Global analysis of qualitative and quantitative metabolism of Notoginsenoside R1 in rat liver-brain-gut axis based on LC-IT-TOF/MS combing mMDF strategy,Phytomedicine

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Global analysis of qualitative and quantitative metabolism of Notoginsenoside R1 in rat liver-brain-gut axis based on LC-IT-TOF/MS combing mMDF strategy
Phytomedicine ( IF 6.7 ) Pub Date : 2022-06-08 , DOI: 10.1016/j.phymed.2022.154261
Kangrui Hu ₁ , Changjian Li ₁ , Tengjie Yu ₁ , Huimin Guo ₁ , Hong Sun ₁ , Shuying Mao ₁ , Zhihao Zhou ₁ , Wei Jin ₁ , Keanqi Liu ₁ , Lin Xie ₁ , Guangji Wang ₁ , Yan Liang ₁

Affiliation

Background

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.

Purpose

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.

Methods

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.

Results

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+CH₂+2H and Rg1+CH₂. 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.

Conclusion

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.

中文翻译：

基于LC-IT-TOF/MS结合mMDF策略的大鼠肝-脑-肠轴三七皂苷R1定性定量代谢的全局分析

背景

中药有效成分在靶器官的代谢研究，有利于明确中药有效成分的真伪。三七皂苷 R1 (NG-R1) 是三七皂苷 (PNS) 的生物活性成分之一，被公认为三七皂苷的特征性标志物。然而，由于缺乏可靠的技术和方法，NG-R1 在靶器官中的代谢尚未阐明。

目的

本研究旨在开发一种可靠有效的策略和技术，以揭示靶器官中中药活性成分的定性和定量代谢，并阐明 NG-R1 在肝-脑-肠轴的生物转化。

方法

在新鲜大鼠脑、肝匀浆和肠道菌群的体外培养系统中研究了 NG-R1 在脑肠轴中的代谢转化。为了快速锁定目标代谢物，我们将质量缺陷过滤器（MDF）设置在不同的范围内，以筛选不同分子量（MW）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了相对定量分析，以克服代谢物因缺乏标准而难以量化的挑战。

结果

当MDF设定在0.50~0.65筛选MW为900~1200 Da的代谢物时，很快就发现了6个新代谢物，然后根据其LC和MS特征鉴定为葡萄糖醛酸结合、氧化、脱氢、甲基化和氢化产物。将MDF设置为0.42-0.52时，有效筛选出6种分子量为600-900 Da的代谢物，分别为Rg1、NG-R2、Rh1、Rg1+CH ₂ +2H和Rg1+CH ₂。筛选MW为300~600 Da的代谢物，MDF设定为0.25~0.42，快速筛选4个新代谢物。定量代谢结果提示肠道菌群是大鼠NG-R1的主要代谢部位，60%以上的NG-R1在肠道菌群中通过去糖基化作用转化为Rg1。