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

Sample preparation for NMR Previously stored O. sinensis and C. militaris were divided into two groups and used for 1H NMR sample preparation. Prior to NMR analysis, O. sinensis and C. militaris were thawed to room temperature. For the 50% water:ethanol (H2O:EtOH) group, 5 mL of the 50% water:ethanol mixture was added to 500 mg O. sinensis or C. militaris and vortexed for 60 s. The mixtures were maintained for 360 days at 25oC. For the water (H2O) group, 5 mL of distilled water was added to 500 mg of O. sinensis or C. militaris. The mixtures were placed in boiling water and heated to 100oC for 30 mins. The extracts were centrifuged (15 min, 13,000 rpm) at 4oC and 3 mL of supernatant was collected. The supernatant was frozen at -80oC and then dried using a vacuum centrifugal evaporator (CHRiST, Alpha 2-4/LSC, Germany). The dried residues were separately dissolved in 3 mL of distilled water. A 600 µL extract was filtered (Millipore Amicon® ULTRA 3 kDa) and filtrates were collected. 3-(Trimethylsilyl) propanesulfonic acid (50 µL) pre-dissolved in D2O (an internal standard) was subsequently added to 450 µL of the filtrates. Finally, aliquots (480 µL) of each extract were transferred to a 5 mm NMR tube (Norell, Morganton, NC, USA) for NMR analysis. The 1H NMR spectra were recorded as detailed below. NMR analysis and data preprocessing All of the 1H NMR spectra were acquired at 298 K on a Bruker AV III 600 MHz NMR spectrometer (Bruker Analytische GmbH, Rheinstetten, Germany) equipped with an inverse cryoprobe operating at a proton NMR frequency of 600.13 MHz. For each sample, other used acquisition parameters were as follows: number of scans = 32, spectral width (SW) = 8,000 Hz, pulse width (PW) = 10 s, and relaxation delay = 1.0 s. A Noesygppr1d pulse sequence was applied to suppress the residual water signal. All of the free induction decay NMR spectra were phased and baseline-correction was performed using Chenomx NMR Suite v.7.7 (Chenomx Inc., Edmonton, Canada). The metabolites were identified by matching spectral signals to the Chenomx 600 MHz Library comprising 330 metabolites. A reference compound DSS-d6 was used as an internal standard for the chemical shifts (set to 0 ppm) and a reference signal for the quantification. Data quantification was performed by comparing the integration of a known reference signal (DSS-d6) with the signals derived from a library of compounds containing chemical shifts and peak multiplicities for all of the resonances of the constituents. The results were exported as an Excel file for further analysis.核磁共振波谱（Nuclear Magnetic Resonance Spectroscopy, NMR）样品前处理：将此前保存的冬虫夏草（Ophiocordyceps sinensis, O. sinensis）与蛹虫草（Cordyceps militaris, C. militaris）分为两组，用于氢谱（1H NMR）样品制备。在开展NMR分析前，需将两种样品解冻至室温。 对于50%水-乙醇（H₂O:EtOH）组：向500 mg冬虫夏草或蛹虫草中加入5 mL 50%水-乙醇混合液，涡旋振荡60秒，随后将混合体系置于25℃下静置360天。 对于纯水（H₂O）组：向500 mg冬虫夏草或蛹虫草中加入5 mL蒸馏水，将混合物置于沸水浴中加热至100℃并保持30分钟。 将上述提取液于4℃下离心（15 min，13000 rpm），收集3 mL上清液。将上清液置于-80℃冷冻后，使用真空离心浓缩仪（CHRiST, Alpha 2-4/LSC，德国）进行干燥。将干燥后的残渣分别用3 mL蒸馏水溶解。取600 μL提取液经超滤管（Millipore Amicon® ULTRA 3 kDa）过滤，收集滤液。向450 μL滤液中加入预先溶于重水（D₂O）的3-(三甲基硅基)丙磺酸钠（50 μL，内标（internal standard））。最后将每份提取液的等分试样（480 μL）转移至5 mm核磁共振管（Norell, Morganton, NC, USA）中，用于NMR分析。 NMR分析与数据预处理：所有氢谱（1H NMR）数据均于298 K下，在配备反向低温探头的布鲁克AV III 600 MHz核磁共振波谱仪（Bruker Analytische GmbH, Rheinstetten, Germany）上采集，质子共振频率为600.13 MHz。每个样品的采集参数如下：扫描次数=32，谱宽（SW）=8000 Hz，脉冲宽度（PW）=10 s，弛豫延迟=1.0 s。采用Noesygppr1d脉冲序列抑制残留水信号。所有自由感应衰减（Free Induction Decay, FID）NMR谱图均进行相位校正，并使用Chenomx NMR Suite v.7.7（Chenomx Inc., Edmonton, Canada）完成基线校正。通过将谱峰信号与包含330种代谢物的Chenomx 600 MHz数据库进行匹配，实现代谢物的定性鉴定。以氘代二甲基硅基丙酸钠（DSS-d6）作为化学位移内标（设定为0 ppm），同时作为定量分析的参考信号。数据定量通过将已知参考信号（DSS-d6）的积分面积与包含所有组分共振峰化学位移及峰多重性的化合物数据库信号进行比对完成。最终分析结果导出为Excel文件，用于后续研究。