. militaris ..No. Metabolite compound Chemical shift (ppm) 1 Leucine 0.95 (t, J = 6.5 Hz), 1.69 (m), 3.76 (m)2 Valine 0.98 (d, J = 6.87 Hz), 1.05 (d,J = 6.0 Hz)3 Ethanol 1.22 (t, J = 7.07 Hz), 3.62 (dd,J 1 = 9.88 Hz,J 2 = 3.80 Hz)4 Lactate 1.3 (d, J = 6.91 Hz)5 Threonine 1.34 (d, J = 6.58 Hz), 4.22 (m)6 Lysine 1.42 (m), 1.74 (m), 3.00 (t, J = 7.5 Hz)7 Alanine 1.46 (d, J = 8.31 Hz)8 Putrescine 1.78 (m) 9 Arginine 1.94 (m), 3.24 (t, J = 6.93 Hz), 3.76 (t,J = 6.11 Hz)10 Glutamate 2.02 (m), 2.14 (m), 2.38 (m) 11 Proline 2.02 (m), 2.06 (m), 4.06 (dd, J 1 = 8.63 Hz,J 2 = 6.42 Hz)12 Methionine 2.14 (s), 2.62 (t, J = 7.58 Hz)13 Acetoacetate 2.3 (s), 3.42 (s) 14 Pyruvate 2.34 (s) 15 Succinate 2.38 (s) 16 2-Oxoglutarate 2.43 (t, J = 6.95 Hz), 2.98 (t,J = 6.84 Hz)17 Glutamine 2.46 (m) 18 Citrate 2.5 (d, J = 15.92 Hz), 2.7 (d,J = 15.59 Hz)19 Methylamine 2.58 (s) 20 Aspartate 2.64 (m), 2.84 (m) 21 Asparagine 2.94 (m), 3.98 (m) 22 Tyrosine 3.02 (dd, J 1 = 4.76 Hz,J 2 = 12.48 Hz), 7.18 (d,J = 8.41 Hz)23 Malonate 3.1 (s) 24 Glucose 3.17 (d, J = 5.57 Hz), 3.19 (d,J = 6.50 Hz), 3.38 (m), 3.46 (m), 3.82 (m), 5.2 (d,J = 4.07 Hz)25 TMAO 3.22 (s) 26 Choline 3.22 (s), 4.02 (m) 27 Betaine 3.26 (s), 3.9 (s) 28 Glycine 3.54 (s) 29 Glycerol 3.54 (m), 3.62 (m) 30 Phenylacetate 3.54 (s), 7.64 (m), 7.38 (m) 31 Xylitol 3.62 (m), 3.72 (m) 32 Xylose 3.66 (m), 3.94 (m), 5.18 (d, J = 3.66 Hz)33 Mannitol 3.66 (dd, J 1 = 11.76 Hz,J 2 = 6.2 Hz), 3.84 (dd,J 1 = 11.87 Hz,J 2 = 2.8 Hz)34 Trehalose 3.85 (m), 5.18 (d, J = 3.8 Hz)35 Guanidoacetate 3.78 (s) 36 Trigonelline 4.46 (s), 8.1 (m), 8.86 (m), 9.14 (s) 37 Cordycepin 6.06 (d, J = 2.50 Hz), 8.27 (s), 8.41 (s)38 Maleate 6.02 (s) 39 Adenosine 6.06 (d, J = 2.50 Hz), 8.27 (s), 8.41 (s)40 Fumarate 6.5 (s) 41 4-Hydroxyphenylacetate 6.86 (d, J = 8.6 Hz), 7.18 (d,J = 8.65 Hz)42 Phenylalanine 7.32 (m), 7.36 (m), 7.42 (m) 43 Xanthine 7.94 (s) 44 Formate 8.44 (s)
-
Figure 2.
Regions of the 600 MHz 1H-NMR spectra identification of C. militaris fruit body.C. militaris metabolites list was described in Table 1. The chemical shift of 44 metabolites was indicated on the NMR spectrum.
-
Figure 3.
PCA and PLS–DA plots of multivariate statistical analyses to differentiate the three development periods of (C. militaris fruit body.A ) PCA score plot based on two principal components (PC1 64.6% and PC2 24.1%). (B ) PLS-DA score plot based on two PLS components: PLS1 component 1 64.6% and PLS2 component 2 24.1% (R2Y = 0.995, Q2Y = 0.990, R2Y intercept = 0.279, Q2Y intercept = -0.314).
Quantification of Cordycepin and β-Glucan in C. militaris Extracts
We have previously reported that cordycepin was enriched in the senescence process of
-
Table 2 . Cordycepin content in
C. militaris extraction..Samples Cordycepin content, mg/g Growth period 13.356 ± 0.541 Mature period 29.014 ± 3.214 Aging period 39.674 ± 4.057 Values (means ± standard deviation, SD;
n = 3)..
In addition to cordycepin, we conducted β-glucan assay because it is one of the most potent mushroom-derived substances and is also known to exhibit favorable biological properties including anti-cancer and immunoenhancement effects [28]. The results of the total glucan and β-glucan contents are presented in Table 3. The total glucan content in the growth, mature, and aging periods of
-
Table 3 . β
- glucan content inC. militaris extraction..Samples Glucan content (dry weight basis) % (w/w) Total-glucan β-glucan Growth period 66.91 ± 5.0 43.41 ± 2.44 Mature period 62.70 ± 4.2 39.84 ± 2.17 Aging period 70.53 ± 3.81 47.86 ± 2.42 Values (means ± standard deviation, SD;
n = 3)..
Metabolic Changes and Cancer Cell Growth Inhibitory Activity of Development Periods of C. militaris Fruiting Bodies
To understand the biochemical pathway of
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Figure 4.
Schematic diagram of the metabolic pathway and relative levels of the compounds in This diagram was modified from pathways in (KEGG) database. ANOVA was performed to assess the statistical significance of differences between groups (*C. militaris of different three development periods.p < 0.05, **p < 0.01, ***p < 0.001, and ns; no significance). Results are presented as means ± standard deviation (SD). Red squares indicate the metabolites which were significanly different in their metabolites in aging period.
Similar to our research, Park
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Figure 5.
Growth inhibition of human hepatic carcinoma HepG2 cells by ethanol extraction of Values are expressed as means ± standard error (SE) of three experiments. *C. militaris . HepG2 cells were treated with various concentration (0, 5, 10, 20, 50, and 100 μg/ml) of theC. militaris extraction according to each of the development periods.p < 0.05, **p < 0.01 and ***p < 0.001 compared with control.
In the present study, we analyzed metabolic profiling and showed the anti-cancer effect of
Supplemental Materials
Acknowledgments
This research was supported by the Bio-industry Technology Development Program (316025-05) of IPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries), the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (No. 2019R1A2C2005157), and a research fund from Catholic Kwandong University (CKURF 201805580001).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.
Fig 2.
Fig 3.
Fig 4.
Fig 5.
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Table 1 . Metabolite assignments and chemical shifts of
C. militaris ..No. Metabolite compound Chemical shift (ppm) 1 Leucine 0.95 (t, J = 6.5 Hz), 1.69 (m), 3.76 (m)2 Valine 0.98 (d, J = 6.87 Hz), 1.05 (d,J = 6.0 Hz)3 Ethanol 1.22 (t, J = 7.07 Hz), 3.62 (dd,J 1 = 9.88 Hz,J 2 = 3.80 Hz)4 Lactate 1.3 (d, J = 6.91 Hz)5 Threonine 1.34 (d, J = 6.58 Hz), 4.22 (m)6 Lysine 1.42 (m), 1.74 (m), 3.00 (t, J = 7.5 Hz)7 Alanine 1.46 (d, J = 8.31 Hz)8 Putrescine 1.78 (m) 9 Arginine 1.94 (m), 3.24 (t, J = 6.93 Hz), 3.76 (t,J = 6.11 Hz)10 Glutamate 2.02 (m), 2.14 (m), 2.38 (m) 11 Proline 2.02 (m), 2.06 (m), 4.06 (dd, J 1 = 8.63 Hz,J 2 = 6.42 Hz)12 Methionine 2.14 (s), 2.62 (t, J = 7.58 Hz)13 Acetoacetate 2.3 (s), 3.42 (s) 14 Pyruvate 2.34 (s) 15 Succinate 2.38 (s) 16 2-Oxoglutarate 2.43 (t, J = 6.95 Hz), 2.98 (t,J = 6.84 Hz)17 Glutamine 2.46 (m) 18 Citrate 2.5 (d, J = 15.92 Hz), 2.7 (d,J = 15.59 Hz)19 Methylamine 2.58 (s) 20 Aspartate 2.64 (m), 2.84 (m) 21 Asparagine 2.94 (m), 3.98 (m) 22 Tyrosine 3.02 (dd, J 1 = 4.76 Hz,J 2 = 12.48 Hz), 7.18 (d,J = 8.41 Hz)23 Malonate 3.1 (s) 24 Glucose 3.17 (d, J = 5.57 Hz), 3.19 (d,J = 6.50 Hz), 3.38 (m), 3.46 (m), 3.82 (m), 5.2 (d,J = 4.07 Hz)25 TMAO 3.22 (s) 26 Choline 3.22 (s), 4.02 (m) 27 Betaine 3.26 (s), 3.9 (s) 28 Glycine 3.54 (s) 29 Glycerol 3.54 (m), 3.62 (m) 30 Phenylacetate 3.54 (s), 7.64 (m), 7.38 (m) 31 Xylitol 3.62 (m), 3.72 (m) 32 Xylose 3.66 (m), 3.94 (m), 5.18 (d, J = 3.66 Hz)33 Mannitol 3.66 (dd, J 1 = 11.76 Hz,J 2 = 6.2 Hz), 3.84 (dd,J 1 = 11.87 Hz,J 2 = 2.8 Hz)34 Trehalose 3.85 (m), 5.18 (d, J = 3.8 Hz)35 Guanidoacetate 3.78 (s) 36 Trigonelline 4.46 (s), 8.1 (m), 8.86 (m), 9.14 (s) 37 Cordycepin 6.06 (d, J = 2.50 Hz), 8.27 (s), 8.41 (s)38 Maleate 6.02 (s) 39 Adenosine 6.06 (d, J = 2.50 Hz), 8.27 (s), 8.41 (s)40 Fumarate 6.5 (s) 41 4-Hydroxyphenylacetate 6.86 (d, J = 8.6 Hz), 7.18 (d,J = 8.65 Hz)42 Phenylalanine 7.32 (m), 7.36 (m), 7.42 (m) 43 Xanthine 7.94 (s) 44 Formate 8.44 (s)
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Table 2 . Cordycepin content in
C. militaris extraction..Samples Cordycepin content, mg/g Growth period 13.356 ± 0.541 Mature period 29.014 ± 3.214 Aging period 39.674 ± 4.057 Values (means ± standard deviation, SD;
n = 3)..
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Table 3 . β
- glucan content inC. militaris extraction..Samples Glucan content (dry weight basis) % (w/w) Total-glucan β-glucan Growth period 66.91 ± 5.0 43.41 ± 2.44 Mature period 62.70 ± 4.2 39.84 ± 2.17 Aging period 70.53 ± 3.81 47.86 ± 2.42 Values (means ± standard deviation, SD;
n = 3)..
References
- Elsayed EA, El Enshasy H, Wadaan MA, Aziz R. 2014. Mushrooms: a potential natural source of anti-inflammatory compounds for medical applications.
Mediators Inflamm. 2014 : 805841. - Sung GH, Hywel-Jones NL, Sung JM, Luangsa-Ard JJ, Shrestha B, Spatafora JW. 2007. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi.
Stud. Mycol. 57 : 5-59. - Shrestha B, Zhang W, Zhang Y, Liu X. 2012. The medicinal fungus Cordyceps militaris: research and development.
Mycological progress. 11 : 599-614. - Das SK, Masuda M, Sakurai A, Sakakibara M. 2010. Medicinal uses of the mushroom Cordyceps militaris: current state and prospects.
Fitoterapia 81 : 961-968. - Hur H. 2008. Chemical Ingredients of Cordyceps militaris.
Mycobiology 36 : 233-235. - Smiderle FR, Baggio CH, Borato DG, Santana-Filho AP, Sassaki GL, Iacomini M,
et al . 2014. Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1-->3)-beta-D-glucan.PLoS One. 9 : e110266. - Reis FS, Barros L, Calhelha RC, Ciric A, van Griensven LJ, Sokovic M,
et al . 2013. The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties.Food Chem. Toxicol. 62 : 91-98. - Jeong JW, Jin CY, Park C, Han MH, Kim GY, Moon SK,
et al . 2012. Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt.Int. J. Oncol. 40 : 1697-1704. - Jeong MH, Lee CM, Lee SW, Seo SY, Seo MJ, Kang BW,
et al . 2013. Cordycepin-enriched Cordyceps militaris induces immunomodulation and tumor growth delay in mouse-derived breast cancer.Oncol. Rep. 30 : 1996-2002. - Lee S, Lee HH, Kim J, Jung J, Moon A, Jeong CS,
et al . 2015. Anti-tumor effect of Cordyceps militaris in HCV-infected human hepatocarcinoma 7.5 cells.J. Microbiol. 53 : 468-474. - Zhu ZY, Pang W, Li YY, Ge XR, Chen LJ, Liu XC,
et al . 2014. Effect of ultrasonic treatment on structure and antitumor activity of mycelial polysaccharides from Cordyceps gunnii.Carbohydr. Polym. 114 : 12-20. - Lee EJ, Kim WJ, Moon SK. 2010. Cordycepin suppresses TNF-alpha-induced invasion, migration and matrix metalloproteinase-9 expression in human bladder cancer cells.
Phytother. Res. 24 : 1755-1761. - Cunningham KG, Manson W, Spring FS, Hutchinson SA. 1950. Cordycepin, a metabolic product isolated from cultures of Cordyceps militaris (Linn.) Link.
Nature 166 : 949. - Yoshikawa N, Yamada S, Takeuchi C, Kagota S, Shinozuka K, Kunitomo M,
et al . 2008. Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.Naunyn Schmiedebergs Arch. Pharmacol. 377 : 591-595. - Song J, Wang Y, Teng M, Cai G, Xu H, Guo H,
et al . 2015. Studies on the antifatigue activities of cordyceps militaris fruit body extract in mouse model.Evid Based Complement Alternat. Med. 2015 : 174616. - Shao LW, Huang LH, Yan S, Jin JD, Ren SY. 2016. Cordycepin induces apoptosis in human liver cancer HepG2 cells through extrinsic and intrinsic signaling pathways.
Oncol Lett. 12 : 995-1000. - Tuli HS, Sandhu SS, Sharma AK. 2014. Pharmacological and therapeutic potential of Cordyceps with special reference to Cordycepin.
3 Biotech. 4 : 1-12. - Zheng P, Xia Y, Xiao G, Xiong C, Hu X, Zhang S,
et al . 2011. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine.Genome Biol. 12 : R116. - Xia Y, Luo F, Shang Y, Chen P, Lu Y, Wang C. 2017. Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin.
Cell Chem Biol. 24 : 1479-1489. - Hasko G, Cronstein BN. 2004. Adenosine: an endogenous regulator of innate immunity.
Trends Immunol. 25 : 33-39. - Wang JH, Byun J, Pennathur S. 2010. Analytical approaches to metabolomics and applications to systems biology.
Semin Nephrol. 30 : 500-511. - Corte L, Tiecco M, Roscini L, De Vincenzi S, Colabella C, Germani R,
et al . 2015. FTIR metabolomic fingerprint reveals different modes of action exerted by structural variants of N-alkyltropinium bromide surfactants onEscherichia coli and Listeria innocua cells.PLoS One 10 : e0115275. - Park SJ, Hyun S-H, Suh HW, Lee S-Y, Sung G-H, Kim SH,
et al . 2013. Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1 H nuclear magnetic resonance spectroscopy.Metabolomics 9 : 236-246. - Hyun SH, Lee SY, Sung GH, Kim SH, Choi HK. 2013. Metabolic profiles and free radical scavenging activity of Cordyceps bassiana fruiting bodies according to developmental stage.
PLoS One 8 : e73065. - Oh J, Yoon DH, Shrestha B, Choi HK, Sung GH. 2019. Metabolomic profiling reveals enrichment of cordycepin in senescence process of Cordyceps militaris fruit bodies.
J. Microbiol. 57 : 54-63. - Shrestha B, Han SK, Sung JM, Sung GH. 2012. Fruiting body formation of cordyceps militaris from multi-ascospore isolates and their single ascospore progeny strains.
Mycobiology 40 : 100-106. - Lee SG, Hyun S-H, Sung G-H, Choi H-K. 2014. Simple and rapid determination of cordycepin in cordyceps militaris fruiting bodies by quantitative nuclear magnetic resonance spectroscopy.
Analytical Lett. 47 : 1031-1042. - Chan GC-F, Chan WK, Sze DM-Y. 2009. The effects of β-glucan on human immune and cancer cells.
J. Hematol. Oncol. 2 : 25. - Cho J-H, Lee J-Y, Lee M-J, Oh H-N, Kang D-H, Jhune C-S. 2013. Comparative analysis of useful β-glucan and polyphenol in the fruiting bodies of Ganoderma spp.
J. Mushrooms 11 : 164-170. - Kang C, Wen T-C, Kang J-C, Meng Z-B, Li G-R, Hyde KD. 2014. Optimization of large-scale culture conditions for the production of cordycepin with Cordyceps militaris by liquid static culture.
ScientificWorldJournal. 2014 : 510627. - Papaspyridi L-M, Zerva A, Topakas E. 2018. Biocatalytic synthesis of fungal β-glucans.
Catalysts 8 : 274. - Huang W, Haferkamp I, Lepetit B, Molchanova M, Hou S, Jeblick W,
et al . 2018. Reduced vacuolar beta-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.Proc. Natl. Acad. Sci. USA 115 : 4791-4796. - Park SE, Kim J, Lee YW, Yoo HS, Cho CK. 2009. Antitumor activity of water extracts from Cordyceps militaris in NCI-H460 cell xenografted nude mice.
J. Acupunct. Meridian Stud. 2 : 294-300. - Lee HH, Lee S, Lee K, Shin YS, Kang H, Cho H. 2015. Anti-cancer effect of Cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis.
Daru 23 : 35. - Jin Y, Meng X, Qiu Z, Su Y, Yu P, Qu P. 2018. Anti-tumor and anti-metastatic roles of cordycepin, one bioactive compound of Cordyceps militaris.
Saudi J. Biol. Sci. 25 : 991-995. - Karaduman D, Eren B, Keles ON. 2010. The protective effect of beta-1,3-D-glucan on taxol-induced hepatotoxicity: a histopathological and stereological study.
Drug Chem. Toxicol. 33 : 8-16. - Yoon TJ, Koppula S, Lee KH. 2013. The effects of beta-glucans on cancer metastasis.
Anticancer Agents Med. Chem. 13 : 699-708. - Cui ZY, Park SJ, Jo E, Hwang IH, Lee KB, Kim SW,
et al . 2018. Cordycepin induces apoptosis of human ovarian cancer cells by inhibiting CCL5-mediated Akt/NF-kappaB signaling pathway.Cell Death Discov. 4 : 62. - Jafaar ZM, Litchfield LM, Ivanova MM, Radde BN, Al-Rayyan N, Klinge CM. 2014. beta-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression.
Int. J. Oncol. 44 : 1365-1375. - Shomori K, Yamamoto M, Arifuku I, Teramachi K, Ito H. 2009. Antitumor effects of a water-soluble extract from Maitake (Grifola frondosa) on human gastric cancer cell lines.
Oncol. Rep. 22 : 615-620.