Flow cytometric estimation on cytotoxic activity of leaf extracts from seashore plants in subtropical Japan: isolation, quantification and cytotoxic action of (-)-deoxypodophyllotoxin.
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 microg/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 microg/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 pM started to inhibit the growth of K562 cells in an all-or-none fashion and at 100 pM or more it produced complete inhibition in all cases. Therefore, the slope of the dose-response curve was very steep. DPT at 100 pM 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.
Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan. masuda@ias.tokushima-u.ac.jp
Toshiya
T
Masuda
Yasuo
Y
Oyama
Shigetomo
S
Yonemori
Yoshio
Y
Takeda
Yuko
Y
Yamazaki
Shinichi
S
Mizuguchi
Mami
M
Nakata
Tomochika
T
Tanaka
Lumi
L
Chikahisa
Yuzuru
Y
Inaba
Yoshihiko
Y
Okada
D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal; D005434:Flow Cytometry; D006801:Humans; D007564:Japan; D020014:K562 Cells; D019684:Magnoliopsida / Q000737:chemistry; D000432:Methanol; D010936:Plant Extracts / Q000737:chemistry* / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry*; D011034:Podophyllotoxin / Q000031:analogs & derivatives*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004365:Drugs, Chinese Herbal; D010936:Plant Extracts; C014451:deoxypodophyllotoxin; D011034:Podophyllotoxin; D000432:Methanol