Abstract

Background

   Rhubarb, containing raw rhubarb (RR) and two processed products
   (steamed rhubarb, SR; carbonized rhubarb, CR), is commonly used in
   high-doses for the treatment of peptic ulcer, especially gastric ulcer
   (GU). However, their active ingredients, therapeutic targets, and
   potential mechanism remain unclear. Meanwhile, the safety of these
   active ingredients is also worth studying.

Methods

   An offline two-dimensional low-pressure liquid
   chromatography/high-performance liquid chromatography coupled with high
   resolution mass spectrometry method was applied to identify the
   chemical constituents of RR, SR, and CR. Then, the plasma and urine
   samples of rats after oral administration of RR, SR, and CR were
   studied for metabolite profiling. Based on the analysis of ingredients
   in vivo, the key active constituents, core therapeutic targets and key
   signaling pathways of RR, SR, and CR against GU were screened via
   network pharmacology and molecular docking. Finally, the efficacy and
   safety of these key active ingredients were evaluated.

Results

   Totally, 183, 120 and 115 compounds were identified or tentatively
   characterized from RR, SR and CR, respectively. Meanwhile, 190, 182 and
   180 components were identified after oral administration of RR, SR and
   CR. By network pharmacology and molecular docking, torachrysone,
   hydroxyemodin, 6-methylrhein, rhein and emodin anthrone might be the
   predominant effective constituents in RR, SR, and CR with AKT1 and EGFR
   being their key targets during the treatment of GU. Moreover,
   EGFR/PI3K/AKT signaling pathway might play a crucial role in the
   therapeutic mechanism of GU. In silio ADMET predictions categorized 5
   compounds as drugs with good oral bioavailability, but these components
   may induce liver injury.

Conclusion

   Overall, our results not only clarified the active substances and
   molecular mechanism for enhancing our understanding about the
   traditional efficacy, but also pay attention to the clinical safety
   issues of raw and prepared rhubarbs.

   Keywords: raw and prepared rhubarbs, chemical analysis in vitro ,
   metabolite profiling in vivo , network pharmacology, molecular docking,
   toxicity prediction

Graphical Abstract

   [43]graphic file with name FPHAR_fphar-2024-1481091_wc_abs.jpg

1 Introduction

   Gastric ulcer (GU) is a common upper gastrointestinal disease
   worldwide, and its symptoms such as epigastric pain and indigestion
   seriously affect the quality of life and work efficiency of about
   10%–15% of the population in the world ([44]Liu et al., 2022;
   [45]Bahramikia and Izadi, 2023). GU is characterized by severe damage
   of gastric mucosa and submucosa, which can cause irritation,
   inflammation, and increase the risk of gastric cancer ([46]Wang et al.,
   2022). Generally, it is caused by several factors such as stress,
   alcohol abuse, over use of nonsteroidal anti-inflammatory drugs
   (NSAIDs), and Helicobacter pylori infection ([47]Shu et al., 2023). At
   present, GU has been treated with antibiotics, antacids, H2 receptor
   blockers, and proton-pump inhibitors ([48]Abd-Eldayem et al., 2022).
   Although these drugs can quickly alleviate clinical symptoms and
   shorten recovery time, they have some adverse effects, such as nausea,
   vomiting, stomach pain, headache, and constipation ([49]Liu et al.,
   2022). Therefore, it is necessary to develop safe, effective, and
   natural agents. In recent decades, traditional Chinese medicines (TCMs)
   have unique advantages in treating GU ([50]Abd-Eldayem et al., 2022;
   [51]Luo J. H. et al., 2023).

   Chinese herb rhubarb, the dried roots and rhizomes of Polygonaceae
   family, comprises Rheum palmatum L., Rheum tanguticum Maxim. ex Balf.
   and Rheum officinale Baill ([52]Zhuang et al., 2020; [53]Zhang et al.,
   2023). Modern pharmacological research has shown that rhubarb has
   various pharmacological effects in the gastrointestinal tract,
   including the protection of intestinal mucosa barrier, the inhibition
   of H. pylori, and the clearance of inflammatory factors ([54]Liu et
   al., 2016). For a long time, it has been used to treat emergencies such
   as gastrointestinal bleeding ([55]Zhang et al., 2018; [56]Xiang et al.,
   2020). Currently, four processed products, namely, raw rhubarb (RR),
   steamed rhubarb (SR), wine-processed rhubarb (WR), and carbonized
   rhubarb (CR), are the most commonly used in clinical practice
   ([57]Zhang J. et al., 2019; [58]Wang et al., 2023). According to the
   processing theory of “wine processing for uplifting” of TCM, WR is
   adept at treating upper energizer diseases, such as eye swelling and
   pain, mouth sores, and ulcers on the tongue. And the stomach belongs to
   the middle energizer area, so there is literature proving that the
   astringent and hemostatic effect of WR is the weakest for
   gastrointestinal bleeding ([59]Wang et al., 2015). Meanwhile, previous
   researches have demonstrated that RR, SR and CR have therapeutic
   effects on experimental GUs ([60]Wang, 2023; [61]Li et al., 2019).
   Therefore, RR, SR and CR were selected as the research objects in this
   paper. However, the active compounds against GU from the above three
   rhubarbs remain unclear.

   Systematic characterization of the chemical constituents in vitro is a
   critical prerequisite for elucidating the active ingredients of TCMs
   ([62]Zheng et al., 2021; [63]Wang, 2023). Nowadays, liquid
   chromatography-high resolution mass spectrometry (LC-HRMS) has become a
   common method for rapid chemical profiling of TCMs ([64]Shi et al.,
   2022; [65]Wang H. D. et al., 2024). However, owing to the chemical
   complexity of TCMs and large differences in content and polarity of
   each component, the traditional LC-MS method remains a challenge to
   comprehensively characterize the chemical components in TCMs,
   especially the minor or even trace existence ([66]Pang et al., 2019;
   [67]Luo W. J. et al., 2023). To address the problem, multidimensional
   LC-MS strategy has been proposed ([68]Zhang et al., 2022). For example,
   offline two-dimensional low-pressure liquid
   chromatography/high-performance liquid chromatography (2D LPLC/HPLC)
   coupled with MS approach could enable the isolation and identification
   of compounds from natural products ([69]Cassien et al., 2021; [70]Zhu
   et al., 2022). LPLC is a separation technique utilizing different
   column packings. Middle chromatogram isolated (MCI) GEL CHP20P is one
   of the most widely used separation materials because of its high
   capacity and stable quality ([71]Fang et al., 2022; [72]Huang et al.,
   2023). Therefore, MCI GEL CHP20P column chromatography was chosen for
   first-dimension separation in our study. We expect that the combination
   of offline 2D LPLC/HPLC and HRMS in this work can provide strong
   technical support for elucidating the active compounds of rhubarb.

   For complex TCMs, it is well known that only the constituents that
   enter the circulatory system, rather than all constituents present, are
   likely to be responsible for the implementation of pharmacological
   effects ([73]Zhang H. Y. et al., 2019; [74]Li et al., 2022). Collected
   blood and urine samples are analyzed before and after administration,
   aiming to discover the exposed substances of TCMs in the body,
   including prototype components and metabolites ([75]Zhong et al., 2023;
   [76]Shi et al., 2024). But metabolic analysis in vivo does not involve
   any correlation with pharmacological activity ([77]Li et al., 2022). In
   recent years, network pharmacology, a “drug-target-disease” interaction
   analysis approach, has become a hotspot. It can make virtual
   predictions of pharmacological mechanisms during the treatment with
   TCMs, so as to make up for the deficiency of metabolite profiling
   ([78]Xiong et al., 2022; [79]Yang et al., 2022). Hence, an integrated
   strategy based on metabolic research and network pharmacology was
   proposed for screening potential active components and targets of TCMs
   ([80]Zhang et al., 2020).

   In the present study, taking RR, SR and CR as examples, we proposed a
   systematic strategy integrating the characterization of in vitro and in
   vivo substances, network pharmacology and molecular docking to reveal
   anti-GU ingredients, potential key targets and pathways in treating GU.
   Additionally, the absorption, distribution, metabolism, excretion, and
   toxicity (ADMET) of these key anti-GU metabolites were further
   predicted by ADMETlab 2.0 and Deep-PK. It is believed that this work
   could screen the active compounds as well as their potential targets
   and pathways, thus laying the foundation for further clarifying the
   anti-GU mechanism of three processed products of rhubarb. In addition,
   the safety assessment aimed to provide clinical guidance and minimize
   the risk of drug-related toxicity for the treatment of GU with rhubarb.

2 Materials and methods

2.1 Chemicals and reagents

   Standard references included emodin, physcion, rhein and chrysophanol,