Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla.

Uncaria rhynchophylla is an evergreen vine plant, belonging to the Rubiaceae family, that is rich in terpenoid indole alkaloids (TIAs) that have therapeutic effects on hypertension and Alzheimer's disease. GATA transcription factors (TF) are a class of transcription regulators that participate in the light response regulation, chlorophyll synthesis, and metabolism, with the capability to bind to GATA cis-acting elements in the promoter region of target genes. Currently the charactertics of GATA TFs in U. rhynchophylla and how different light qualities affect the expression of GATA and key enzyme genes, thereby affecting the changes in U. rhynchophylla alkaloids have not been investigated. In this study, 25 UrGATA genes belonging to four subgroups were identified based on genome-wide analysis. Intraspecific collinearity analysis revealed that only segmental duplications were identified among the UrGATA gene family. Collinearity analysis of GATA genes between U. rhynchophylla and four representative plant species, Arabidopsis thaliana, Oryza sativa, Coffea Canephora, and Catharanthus roseus was also performed. U. rhynchophylla seedlings grown in either red lights or under reduced light intensity had altered TIAs content after 21 days. Gene expression analysis reveal a complex pattern of expression from the 25 UrGATA genes as well as a number of key TIA enzyme genes. UrGATA7 and UrGATA8 were found to have similar expression profiles to key enzyme TIA genes in response to altered light treatments, implying that they may be involved in the regulation TIA content. In this research, we comprehensively analyzed the UrGATA TFs, and offered insight into the involvement of UrGATA TFs from U. rhynchophylla in TIAs biosynthesis.

Effect of nitrogen reduction by chemical fertilization with green manure (Vicia sativa L.) on soil microbial community, nitrogen metabolism and and yield of Uncaria rhynchophylla by metagenomics.

Uncaria rhynchophylla is an important herbal medicine, and the predominant issues affecting its cultivation include a single method of fertilizer application and inappropriate chemical fertilizer application. To reduce the use of inorganic nitrogen fertilization and increase the yield of Uncaria rhynchophylla, field experiments in 2020-2021 were conducted. The experimental treatments included the following categories: S1, no fertilization; S2, application of chemical NPK fertilizer; and S3-S6, application of chemical fertilizers and green manures, featuring nitrogen fertilizers reductions of 0%, 15%, 30%, and 45%, respectively. The results showed that a moderate application of nitrogen fertilizer when combined with green manure, can help alleviate soil acidification and increase urease activity. Specifically, the treatment with green manure provided in a 14.71-66.67% increase in urease activity compared to S2. Metagenomics sequencing results showed a decrease in diversity in S3, S4, S5, and S6 compared to S2, but the application of chemical fertilizer with green manure promoted an increase in the relative abundance of Acidobacteria and Chloroflexi. In addition, the nitrification pathway displayed a progressive augmentation in tandem with the reduction in nitrogen fertilizer and application of green manure, reaching its zenith at S5. Conversely, other nitrogen metabolism pathways showed a decline in correlation with diminishing nitrogen fertilizer dosages. The rest of the treatments showed an increase in yield in comparison to S1, S5 showing significant differences (p < 0.05). In summary, although S2 demonstrate the ability to enhance soil microbial diversity, it is important to consider the long-term ecological impacts, and S5 may be a better choice.

Triterpenoids from the hook-bearing stems of Uncaria rhynchophylla.

An unprescribed nortriterpenoid with an aromatic E ring, uncanortriterpenoid A (1), together with fourteen known triterpenoids (2-15), were isolated from the hook-bearing stems of Uncaria rhynchophylla Miq. Based on extensive spectroscopic analyses, the NMR data of 2, 5, and 10 in CD3OD were assigned for the first time, and the wrongly assigned δC of C-27 and C-29 of 2 were revised. Among the known compounds, 7, 13, and 15 were isolated from this species for the first time, and 15 represents the first lanostane triterpenoid bearing an extra methylidene at C-24 for the Rubiaceae family. Additionally, compounds 6 and 14 exhibited moderate ferroptosis inhibitory activity, with an EC50 value of 14.74 ± 0.20 µM for 6 and 23.11 ± 1.31 µM for 14.

Minor alkaloids from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new monoterpene alkaloids (1 - 7), along with 16 known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis, single crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are stereoisomers belonging to a novel type of pseudoindoxyl monoterpene alkaloids, 3 is the first monoterpene furoindole alkaloid from nature, and 4 - 7 are derivatives of the known monoterpene alkaloids featuring different structures.

The basic chemical substances of total alkaloids of Uncaria rhynchophylla and their anti-neuroinflammatory activities.

To clarify the chemical basis of the total alkaloids of Uncaria rhynchophylla, HPLC-VWD chromatogram of total alkaloids was established. Under its guidance, modern chromatographic and spectroscopic techniques were used to track, isolate and identify the representative principal components. As a result, one new monoterpenoid indole alkaloid, 3S,15S-N4-methoxymethyl-geissoschizine methyl ether (1), together with 20 known alkaloids (2-21), and 5 other known compounds (22-26) were obtained. Meanwhile, sixteen characteristic peaks were identified from the total alkaloids using HPLC analysis. Then, the anti-neuroinflammatory effect of compounds 1-21 was assessed through inhibiting nitric ---oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Among them, compounds 1, 3, 7, 8, 11, 12, 19 and 21 showed potent inhibitory activities with IC50 values of 5.87-76.78 µM.

Genome-Wide Identification of Seven in Absentia E3 Ubiquitin Ligase Gene Family and Expression Profiles in Response to Different Hormones in Uncaria rhynchophylla.

SINA (Seven in absentia) E3 ubiquitin ligases are a family of RING (really interesting new gene) E3 ubiquitin ligases, and they play a crucial role in regulating plant growth and development, hormone response, and abiotic and biotic stress. However, there is little research on the SINA gene family in U. rhynchophylla. In this study, a total of 10 UrSINA genes were identified from the U. rhynchophylla genome. The results of multiple sequence alignments and chromosomal locations show that 10 UrSINA genes were unevenly located on 22 chromosomes, and each UrSINA protein contained a SINA domain at the N-terminal and RING domains at the C-terminal. Synteny analysis showed that there are no tandem duplication gene pairs and there are four segmental gene pairs in U. rhynchophylla, contributing to the expansion of the gene family. Furthermore, almost all UrSINA genes contained the same gene structure, with three exons and two introns, and there were many cis-acting elements relating to plant hormones, light responses, and biotic and abiotic stress. The results of qRT-PCR show that most UrSINA genes were expressed in stems, with the least expression in roots; meanwhile, most UrSINA genes and key enzyme genes were responsive to ABA and MeJA hormones with overlapping but different expression patterns. Co-expression analysis showed that UrSINA1 might participate in the TIA pathway under ABA treatment, and UrSINA5 and UrSINA6 might participate in the TIA pathway under MeJA treatment. The mining of UrSINA genes in the U. rhynchophylla provided novel information for understanding the SINA gene and its function in plant secondary metabolites, growth, and development.

Identification of three key enzymes involved in the biosynthesis of tetracyclic oxindole alkaloids in Uncaria rhynchophylla.

Tetracyclic oxindole alkaloids (TOAs), main active ingredients of Uncaria rhynchophylla (UR), has inspired the interest of pharmacologists and chemists because of its great potential in the treatment of the diseases of the nervous system and cardiovascular system and its special spirooxindole scaffold, but the biosynthetic pathway of this compounds is still unknown. In this work, the metabolomics and transcriptomics of hook, leaf and stem of UR were analyzed, and 31 alkaloids and 47,423 unigenes were identified, as well as the relative contents of these alkaloids were evaluated. Based on the above results and literatures, a proposal biosynthetic pathway for TOAs was devised. Furthermore, three unigenes were suggested mediating the biosynthesis of TOAs through the integrated analysis of metabolomics and transcriptomics, and three enzymes, tryptophan decarboxylase, strictosidine synthase and strictosidine-ß-d-glucosidase, were identified as important catalytic enzymes for the synthesis of tryptamine, strictosidine (7) and 4,21-dehydrogeissochizine, respectively, which are considered as the important precursors of TOAs.

Identification of Uncaria rhynchophylla in the Potential Treatment of Alzheimer's Disease by Integrating Virtual Screening and In Vitro Validation.

Uncaria rhynchophylla (Gouteng in Chinese, GT) is the main medicine in many traditional recipes in China. It is commonly used to alleviate central nervous system (CNS) disorders, although its mechanism in Alzheimer's disease is still unknown. This study was designed to predict and validate the underlying mechanism in AD treatment, thus illustrating the biological mechanisms of GT in treating AD. In this study, a PPI network was constructed, KEGG analysis and GO analysis were performed, and an "active ingredient-target-pathway" network for the treatment of Alzheimer's disease was constructed. The active ingredients of GT were screened out, and the key targets were performed by molecular docking. UHPLC-Q-Exactive Orbitrap MS was used to screen the main active ingredients and was compared with the network pharmacology results, which verified that GT did contain the above ingredients. A total of targets were found to be significantly bound up with tau, Aß, or Aß and tau through the network pharmacology study. Three SH-SY5Y cell models induced by okadaic acid (OA), Na2S2O4, and H2O2 were established for in vitro validation. We first found that GT can reverse the increase in the hyperphosphorylation of tau induced by OA to some extent, protecting against ROS damage. Moreover, the results also indicated that GT has significant neuroprotective effects. This study provides a basis for studying the potential mechanisms of GT in the treatment of AD.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis.

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

Transcriptome revealing the dual regulatory mechanism of ethylene on the rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN) are extracted from Uncaria rhynchophylla, which are used to treat Alzheimer's disease. However, the massive accumulation of RIN and IRN in U. rhynchophylla requires exogenous stimulation. Ethylene is a potential stimulant for RIN and IRN biosynthesis, but there is no study on the role of ethylene in RIN or IRN synthesis. This study investigated the regulation of ethylene in RIN and IRN biosynthesis in U. rhynchophylla. An increase in the content of RIN and IRN was observed that could be attributed to the release of ethylene from 18 mM ethephon, while ethylene released from 36 mM ethephon reduced the content of RIN and IRN. The transcriptome and weighted gene co-expression network analysis indicated the up-regulation of seven key enzyme genes related to the RIN/IRN biosynthesis pathway and starch/sucrose metabolism pathway favored RIN/IRN synthesis. In comparison, the down-regulation of these seven key enzyme genes contributed to the reduction of RIN/IRN. Moreover, the inhibition of photosynthesis is associated with a reduction in RIN/IRN. Photosynthesis was restrained owing to the down-regulation of Lhcb1 and Lhcb6 after 36 mM ethephon treatment and further prevented supply of primary metabolites (such as α-D-glucose) for RIN/IRN synthesis. However, uninterrupted photosynthesis ensured a normal supply of primary metabolites at 18 mM ethephon treatment. AP2/ERF1, bHLH1, and bHLH2 may positively regulate the RIN/IRN accumulation, while NAC1 may play a negative regulatory role. Our results construct the potential bidirectional model for ethylene regulation on RIN/IRN synthesis and provide novel insight into the ethylene-mediated regulation of the metabolism of terpenoid indole alkaloids.

Safety and biological activity evaluation of Uncaria rhynchophylla ethanolic extract.

Uncaria rhynchophylla (UR) belongs to the Rubiaceae family, and its dried hooks are usually used in traditional medicine. It is effective in treating diseases related to the central nervous system. This study aimed to evaluate the safety of UR extract, investigate its antimutagenic and antioxidative activities, and elucidate its active components. Extraction and fractionation of the UR extract resulted in yields of 6.71%, 0.037%, 0.042%, 0.152%, 0.332%, and 5.132%, for hexane, ether, DCM, EtOAC, and aqueous fractions, respectively. The four indole alkaloids, total phenolic content (TPC), and total flavonoid content (TFC) of UR extract and its subfractions were measured. Alkaloid content was highest in the UR extract. TPC was the highest in the EtOAC fraction (373.7 ± 20.9 mg gallic acid equivalent (GAE)/g), whereas TFC was the highest in the UR extract (33.5 ± 2.4 mg quercetin equivalent (QE)/g). To assess the safety of UR extract mutagenicity, cytotoxicity, and oxidative stress inducibility assays were performed. The UR extract (2000 µg/plate) showed excellent antimutagenic activity (above 90%) against BaP in both TA98 and TA100 strains. The UR extract exhibited efficient DPPH (RC50 239.2 ± 16.5 µg/mL) and ABTS scavenging activity (RC50 458.7 ± 25.0 µg/mL). The UR extract (150 µg/mL) showed cytoprotective activity (65.6% ± 9.2%) against t-BHP. Among the subfractions, the EtOAC fraction possessed the strongest activities, overall. UR generally showed excellent biological activity at nontoxic concentrations (determined in vitro in current work), although the chemical composition of UR requires further investigation prior to its potential future use.

Minor monoterpene derivatives from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new minor monoterpene derivatives (1-7), together with six known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations, of which 1 was confirmed by single crystal X-ray diffraction.

Comparative transcriptome analysis revealed the molecular mechanism of the effect of light intensity on the accumulation of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN), the main medicinal components in plant Uncaria rhynchophylla, have potential effects on Alzheimer's disease. Understanding the influence of environmental factors, especially light intensity, on the production of these active ingredients will help to improve cultivation techniques. Compared with the 100% light intensity (CK), the contents of RIN and IRN in U. rhynchophylla leaves significantly increased at 20% light intensity (HS) after 7 and 21 days. Short-term shading (21d) changed some morphological indicators of U. rhynchophylla, but did not affect its biomass. Transcriptome profile analysis was performed on data from two groups (7 and 21 days) of CK and HS samples and yielded 79,817 unigenes with an average length of 1023 bp. Concurrently, 2391 and 2136 differentially expressed genes were identified in the transcriptome data for, respectively, 7 and 21 days of shade treatment. Notably, unigenes known to be involved upstream in the biosynthesis of RIN and IRN, such as G8O, IO, 7-DLGT, LAMT, TDC, and STR, were mostly upregulated. In addition, 1065 putative transcription factors (TFs) were identified and grouped into 55 TF families; 26 TFs showed differential expression in the shade treatment after 7 and 21 days. HY5 and PIFs, two important TFs of the light signaling pathway, also showed differential expression. This study provides insight into how gene expression was affected by light intensity during RIN and IRN accumulation in U. rhynchophylla. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01142-2.

[Steroids from Uncaria rhynchophylla].

Thirteen steroids(1-13) were isolated from the non-alkaloid constituents of Uncaria rhynchophylla by column chromatography on silica gel, ODS, Sephadex LH-20, and preparative HPLC chromatography, and their structures were elucidated by analyses of the MS and NMR spectral data. All the compounds were isolated from the genus Uncaria for the first time, and 1 was a new compound. The ~1H-NMR and ~(13)C-NMR data of two compounds(12 and 13) in deuteron-chloroform were completely assigned. This study enriched the steroid constituents of U. rhynchophylla and provided scientific references for the elucidation of active constituents and further development and utilization of U. rhynchophylla.

Minor triterpenes from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Six new triterpenes, uncarinic acids KP (1-6), along with 24 known analogues, were isolated as minor constituents of an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). By comprehensive spectroscopic data analysis, their structures were elucidated as derivatives of olean-12-en-28-oic acid and urs-12-en-28-oic acid with different oxidized forms at C-3, C-6, and/or C-23, respectively. Cell-based preliminary bioassay showed that the (E)-/(Z)-coumaroyloxy and (E)-/(Z)-feruloyloxy units at C-27 of olean-12-en-28-oic acid and urs-12-en-28-oic acid played roles in their bioactivities.[Formula: see text].

Minor alkaloids from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new monoterpene alkaloids (1-7), along with 18 known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Compound 1 is the first monoterpene 22-norindoloquinolizidine alkaloid with a ketene unit, while 2 and 3 are unusual indoloquinolizidine alkaloids having an oxazinane ring.[Formula: see text].

The Main Alkaloids in Uncaria rhynchophylla and Their Anti-Alzheimer's Disease Mechanism Determined by a Network Pharmacology Approach.

Alzheimer's disease (AD) is a growing concern in modern society, and effective drugs for its treatment are lacking. Uncaria rhynchophylla (UR) and its main alkaloids have been studied to treat neurodegenerative diseases such as AD. This study aimed to uncover the key components and mechanism of the anti-AD effect of UR alkaloids through a network pharmacology approach. The analysis identified 10 alkaloids from UR based on HPLC that corresponded to 90 anti-AD targets. A potential alkaloid target-AD target network indicated that corynoxine, corynantheine, isorhynchophylline, dihydrocorynatheine, and isocorynoxeine are likely to become key components for AD treatment. KEGG pathway enrichment analysis revealed the Alzheimers disease (hsa05010) was the pathway most significantly enriched in alkaloids against AD. Further analysis revealed that 28 out of 90 targets were significantly correlated with Aß and tau pathology. These targets were validated using a Gene Expression Omnibus (GEO) dataset. Molecular docking studies were carried out to verify the binding of corynoxine and corynantheine to core targets related to Aß and tau pathology. In addition, the cholinergic synapse (hsa04725) and dopaminergic synapse (hsa04728) pathways were significantly enriched. Our findings indicate that UR alkaloids directly exert an AD treatment effect by acting on multiple pathological processes in AD.

Pharmacokinetic interaction between rhynchopylline and pellodendrine via CYP450 enzymes and P-gp.

CONTEXT: Rhynchopylline and pellodendrine are major extractions of commonly used Chinese medicine in gynaecology. The interaction between these two compounds could affect treatment efficiency and even result in toxicity during their co-administration in gynaecological prescription. OBJECTIVE: The pharmacokinetic interaction between rhynchopylline and pellodendrine and the potential mechanism were investigated in this study. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into four groups to investigate the pharmacokinetic interaction between rhynchopylline (30 mg/kg) and pellodendrine (20 mg/kg) with single dose of these two drugs as the control. The transport of rhynchopylline was evaluated in the Caco-2 cell model. Additionally, the metabolic stability and the activity of corresponding CYP450 enzymes were assessed in rat liver microsomes. RESULTS: The pharmacokinetic profile of rhynchopylline was dramatically affected by pellodendrine with the increased area under the pharmacokinetic curve (3080.14 ± 454.54 vs. 1728.08 ± 220.598 µg/L*h), Cmax (395.1 ± 18.58 vs. 249.1 ± 16.20 µg/L), prolonged t1/2 (9.74 ± 2.94 vs. 4.81 ± 0.42 h) and the reduced clearance rate (from 11.39 ± 1.37 to 5.67 ± 1.42 L/h/kg). No significant changes were observed in the pharmacokinetics of pellodendrine. The transport of rhynchopylline was significantly inhibited by pellodendrine with a decreasing efflux ratio (1.43 vs. 1.79). Pellodendrine significantly inhibited the activity of CYP1A2 and CYP2C9 with IC50 values of 22.99 and 16.23 µM, which are critical enzymes responsible for the metabolism of rhynchopylline. DISCUSSION AND CONCLUSIONS: The adverse interaction between rhynchopylline and pellodendrine draws attention to the co-administration of these two herbs and provides a reference for further investigations with a broader study population.

Systematic comparison of metabolic differences of Uncaria rhynchophylla in rat, mouse, dog, pig, monkey and human liver microsomes.

Metabolites have a close relationship with the efficacy and safety of herbal medicines. However, ubiquitous matrix interferences, complex co-elution, and minor or trace amounts in plasma restrict the comprehensive identification of metabolites. In this study, an efficient strategy comprising a mass defect filter and time-staggered targeted ion lists was established to characterize the metabolites of alkaloids of Uncaria rhynchophylla (UR) for the systematic comparison of metabolic differences in rat, mouse, dog, pig, monkey and human liver microsomes. The mass defect filter model effectively decreased interfering ions by 63-68%, and time-staggered precursor ion lists significantly increased the number of triggered MS/MS fragmentation by 65-120% in liver microsomes of six species. Ultimately, a total of 165 metabolites in the liver microsomes of six species were tentatively characterized, and the main metabolic pathways were demethylation, isomerization, hydrolysis, oxygenation and dehydrogenation. The results showed that the mouse liver microsomes exhibited metabolic behavior most similar to human metabolism of UR alkaloids. We hope that these results provide basic data for further investigation of UR metabolism in different species, and that the strategy can provide a reference for metabolite characterization of herbal medicines in complex biological matrix. Graphical abstract.