Insights into Euphorbia diversity: Probing the contrasts between Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata

Graphical abstract Image 1

In traditional Chinese medicine (TCM), Euphorbia fischeriana Steud (E.fischeriana) and Euphorbia ebracteolata Hayata (E.ebracteolata), commonly referred to as "Langdu", are widely extensively utilized for treating lymphatic tuberculosis and ringworm [1].Both plant species are perennial herbaceous plants mainly distributed in northeastern China, Mongolia, Russia (Siberia), and Republic of Korea [2].There have been many reports on the chemical constituents and pharmacological effects of the two plant species, which has made more and more researchers realize that there may be differences between E. fischeriana and E. ebracteolata.In some cases, long-term improper use of herbal medicines can even lead to life-threatening conditions [3,4].Therefore, it is essential to employ an effective technology to differentiate between these two plants based on their chemical constituents and biological activities, so as to reduce the harm caused by the mixing and misuse of medicinal materials.Therefore, the present paper describes a study of the differences between E. ebracteolata and E. fischeriana, using untargeted plant metabolomics and biological activity evaluations.This study aims to provide valuable insight into their equivalence and potential interchangeability in TCM and clinical medication.
To better understand the chemical constitutions of the E. fischeriana and E. ebracteolata, the primary and secondary metabolites were identified by the ultra-high-performance liquid chromatography-Q Exactive mass spectrometry (UHPLC-QE-MS) platform (Supplementary data).By comparing the MS/MS spectral data with the standard compounds, literature, and METLIN database, peaks 1e31 were identified and annotated, as presented in Table S1 [5].Chemotaxonomically, the identified or annotated metabolites mainly belonged to terpenoids and acetophenone derivatives (Fig. 1A).Nearly half of the terpenoids are abietane diterpenoids, and the remaining consist of three atisane diterpenoids, one pimarane diterpenoid, three tigliane diterpenoids, one triterpenoid, and two unclassified diterpenes (Fig. 1B).Acetophenone derivatives include six acetophenone glycosides, two ordinary acetophenones, and one acetophenone dimer (Fig. 1C).
The identification results showed unique chemical components for each species.Therefore, we used untargeted plant metabolomics techniques to study the metabolite differences between them.As illustrated in Fig. 1D, samples were divided into two clusters on the PC1 (73.9 %) vs. PC2 (11.3 %) plane.In addition, to further obtain the metabolites that made important contributions to distinguish the two species, a partial least-squares discriminant analysis (PLS-DA) model was performed (Fig. 1E).When the top three components are generated, the R 2 (cumulative) and Q 2 (cumulative) are 0.985 and 0.972, respectively (Fig. S1), suggesting the applicability and predictability of the model.Similar results were also obtained from PLS-DA, two distinct clusters had variance values of 73.9% and 9% on component 1 and component 2, respectively.The difference in metabolites between the two species was also obvious by heatmap analysis (Fig. S2).Then, the variable importance value (VIP) and fold change (FC) value were calculated, and the key components were screened according to the condition of VIP > 1.2 and FC > 2.0 (Fig. S3 and Table S2).The relative contents of these markers are shown in Fig. 1F, among which peaks 12, 14, 13, and 28 are more abundant in E. fischeriana, and corresponding peaks 19, 15, 24, and 27 are more abundant in E. ebracteolate.According to the results of plant metabolomics, we found that the metabolic components of E. fischeriana and E. ebracteolata were significantly different, and selected eight key marker metabolites to distinguish the two species.In addition, the results of plant metabolomics on the same species in different regions indicated that different habitats have little effect on the composition of metabolites (Table S3 and Figs.S4 and S5).
A systematic phytochemical study was conducted to better understand the differences in chemical compositions and biological activities between the two plants.Twelve representative compounds (compounds 3e4 and 9e12 from E. fischeriana; compounds 1e2 and 5e8 from E. ebracteolata) were isolated from two plants using various chromatographic methods such as negative pressure silica gel, octadecylsilyl, Sephadex LH-20, and preparative and semi-preparative high performance liquid chromatography (HPLC) (Figs.S6eS31).Structurally, compounds 1e12 (Fig. 2A) could be mainly classified into three structural types, including acetophenone and acetophenone glycosides (compounds 1e5), abietane diterpenoids (compounds 6e9), and tigliane diterpenoids (compounds 10e12).Compounds 5 and 9e11 were the iconic different metabolites of E. fischeriana and E. ebracteolata, selected by previous metabolomics research on plants.This work of extraction and isolation provided the material basis for further comparison of the biological activities of the two plants.
To clarify whether there is any difference in antitumor activity between E. fischeriana and E. ebracteolata, the inhibitory effects of compounds 1e12 and extracts against Hep-G2, A549, HCT116, and AGS were tested (Table 1 and Fig. 2B).The results of the antitumor activity test in this study showed that the extract of E. fischeriana exhibited stronger antitumor activity than those of E. ebracteolate.Combined with literature reports and results of compounds 1e12, diterpenoids had the most significant antitumor activity.Metabolomics studies have shown that E. fischeriana contains more diterpenoids, especially tigliane diterpenoids, which may account for the difference in antitumor activity between the two species.
Antioxidant activity plays a key role in studying the difference in pharmacological activity between E. ebracteolate and E. fischeriana.The free radical scavenging activities of compounds and extracts, along with vitamin C were evaluated by using 2,2 0 -azino-bis(3ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay methods, respectively (Figs. 2C and D).Analyzing the results of the antioxidant activity of compounds 1e12, it was found that acetophenone derivatives (compounds 1e5) have the strongest antioxidant capacity, which also suggested that they were the key secondary metabolites with significant antioxidant activity of the two species.In addition, the experimental results showed that extracts of E. ebracteolata had stronger antioxidant activity than extracts of E. fischeriana.12.50 12.50 3.12 6.25 2,4-Dihydroxy-6-methoxy-3-methyl acetophenone (compound 5) 6.25 12.50 6.25 3.12 17-Hydroxyjolkinolide B (compound 6) 12.50 6.50 3.12 3.12 Jolkinolide B (compound 7) 6 The antibacterial activity of compounds 1e12 and extracts were tested employing Gram-positive bacteria Staphylococcus aureus (S. aureus) and Bacillus subtilis (B.subtilis) and Gram-negative bacteria Escherichia coli (E.coli) and Pseudomonas aeruginosa (P.aeruginosa), respectively (Table 2 and Fig. S32).Berberine and penicillin were selected as positive drugs.From the minimum inhibitory concentration (MIC) results of compounds 1e12, it could be seen that the abietane diterpenoids, the common component of the two plants, have the most significant antibacterial activity, which indicated that abietane diterpenes may be the material basis for their antibacterial activity.Further experimental results showed the extracts of E. fischeriana and E. ebracteolata showed strong antibacterial activity, which also verified the above conclusions.More detailed analysis and discussion of the activity results are shown in the Supplementary data.
In this study, non-targeted metabolomics techniques were used to analyze the metabolite differences between E. ebracteolata and E. fischeriana, which were not obvious by the simple analysis of phytochemical research and chromatograms.In addition, the difference in antibacterial, antitumor, and antioxidant activities of the two species was evaluated.These results showed that E. ebracteolata and E. fischeriana not only have significant differences in chemical composition but also show different effects in antibacterial, antitumor, and antioxidant.In summary, this study demonstrated that the mixture and misuse of E. ebracteolata and E. fischeriana in industrial production and clinical use should be avoided.Moreover, this study demonstrated the efficacy of integrating metabolomic and bioactive strategies for distinguishing medicinal materials with similar appearances and morphologies, ultimately providing promising prospects in the identification and utilization of medicinal plants.

Declaration of competing interest
The authors declare that there are no conflicts of interest.

Fig. 1 .
Fig. 1. Results of plant metabolomics analysis.(A) Structural classification of the 31 metabolites identified.(B) Structural classification of terpenoids.(C) Structural classification of acetophenones.(D) Scores plot of principal component analysis (PCA) for liquid chromatography-mass spectrometry (LC-MS) metabolomics.(E) Scores plot of partial least-squares discriminant analysis (PLS-DA) for LC-MS metabolomics.(F) Relative content of eight metabolites that make the most significant contribution to distinguishing the two species.PC: principal component; EE: Euphorbia ebracteolata Hayata.EF: Euphorbia fischeriana Steud.