Cardenolide-rich fraction of Pergularia tomentosa as a novel Antiangiogenic agent mainly targeting endothelial cell migration

Abstract

Purpose

Angiogenesis related abnormalities underlie several life-threatening disorders. Despite approved therapies, scientists have yet to develop highly efficient, low cost approaches with minimal side effects.

Methods

We evaluated the antiangiogenic activity of 50% hydroalcoholic extracts of Pergularia tomentosa L. root and aerial parts along with their EtOAc and water fractions, in vivo and in vitro. Transgenic zebrafish line Tg(fli1:EGFP) was used for in vivo assay and human umbilical vein endothelial cell (HUVEC) migration test along with possibility of tube formation were performed as in vitro tests. Furthermore, microvasculature in chicken chorioallantoic membrane (CAM) was assessed under P. tomentosa treatment. The fractionation of the 50% hydroalcoholic extracts was led to the identification of the best active fraction in this study. The metabolite profiling of the active fraction was also carried out using LC-HRESIMS analysis.

Results

Pergularia tomentosa markedly inhibited intersegmental vessel (ISV) formation at 48 h post-fertilization (hpf) embryos in zebrafish. The water fraction of root hydroalcoholic extract (PtR2), showed strong antiangiogenic effect with minimal adverse viability impacts. Over 80% of embryos showed more than 50% inhibition in their ISV development at 20 and 40 μg/mL. PtR2 at 20 μg/mL substantially reduced human umbilical vein endothelial cell (HUVEC) migration up to 40%, considerable destruction of the formed tubes in the tube formation and microvasculature in CAM assays. Immunocytochemistry showed a marked reduction in vascular endothelial cadherin (VE-cadherin) abundance at cell junctions concurrent with substantial reduction of phospho-Akt (p-Akt) and β-catenin protein expressions. Phytochemical profile of PtR2 showed a rich source of cardenolide structures, including ghalakinoside, calactin and calotropin derivatives.

Conclusion

Thus, the P. tomentosa cardenolide-rich fraction (PtR2) may hold a considerable promise for an antiangiogenic impact by impairment of endothelial cell (EC) migration and viability.

Graphical abstract

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Abbreviations

CAM:

Chorioallantoic membrane

CGs:

Cardiac glycosides

Dil-Ac-LDL:

Acetylated low-density lipoprotein

EC:

Endothelial cells

EtOAc:

Ethyl acetate

HUVECs:

Human umbilical vein endothelial cells

ISV:

Intersegmental vessel

LC:

Liquid chromatography

LC-HRESIMS:

LC coupled to electrospray ionization and high resolution mass spectrometry

VE-cadherin:

Vascular endothelial cadherin

VEGF:

Vascular endothelial growth factor

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Acknowledgements

The authors would like to thank Fatemeh Radmanesh, Mohammad Rezaei, and Saeed Yakhkeshi for their assistance. We would like to thank Prof. Hossein Baharvand for his kind advices.

Availability of data and material

The data are available upon request.

Code availability

Not applicable

Funding

This work was supported financially by Research and Technology Council of Royan Institute.

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Authors

Contributions

MH performed the experiments and wrote the manuscript. MA helped for phytochemical parts, plant extracts, fractions and manuscript writing. AM performed the Western blot experiments. SPI and AnC performed the LC-HRESIMS analyses and metabolite profiling. AlC helped in design of zebrafish screenings. SPA designed and supervised the research project and wrote the manuscript.

Corresponding author

Correspondence to Sara Pahlavan.

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All animal studies were performed in accordance with guidelines approved by the Ethics Committee of Royan Institute in conformity with the NIH Guide for the Care and Use of Laboratory Animals.

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Electronic supplementary material

Fig 1S
figure6

Zebrafish screening bioassay for extracts obtained from root of Pergularia tomentosa (P. tomentosa). (PNG 6098 kb)

Fig 2S
figure7

Cell viability assay in HUVECs treated with serial concentrations of PtR2. (PNG 6098 kb)

Fig 3S
figure8

LC-HRESIMS profile (negative-ion mode) of the water fraction from the hydroalcoholic root extract of Pergularia tomentosa (P. tomentosa). (PNG 6098 kb)

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High resolution (TIF 24397 kb)

High resolution (TIF 24393 kb)

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Hosseini, M., Ayyari, M., Meyfour, A. et al. Cardenolide-rich fraction of Pergularia tomentosa as a novel Antiangiogenic agent mainly targeting endothelial cell migration. DARU J Pharm Sci (2020). https://doi.org/10.1007/s40199-020-00356-7

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Keywords

  • Pergularia tomentosa
  • Asclepiadaceae
  • Antiangiogenesis
  • Cardenolide
  • VE-cadherin
  • Zebrafish