Key Lab of Drug Metabolism & Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, PR China.
Key Lab of Drug Metabolism & Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, PR China.
Phytomedicine. 2022 Sep;104:154261. doi: 10.1016/j.phymed.2022.154261. Epub 2022 Jun 8.
The metabolism study of active components for traditional Chinese medicine (TCM) in target organs is conducive to clarify the authentic active ingredients. Notoginsenoside R1 (NG-R1), one of the bioactive components of Panax notoginsenoside (PNS), is commonly acknowledged as the characteristic marker of PNS. However, the metabolism of NG-R1 in target organs has not been clarified yet due to the lack of robust technique and approach.
The present study aimed to develop a reliable and efficient strategy and technology for revealing the qualitative and quantitative metabolism of active components of TCMs in target organs, and to clarify the biotransformation of NG-R1 in liver-brain-intestinal axis.
The metabolic transformation of NG-R1 in the brain gut axis was investigated in the in vitro incubation system of fresh rat brain, liver homogenate, and intestinal flora. To quickly lock the target metabolites, we set the mass defect filter (MDF) in different ranges to screen metabolites with different molecular weight (MW). This strategy was defined as multi-stage MDF (mMDF). In addition, we performed relative quantitative analysis on all metabolites according to the peak area acquired by LC-IT-TOF/MS to overcome the challenge that metabolites are difficult to be quantified due to the lack of standards.
When MDF was set at 0.50 to 0.65 to screen metabolites with MW of 900 to 1200 Da, 6 novel metabolites were quickly found, and then identified as glucuronic acid binding, oxidation, dehydrogenation, methylation and hydrogenation products according to their LC and MS characteristics. When setting MDF at 0.42 - 0.52, 6 metabolites with MW of 600 to 900 Da were effectively screened and identified as Rg1, NG-R2, Rh1, Rg1+CH+2H and Rg1+CH. To screen the metabolites with MW of 300 to 600 Da, MDF was set at 0.25 - 0.42, and 4 novel metabolites were screened rapidly. The results of quantitative metabolism suggested that intestinal flora was the main metabolic site of NG-R1 in rat, and more than 60% of NG-R1 was converted to Rg1 by deglycosylation in the intestinal flora.
The mMDF strategy can significantly improve the research efficiency of qualitative metabolism of saponins. Although NG-R1 could be transformed into a variety of metabolites in rat liver and brain homogenate, it still existed mainly in prototype form. In the rat flora, NG-R1 mainly existed in the form of deglycosylated metabolite Rg1.
研究中药（TCM）活性成分在靶器官中的代谢情况，有助于阐明其真实的活性成分。三七总皂苷（PNS）中的生物活性成分之一——人参皂苷 R1（NG-R1），通常被认为是 PNS 的特征性标志物。然而，由于缺乏强大的技术和方法，NG-R1 在靶器官中的代谢情况仍不清楚。
本研究旨在开发一种可靠、高效的策略和技术，用于揭示 TCM 活性成分在靶器官中的定性和定量代谢情况，并阐明 NG-R1 在肝肠脑轴中的生物转化情况。
在新鲜大鼠脑、肝匀浆和肠道菌群的体外孵育体系中，研究 NG-R1 在脑肠轴中的代谢转化情况。为了快速锁定目标代谢物，我们在不同范围内设置质量亏损滤波器（MDF），以筛选分子量（MW）不同的代谢物。该策略被定义为多阶段 MDF（mMDF）。此外，我们根据 LC-IT-TOF/MS 获得的峰面积对所有代谢物进行相对定量分析，以克服由于缺乏标准而导致代谢物难以定量的挑战。
当 MDF 设置为 0.50 至 0.65 以筛选 MW 为 900 至 1200 Da 的代谢物时，快速发现了 6 种新的代谢物，并根据其 LC 和 MS 特征鉴定为葡萄糖醛酸结合物、氧化产物、脱氢产物、甲基化产物和加氢产物。当 MDF 设置为 0.42 至 0.52 时，有效筛选并鉴定出 MW 为 600 至 900 Da 的 6 种代谢物，分别为 Rg1、NG-R2、Rh1、Rg1+CH+2H 和 Rg1+CH。为了筛选 MW 为 300 至 600 Da 的代谢物，MDF 设置为 0.25 至 0.42，快速筛选出 4 种新的代谢物。定量代谢研究结果表明，肠道菌群是 NG-R1 在大鼠体内代谢的主要部位，肠道菌群中超过 60%的 NG-R1 通过去糖基化转化为 Rg1。
mMDF 策略可以显著提高皂苷类化合物定性代谢研究的效率。尽管 NG-R1 在大鼠肝和脑匀浆中可以转化为多种代谢物，但仍以原型为主。在大鼠菌群中，NG-R1 主要以去糖基化代谢物 Rg1 的形式存在。
