Planta Med 2006; 72 - S_025
DOI: 10.1055/s-2006-949758

Biological activity of a putative 50-kDa protein purified from Tinospora rumphii Boerl

J Charmain 1, O Bonifacio 1, RR Matias 2, FF Natividad 2
  • 1Philippine Institute of Traditional and Alternative Health Care, Atlanta Centre, 31 Annapolis St, San Juan, Metro Manila, Philippines
  • 2Research and Biotechnology Division-St. Luke's Medical Center, E. Rodriguez Blvd., Quezon City, Philippines

Tinospora rumphii Boerl. locally known in the Philippines as makabuhay is one of the most common plants being used to treat various ailments. Aqueous plant extracts are prescribed in the treatment of indigestion, diarrhea, scabies and topical ulcers. A 50-kDa protein purified from the stem was assayed in vitro for its cytotoxic activity in five human cell lines (HeLa, LIM 1215, HT-29, Jurkat and a normal cell line – fetal skin fibroblast). The apoptosis-inducing activity was likewise investigated by flow cytometry, DNA staining and DNA fragmentation using the same set of cell lines as the target cells. Genes that are upregulated in the cells treated with the purified 50-kDa protein were identified by differential display reverse transcription polymerase chain reaction (DDRT-PCR). Five clones from each sample were sequenced and analyzed.

In all cell lines studied, the 50-kDa protein demonstrated strong cytotoxicity (IC50 from 4 to 6.5 ng/µl) and induced cell death in a dose-dependent manner. Typical morphological and biochemical features of apoptosis including cell shrinkage, chromatin condensation, DNA ladder formation, phosphatidylserine expression using Annexin V were observed in all cell lines used in the study. From DDRT-PCR, a total of 176 genes were differentially expressed, 65 of which were upregulated and 111 were down regulated. Four cDNAs were successfully cloned and sequenced. The sequences showed homology to transcription factors, a chemokine receptor and a voltage-dependent anion channel. The identification of these genes may lead to the elucidation of the molecular mechanism of action of the cytotoxic activity of the protein.

Acknowledgments:

Philippine Council for Health Research and Development – Department of Science and Technology

Research and Biotechnology Division – St. Luke's Medical Center

Institute of Biology – University of the Philippines Diliman

References: 1. Mosmann, T. (1983), J. Immunol. Methods 65: 55–63. 2. Liang, P., Pardee, A. (1992), Science 257: 967–971.