Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

Moe Nishimura,Nobuo Kawahara,Hiroko Nakayama,Hitomi Kawakami,Yasuhito Shimada,Kaoru Takayanagi,Hiroyuki Fuchino

doi:10.3390/ijms22126354

Abstract

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 μM, and 20.2 μM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.

Highlights

Cell-based screening identified that methanol extracts of Styrax japonica Siebold et al Zuccarini (SJSZ) (10 μg/mL) strongly suppressed PC-3 cell viability
1.6 ± 0.3 μM, respectively (Figure 1d). We confirmed that these jegosaponins suppressed the viability of other human prostate cancer cell lines, namely DU145 and
SJSZ extract contains anti-cancer constituents, which act against human prostate cancer cells, as subsequently identified them as two jegosaponins, jegosaponin A and B

Summary

Introduction

Drug discovery from plants has been ongoing since ancient times and is still undergoing various modified protocols, such as high-throughput screening and in vivo testing, including in zebrafish [3,4,5]. Styrax japonica Siebold et al Zuccarini (SJSZ) is a shrub of the family Syracaceae, native to Japan, China, and Korea. The species possesses high commercial, nutritional, and therapeutic values, and has long been used conventionally by many communities for treating oral and dental diseases and respiratory ailments [6]. Several bioactive molecules have been identified in SJSZ in previous studies. Phytoglycoprotein (38 kDa) from SJSZ suppresses cell proliferation in human carcinomatous cells [9,10] and mice [11]

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Conclusion