Guduchyadi medhya yoga-An HRLCMS-orbitrap data

Background and rationale. Guduchyadi Yoga (GY) is a classical eight-drug Ayurvedic formulation traditionally indicated for cognition. Constituents relevant to cognitive impairment and stress include: Tinospora cordifolia (anti-stress; memory support), Achyranthes aspera (antidepressant-like effects), Embelia ribes/embelin (anticonvulsant/CNS-depressant), Convolvulus pluricaulis (memory/stress benefits), Acorus calamus (CNS activity), Terminalia chebula (in-vitro neuroprotection via ROS-scavenging phenolics/tannins), and Asparagus racemosus (neuroprotection; memory support). Classical Rasapañcaka and medhya (cognitive) indications, together with small clinical observations (e.g., MMSE and HAM-A/HAM-D improvements), motivate systematic chemical profiling and computational evaluation. Hydro-alcoholic extract (HAE). Solvent: ethanol:water (70:30, v/v; LC–MS grade). Weighed 25.00 g GY into a Type-I glass bottle; added 250 mL solvent (1:10 w/v). Extraction: magnetic stirring 300 rpm at 20–25 °C for 8 h (covered/amber-wrapped), then 16 h static maceration. Clarification: Whatman No. 42 filtration followed by 0.22 µm PTFE filtration. Solvent removal: rotary evaporation at 40 °C (reduced pressure) to near-dryness. The dried extract was transferred to sterile amber vials, yield (%) = (extract mass/25 g)×100, optionally N₂-flushed, and stored at −20 °C, protected from light. For LC–MS, aliquots were reconstituted/diluted and filtered per SOP. UHPLC–HRMS conditions. Thermo Scientific Vanquish UHPLC (autosampler 4 °C; column oven 40 °C) with RP-C18 column (2.1 × 100 mm, 1.9–2.6 µm). Injection: 5 µL. Total run: 30.0 min at 0.300 mL min⁻¹. Mobile phases: A = water (0.1% formic acid); D = programmed organic channel (B,C = 0%). Gradient (A complements D): 0–2 min, 5% D; 2–15 min, 5→95% D; 15–20 min, 95% D; 21 min, return to 5% D; 21–30 min, re-equilibrate. MS: Q Exactive™ Plus (HESI), Full MS with data-dependent MS² (Top-5), acquired in separate positive/negative runs. Full MS: 70,000 (m/z 200), AGC 1e6, max IT 100 ms; dd-MS²: 17,500, AGC 1e5, max IT 100 ms, isolation 1.5 m/z, stepped NCE 30, dynamic exclusion 10 s. Tune files: Cal_positive_29082024.mstune; Cal_negative_29082024.mstune. Divert Valve A: Start in 1–2 (True). Data acquisition and processing. Data were acquired in Xcalibur/SII (profile). Sequence: blank → system suitability → three QCs → study samples (QC every 8–10) → final blanks. Autosampler: puncture offset 0 µm; tray 4 °C. Raw files were processed in ⟨software/version⟩ with centroiding; mass error ≤ 5 ppm; minimum peak width 6–8 s; RT alignment 0.2–0.3 min; gap-filling enabled. MS² matches were queried against ⟨mzCloud/MassBank/in-house⟩ at predefined score thresholds. Features with QC RSD > 20% were flagged/excluded unless justified. Putative annotations were reported at MSI Level 2; Level 1 required |Δm| ≤ 5 ppm, RT within ±0.20 min of an authentic standard, and concordant MS² fragments.研究背景与依据。古杜查迪亚瑜伽方（Guduchyadi Yoga, GY）是经典的八味阿育吠陀方剂，传统用于认知调理。与认知障碍及应激相关的组方成分包括：青牛胆（Tinospora cordifolia）——具有抗应激、改善记忆的作用；倒扣草（Achyranthes aspera）——具备类抗抑郁效应；酸藤子/酸藤素（Embelia ribes/embelin）——可发挥抗惊厥、中枢神经系统（central nervous system, CNS）抑制作用；多花打碗花（Convolvulus pluricaulis）——能改善记忆、缓解应激；菖蒲（Acorus calamus）——具有中枢神经活性；诃子（Terminalia chebula）——可通过清除活性氧（reactive oxygen species, ROS）的酚类/单宁类成分实现体外神经保护；天门冬（Asparagus racemosus）——具备神经保护、改善记忆的功效。传统Rasapañcaka与益智（medhya）适应症，结合小型临床观察结果（如简易精神状态检查表（Mini-Mental State Examination, MMSE）、汉密尔顿焦虑量表（Hamilton Anxiety Rating Scale, HAM-A）/汉密尔顿抑郁量表（Hamilton Depression Rating Scale, HAM-D）评分改善），推动了系统性化学表征与计算评估研究的开展。 水醇提取物（HAE）。溶剂：乙醇-水（70:30，体积比；液相色谱-质谱级）。称取25.00 g GY置于Ⅰ型玻璃瓶中，加入250 mL溶剂（料液比1:10，w/v）。提取工艺：20~25 ℃下以300 rpm磁力搅拌8 h（避光密封），随后静置浸渍16 h。澄清步骤：先经Whatman No.42滤纸过滤，再经0.22 µm聚四氟乙烯（polytetrafluoroethylene, PTFE）滤膜过滤。溶剂去除：40 ℃减压旋转蒸发至近干。将干燥后的提取物转移至无菌棕色瓶中，得率（%）=（提取物质量/25 g）×100，可选择充入氮气（N₂）保护，于-20 ℃避光储存。用于液相色谱-质谱（liquid chromatography-mass spectrometry, LC-MS）分析的等分试样需按照标准操作规程（SOP）复溶、稀释并过滤。 超高效液相色谱-高分辨质谱（UHPLC-HRMS）分析条件。采用赛默飞世尔科技（Thermo Scientific）Vanquish UHPLC系统（自动进样器温度4 ℃；柱温箱温度40 ℃），搭配反相C18色谱柱（2.1 × 100 mm，1.9~2.6 µm）。进样体积：5 µL。总运行时间：30.0 min，流速0.300 mL·min⁻¹。流动相：A相为含0.1%甲酸的水；D相为预设有机相通道（B、C相初始占比为0%）。梯度洗脱程序（A相与D相互补）：0~2 min，5% D；2~15 min，5%→95% D；15~20 min，95% D；21 min，恢复至5% D；21~30 min，体系平衡。质谱（MS）检测采用Q Exactive™ Plus静电场轨道阱质谱仪（加热电喷雾电离（heated electrospray ionization, HESI）源），采用全扫描质谱结合数据依赖性二级质谱（Top-5模式），分别在正、负电离模式下采集数据。全扫描模式：分辨率70,000（m/z 200处），自动增益控制（automatic gain control, AGC）目标值1×10⁶，最大离子注入时间100 ms；数据依赖性二级质谱（dd-MS²）模式：分辨率17,500，AGC目标值1×10⁵，最大离子注入时间100 ms，隔离窗口1.5 m/z，阶梯式碰撞能量（stepped NCE）30，动态排除时间10 s。调谐文件：Cal_positive_29082024.mstune；Cal_negative_29082024.mstune。分流阀A：1~2 min开启（True）。 数据采集与处理。数据采用Xcalibur/SII软件以profile模式采集。分析序列：空白对照→系统适用性样品→三份质量控制（QC）样品→研究样品（每8~10个研究样品插入QC样品）→最终空白对照。自动进样器：穿刺偏移0 µm；样品托盘温度4 ℃。原始数据采用<软件/版本>软件进行质心化处理，质量误差阈值≤5 ppm，最小峰宽6~8 s，保留时间（retention time, RT）对齐偏差0.2~0.3 min，启用缺口填充。二级质谱匹配结果通过<mzCloud/MassBank/in-house>按照预设评分阈值进行检索。质量控制相对标准偏差（relative standard deviation, RSD）>20%的特征峰将被标记或排除，除非有合理解释。推定注释按照代谢物标准倡议（Metabolomics Standards Initiative, MSI）二级（Level 2）进行报告；一级注释（Level 1）需满足：质量偏差|Δm|≤5 ppm，保留时间与对照品偏差±0.20 min以内，且二级质谱碎片谱图匹配一致。