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 KoreaCorrespondence to:
J. Microbiol. Biotechnol. 2019; 29(8): 1212-1220
Published August 28, 2019
Copyright © The Korean Society for Microbiology and Biotechnology.
Medicinal mushrooms have long been used in Asian culture and recently received considerable attention through discovery of useful natural products with biological activities .
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
Characteristics of the development periods of( C. militarisfruit 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
Fig. 2 and Table 1 showed that 44 metabolites were identified in 50% ethanol extracts from different development periods (
Regions of the 600 MHz 1H-NMR spectra identification of C. militarisfruit body. C. militarismetabolites list was described in Table 1. The chemical shift of 44 metabolites was indicated on the NMR spectrum.
PCA and PLS–DA plots of multivariate statistical analyses to differentiate the three development periods of( C. militarisfruit 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
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 . 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
Metabolic Changes and Cancer Cell Growth Inhibitory Activity of Development Periods of
C. militaris Fruiting Bodies
To understand the biochemical pathway of
Schematic diagram of the metabolic pathway and relative levels of the compounds inThis diagram was modified from pathways in (KEGG) database. ANOVA was performed to assess the statistical signiﬁcance of differences between groups (* C. militarisof 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
Growth inhibition of human hepatic carcinoma HepG2 cells by ethanol extraction ofValues 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 the C. militarisextraction 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
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
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.
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