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1H NMR spectroscopy-based metabolic profiling of Ophiocordyceps sinensis and Cordyceps militaris of water-boiled and 50% ethanol-soaked extracts

ABSTRACT: Introduction Ophiocordyceps sinensis, a well-known Chinese complementary herb, is a rare and valuable therapeutic resource. Cordyceps militaris (C. militaris) is a commonly used substitute for O. sinensis. A metabolomic-based approach for exploring the similarities and differences in the metabolites of O. sinensis and C. militaris in water-boiled and 50% ethanol-soaked extracts is of great significance. Objectives To determine a vital role of extraction methodologies in influencing the metabolic composition of herbs, 1HNMR-based profiling was used to characterize the metabolic fingerprints of O. sinensis and C. militaris. Methods To make a distinction between the global metabolite profiling of O. sinensis and C. militaris extracts obtained from either the water-boiled or 50% ethanol-soaked methods, we screened the herbs samples using 1HNMR-based metabolic fingerprints combined with multivariate statistical analysis. Results This study revealed that a total of 43 (82.69% of 52) metabolites were detectable in both O. sinensis and C. militaris. According to the variable importance in projection (VIP) value and p-value from the Mann-Whitney test, 7 metabolites (alanine, aspartate, glutamate, mannitol, ornithine, serine, and trehalose) differed between O. sinensis and C. militaris. Arginine, glucose, putrescine, pyroglutamate, betaine, O-phosphocholine, and xylose differed significantly between the water-boiled and 50% ethanol-soaked methods used to prepare the herb extracts. Conclusion A total of 52 primary metabolites were identified and quantified from O. sinensis and C. militaris samples. The study suggests that a water-boiled extraction is much faster method and strongly recommended over the 50% ethanol-soaked method for both O. sinensis and C. militaris.

ORGANISM(S): Ophiocordyceps Sinensis Cordyceps Militaris Ophiocordyceps Ophiocordyceps Sinensis;cordyceps Militaris Cordyceps

TISSUE(S): Fungal Cells

SUBMITTER: Xin Zhong

PROVIDER: ST001189 | MetabolomicsWorkbench | Sun Apr 28 00:00:00 BST 2019

REPOSITORIES: MetabolomicsWorkbench

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Project description:Samples for metabolomic analysis were prepared as follows. A 200 uL aliquot of bacterial cultures or control medium (prepared, as described in the above section) was mixed with 800 uL of 99.5 percent ethanol (Fisher Scientific) and subjected to 3 rounds of freeze thaw cycles: 10 minutes at -80C, 8 minutes at room temperature. Next, samples were spun down for 10 minutes at 16,000g, 4C to remove cell debris, and supernatants were transferred to new tubes. Ethanol supernatants were stored at -80C until further extraction. Each ethanol extraction was performed in two technical duplicates. The solid phase extraction (SPE) of the ethanol extracts was performed with the Oasis HLB 96-well plate (30 mg Sorbent per Well, 30 um Particle Size) (Waters Corporation) set up with a vacuum pump manifold. Each well of the plate was washed with 700 uL of 100 percent HPLC-grade methanol and equilibrated with 700 uL of HPLC-grade water. Next, 400 uL of ethanol extracts were added to the wells and allowed to slowly elute. Wells were next washed with 800 uL of 5 percent methanol in water. Metabolites from bacterial cultures or control media were eluted with 200 uL of 100 percent methanol. Vacuum up to 20 psi was applied for the wells that did not elute within one hour. The collected eluted metabolites were stored at -20 until the HPLC-MS analysis. The HPLC-MS analysis of extracted metabolites was performed with the use of a Dionex UltiMate 3000 UHPLC system (Fisher Scientific) coupled to a Bruker impact HD quadrupole time-of-flight mass spectrometer (Bruker). The chromatographic separation was performed on a Kinetex C18 1.7 um, 100A UHPLC column (50 mm x 2.1 mm) (Phenomenex), held at 40C during analysis. 5 uL of each sample was injected. Mobile phase A was water, 0.1 percent v:v formic acid, and mobile phase B was acetonitrile, 0.1 percent v:v formic acid. The solvent gradient table was set as follows: 2 percent B, increased to 10 percent B over 0.2 min, then to 100 percent B at 12 min, held at 100 percent B for 1.5 min, decreased back to 2 percent B in 0.5 min, followed by washout cycle and equilibration; total analysis time of 15 min. The scanned m/z range was 80-2000 Th; capillary voltage 4,500 V; nebulizer gas pressure 2 bar, drying gas flow rate 9 l min-1 and temperature 200C. The full MS scan was followed by a tandem mass spectrometry fragmentation of the seven most abundant ions within the spectrum. For tandem mass spectrometry, the collision cell collision energy was set at 3 eV and the collision energy was stepped 50 percent, 75 percent, 150 percent, and 200 percent. The scan rate was 3 Hz. A HP-921 lock mass compound was infused during the analysis for post processing mass correction.

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1H NMR spectroscopy-based metabolic profiling of Ophiocordyceps sinensis and Cordyceps militaris of water-boiled and 50% ethanol-soaked extracts

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