ABSTRACT
One hundred thirty-seven species of traditional Chinese herbal medicines belonging to 61 families were collected from Yunnan and Sichuan province in the People's Republic of China and tested for cell cycle inhibitory activity against tsFT210 cell line. Thirty-one traditional Chinese medicines from 18 families showed bioactivity, and the ethanol extract of Rubus aleaefolius. Poir. rhizome and Trewia nudiflora. L. rhizome exhibited the strongest inhibitory activity on cell cycle progression at the G0/G1 and G2/M phases, respectively. The screening results indicated that this bioassay is suitable for screening bioactive natural products from herbal medicines.
Introduction
In the general condition, if the control process of the cell cycle were deregulated, the undesired and unlimited proliferation of cancerous cells would occur, leading to an imbalance and subsequently developing to cancer. Therefore, chemical agents inhibiting the cell cycle might be useful to regulate the balance and thus possess the potential to cure cancer. From this viewpoint, a screening bioassay of detecting cell cycle inhibitory activity using mammalian tsFT210 cells (Th'ng et al., Citation1990; Osada et al., Citation1997) was developed. In this paper, we report the successful application of the bioassay in screening anticancer compounds from traditional Chinese herbal medicines on a large scale. One hundred thirty-seven species of herbal medicines belonging to 61 families were collected from Yunnan and Sichuan province in the People's Republic of China. The ethanol and aqueous extracts of each plant material have been tested.
Materials and Methods
Traditional Chinese medicines
One hundred thirty-seven species of traditional Chinese herbal medicines belonging to 61 families were collected from various areas in China and were identified by Prof. Qishi Sun, Division of Pharmacognosy, Shenyang Pharmaceutical University, where voucher specimens are deposited.
Preparation of extracts
Aqueous extracts: Powdered dry material (5 g) was boiled with 50 ml water for 30 min and the filtrate was freeze-dried. Ethanol extracts: Powdered dry material (5 g) was refluxed with 95% aqueous EtOH for 45 min at 90°C. Extraction was repeated twice. The extract was concentrated and freeze-dried.
Sample preparation
Aqueous extract was dissolved in 50% MeOH and ethanol extract was dissolved in 90% MeOH in a final sample concentration of 10.0 mg/ml. If necessary, the test sample was first dissolved in 100 µl. DMSO and then diluted with 900 µl 90% MeOH to given a final concentration of 10.0 mg/ml.
Bioassay
The mouse ts-FT210 cell line, which is a temperature-sensitive p34cdc2. mutant isolated from the mouse mammary carcinoma cell line FM3A (Th'ng et al., Citation1990; Osada et al., Citation1997), was used for the bioassay.
The cells were routinely maintained at 32°C in RPMI-1640 medium supplemented with 10% fetalbovine serum (FBS) under a humidified atmosphere of 5% CO2. During the testing, 5 µl of the sample solution was added into each well of a 24-well plate containing the cells at the density of 2 × 105 cell/ml in 0.5 ml of fresh medium, and the final concentration was 100 µg/ml: then, the cells were asynchronousy incubated at 32°C for 17 h. The cells in each well of the 24-well plate were respectively transferred into 1.5-ml; Eppendorf centrifuge tubes and harvested by centrifugation at 3000 rpm for 3 min at 4°C. The cells were washed once with cold phosphate-buffered saline (PBS) and harvested again by centrifugation under the same conditions. Then, 150 µl of propidium iodide solution (propidium iodide 50 µg/ml, sodium citrate 0.1%, and Nonidet P-40 0.2%) was added into the tubes, and the cells were stained at 4°C for 30 min. The cells were then subjected to a flow cytometric analysis, and the distribution within the cell cycle was determined The activities were evaluated by comparing the change of percentage content of the DNA in the cell cycle progression, such as G0/G1 and G2/M phases. The bioactivity index “+++,” “++” “+,”was represented as the increasing rates of DNA content of above 50%, between 25–50%, and 10–25% in the various cell cycle distributions, respectively. All the active samples were determined twice.
Results
Different organs of 137 traditional Chinese medicines (TCMs) belonging to 61 families were tested for cell cycle inhibitory activity against tsFT210 cell line. The results are shown in . Four aqueous extracts and 28 ethanol extracts showed various cell cycle inhibitory activities against the mouse ts-FT210 cell line. Ten extracts belonging to nine families possessed inhibitory activities on cell cycle progression of tsFT210 cells at the G0/G1 phase, including Fissistigma maclurei. stem (Annonaceae) (no. 8, +), Pofyathia chilensis. bark (Annonaceae) (no. 12, +), Betula alnoides. bark (Betulaceae) (no. 21, +), Radermachera microcalyx. bark (Bignonaceae) (no. 24, +), Securinega virosa. root (Euphorbiaceae) (no. 48, +), Engelhardtia serrata. bark (Juglandaceae) (no. 56, +), Crotalaria acicularia. branch (Leguminosae) (no. 75, +), Rubus aleaefolius. rhizome (Rosaceae) (no. 109, ++), Helicteres angustifolia. whole plant (Sterculiaceae) (no. 122, +), and Syzygium szemaoense. bark (Uyrtaceae) (no. 127, +). Among all the tested samples, the ethanol extract of it R. aleaefolius. rhizome showed the strongest activity. Twenty-one extracts belonging to 13 families showed inhibitory activities on cell cycle at G2/M phase, including Fissitigma acuminatissimum. branch (Annonaceae) (no. 7, +), Fissistigma oldhamii. stem (Annonaceae) (no. 9, +), Fissistigma retusum. bark (Annonaceae) (no. 10, +), Goniothalamus chilensis. bark (Annonaceae) (no. 11, ++), Dioscorea camposita. rhizome (Dioscoreaceae) (no. 37, ++), Euphorbia hirta. whole plant (Euphorbiaceae) (no. 44, +), Trewia nudiflora. rhizome (Euphorbiaceae) (no. 49, +++), Gnetum montanum. stem (Gnetaceae) (no. 50, +), Aesculus assamica. root (Hippocastanaceae) (no. 54, ++), Pterocarya tonkinensis. rhizome (Juglandaceae) (no. 57, +), Lindera strychnifolia. branch (Lauraceae) (no. 65, +), Albizzia lucidior. bark (Leguminosae) (no. 70, +), Etythrina lithosperma. stem (Leguminosae) (no. 76, +), Millettia dielsiana. trig (Leguminosae) (no. 79, +), Diploclisia glaucescens. trig (Menispermaceae) (no. 91, +), Pericampylus glaucus. branch (Menispermaceae) (no. 92, ++), Measa indica. root (Myrsinaceae) (no. 96, +), Canarium bengalensis. bark (Polygalaceae) (no. 104, +), Zanthoxylum acanthopodium. bark (Rutaceae) (no. 114, ++), Zanthoxylum planispinum. rhizome (Rutaceae) (no. 116, +), and Selaginella referi. whole plant (Selaginellaceae) (no. 117, +). Among all the tested samples, the ethanol extract of T. nudiflora. rhizome exhibited the strongest activity.
Table 1.. The effect of ethanol and aqueous extracts of traditional Chinese medicines (TCMs) against tsFT210 cell line.
Discussion
The need for antitumor and anticancer agents from natural sources contributes to the development of effective, rapid screening systems. The bioassay detecting the cell cycle progression of tsFT210 cell has proved successful in screening TCMs on a larg scale. With the bioassay, 19 bioactive compounds have been isolated from R. aleaefolius. by bioactivity-guided fractionation (Zhao et al., Citation2001; Cui et al., Citation2002Citation2003). Other active extracts are to be applied for further investigation to isolate bioactive components.
Acknowledgments
We are grateful to Prof. Qishi Sun (Division of Pharmacognosy, Shenyang Pharmaceutical University, People's Republic of China) for his contributions on the collection and identification of the plant material. We also thank Mrs. Gexia Qu (Department of Natural Products Chemistry, Shenyang Pharmaceutical University, People's Republic of China) for preparing the ethanol and aqueous extract. Great thanks are also given to Dr. Y.Z. Tian (Antibiotic Research Group of Physico-chemical Institute, Japan) for providing the tsFT210 cell.
References
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