Distribution of ingenol derivatives in Euphorbia plants and UV-dependent promotion of ingenol production in greenhouse-grown Euphorbia plants
Introduction
Euphorbia L., belonging to the spurge family (Euphorbiaceae), is one of the largest genera in the flowering-plant kingdom. It contains more than 2000 species worldwide, which are particularly distributed across drier areas of the tropics and subtropics. Taxon of Euphorbia is difficult and complex due to inadequate taxon sampling and morphological diversity. The morphological diversity in this genus includes herbs, shrubs, trees, and various xerophytic and succulent forms. Many Euphorbia species are widely utilized as biofuel plants (Escrig et al., 2013; Hastilestari et al., 2013) or folk medicine (Ernst et al., 2015; Yener et al., 2018;) and are also well-known to be toxic and irritant to skin. Ingenane diterpenoids are considered as the major irritants and tumor-promoting constituents in the spurge family (Fürstenberger and Hecker, 1985; Opferkuch and Hecker, 1974; Zayed et al., 2001) and have shown considerable potential as lead compounds in drug discovery (Huang et al., 2019; Jin et al., 2015; Liu et al., 2018). Ingenane-type diterpenoids have a complex skeleton of 5/7/7/3 fused-ring featuring a ketone bridge between C-8 and C-10. Generally, there may be some functionalities (hydroxyl, benzoyl, or long-chain alkyl ester) located at C-3, C-5, C-13 or C-17. The distribution of ingenane diterpenoids is known limited to Euphorbia and Mabea Aubl. (Brooks et al., 1990), the two genera in the Euphorbiaceae family. To date, about 180 ingenane diterpenoids have been isolated and characterized from more than 60 Euphorbia species (Appendino, 2016). The ingenol ester, ingenol mebutate (ingenol-3-angelate), first isolated from E. peplus L., is one of the most well-studied macrocyclic diterpenoids for its remarkable pharmaceutical activity. Ingenol mebutate gel has been approved by the U.S. Food and Drug Administration for the treatment of actinic keratosis (Gras, 2013; Lebwohl et al., 2012) and applied to treat basal cell carcinoma (Bettencourt, 2016; Izzi et al., 2016) and non-melanoma skin cancer (Siller et al., 2010). Currently, ingenol mebutate is mainly produced by semi-synthesis from ingenol instead of direct isolation from E. peplus for the low yield of only 1.1 mg/kg from the plant tissues (Hohmann et al., 2000). Ingenol production is mainly isolated from natural materials, such as seeds of E. lathyris L. at yields exceeding 100 mg/kg (287.35 μmol/kg) (Appendino, 2016).
Many reliable methods were developed to determine the ingenol derivatives content in plant samples considering the biomedical importance and commercial value of ingenol. The total content of ingenol derivatives can be quantified by determination of the free ingenol content in their hydrolysates (Girin et al., 1993). Béres et al. (2018) developed a method for quantitative analysis of hydrolyzed ingenol with 18O-labelled ingenol analogue via UHPLC-MS/MS and analyzed 38 species of the Euphorbia genus. Ingenol derivatives also can be identified by the implementation of different mass spectrometric techniques including ion trap MS/MS, high-resolution MS/MS and ultra-high resolution quadrupole-time-of-flight (Q-TOF) MS/MS (Nothias-Scaglia et al., 2015; Sobottka et al., 2016; Vogg et al., 1999; Yener et al., 2019).
The aim of this study is to search more applicable materials for ingenol production. A sensitive, robust method utilizing UHPLC with tandem MS was applied to determine the total content of ingenol derivatives in 61 selected species from Euphorbia genus and the relative taxa and 13 Euphorbia plants cultivated under different greenhouse covering materials. Metabolite patterns of different plants were obtained and further analyzed the similarities and differences of the chemical composition among these Euphorbia plants using principal component analysis (PCA) and hierarchical cluster analysis (HCA). Additionally, the results provided a meaningful basis for the industrial production of ingenol and comprehensive utilization and subsequent research of Euphorbia plants.
Section snippets
Chemicals
All the chemicals used in preparation for sample solution, including methanol, acetic acid and sodium hydroxide (NaOH), were analytical grade. Ultrapure water was obtained from a Miaozhiyi MZY-U system (Miaozhiyi Electronic Technology co., Ltd, Nanjing, China). Acetonitrile (HPLC grade) and formic acid (HPLC grade) used for UHPLC/Q-TOF-MS were purchased from Merck (Darmstadt, Germany) and Roe Scientific Inc. (Newark, DE, USA), respectively. Ingenol (purity > 98 %, checked by UHPLC-MS and 1H
Method validation
To analyze the total content of ingenol derivatives in Euphorbia plants, reliable methods for sample-preparation and determination were developed and validated. In positive MS mode, the protonated molecular ion (m/z 349 [M+H]+) of ingenol molecule was formed and characteristic fragment ions of ingenol were produced by H2O loss (m/z 18) from molecular ion, including m/z 331 [M+H - H2O]+, 313 [M+H - 2H2O]+, 295 [M+H - 3H2O]+, and 277 [M+H- 4H2O]+. In the MS spectrum, m/z 366 [M + NH4]+, m/z 371
Conclusion
In this study, hydrolysates of 50 Euphorbia plants, 9 plants from other Euphorbiaceae genera and 2 Phyllanthaceae plants were prepared by uniform procedure and analyzed by UHPLC-Q-TOF-MS under the same conditions. The contents of total ingenol derivatives in the methanol extracts of 61 Euphorbia species were reported for the first time. Contents of the free ingenol produced from ingenol esters were calculated, with the highest content (527.82 ± 38.19 μmol/kg of dry herb weight) in E.
CRediT authorship contribution statement
Meixiang Chen: Investigation, Methodology, Analysis, Resources. Mei Tian: Resources, Analysis, Validation, Writing - original draft, Writing - review & editing, Funding acquisition. Fei Liu: Resources, Validation, Supervision, Funding acquisition. Wanli Zhao: Analysis, Writing – review. Jian Peng: Methodology, Analysis. Linwei Li: Resources, Supervision, Funding acquisition. Min Yin: Supervision, Resources, Software. Yonghua Gu: Resources, Supervision, Writing – review. Xu Feng:
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported financially by the National Natural Science Foundation of China [grant number: 31770383, 31970375] and Natural Science Foundation of Jiangsu Province [grant number: BK20200295, BK20180311, BK20200287].
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Integrating metabolomics and transcriptomics to unveil the spatiotemporal distribution of macrocyclic diterpenoids and candidate genes involved in ingenol biosynthesis in the medicinal plant Euphorbia lathyris L.
2022, Industrial Crops and ProductsCitation Excerpt :Therefore, ingenol, the representative compound of ingenane-type diterpenoids with multiple hydroxyl groups, is of great value in research, development and application due to their ability to form a variety of active ingredients after esterification. However, ingenol is mainly distributed in trace amounts in Euphorbia plants (Appendino et al., 1999; Chen et al., 2021; King et al., 2014). The total chemical synthesis of ingenol is also complicated, giving a yield of less than 1% (Jørgensen et al., 2013).
- 1
These two authors contributed equally to this work.