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.
Masuda
Toshiya
T
Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan. masuda@ias.tokushima-u.ac.jp
Oyama
Yasuo
Y
Yonemori
Shigetomo
S
Takeda
Yoshio
Y
Yamazaki
Yuko
Y
Mizuguchi
Shinichi
S
Nakata
Mami
M
Tanaka
Tomochika
T
Chikahisa
Lumi
L
Inaba
Yuzuru
Y
Okada
Yoshihiko
Y
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