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Flow cytometric estimation on cytotoxic activity of leaf extracts from seashore plants in subtropical Japan: isolation, quantification and cytotoxic action of (‐)‐deoxypodophyllotoxin
Author(s) -
Masuda Toshiya,
Oyama Yasuo,
Yonemori Shigetomo,
Takeda Yoshio,
Yamazaki Yuko,
Mizuguchi Shinichi,
Nakata Mami,
Tanaka Tomochika,
Chikahisa Lumi,
Inaba Yuzuru,
Okada Yoshihiko
Publication year - 2002
Publication title -
phytotherapy research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.019
H-Index - 129
eISSN - 1099-1573
pISSN - 0951-418X
DOI - 10.1002/ptr.902
Subject(s) - cytotoxic t cell , bioassay , apoptosis , biology , cytotoxicity , cell culture , ethidium bromide , traditional medicine , chemistry , botany , biochemistry , in vitro , medicine , dna , genetics
The cytotoxic activity of methanol extracts of leaves collected from 39 seashore plants in Iriomote Island, subtropical Japan was examined on human leukaemia cells (K562 cells) using a flow cytometer with two fluorescent probes, ethidium bromide and annexin V‐FITC. Five extracts (10 μg/mL) from Hernandia nymphaeaefolia, Cerbera manghas, Pongamia pinnata, Morus australis var. glabra and Thespesia populnea greatly inhibited the growth of K562 cells. When the concentration was decreased to 1 μg/mL, only one extract from H. nymphaeaefolia still inhibited the cell growth. A cytotoxic compound was isolated from the leaves by bioassay‐guided fractionation and was identified as (‐)‐deoxypodophyllotoxin (DPT). The fresh leaves of H. nymphaeaefolia contained a remarkably high amount of DPT (0.21 ± 0.07% of fresh leaf weight), being clarified by a quantitative HPLC analysis. DPT at 70–80 p M started to inhibit the growth of K562 cells in an all‐or‐none fashion and at 100 p M or more it produced complete inhibition in all cases. Therefore, the slope of the dose‐response curve was very steep. DPT at 100 p M or more decreased the cell viability to 50%–60% and increased the number of cells undergoing apoptosis (annexin V‐positive cells). The results indicate that DPT contributes to the cytotoxic action of the extract from the leaves of H. nymphaeaefolia on K562 cells. Copyright © 2002 John Wiley & Sons, Ltd.