Medicinal Plant Properties

About the Thailand Institute of Scientific and Technological Research

The Thailand Institute of Scientific and Technological Research (TISTR) was originally set up by the Applied Scientific Research Corporation of Thailand in 1963. In 1979 this was completely replaced by the TISTR- a non-profit making state enterprise under the Ministry of Science and Technology . Its mission is "to create research facilities for developments of the country, natural resources, industry and national projects."

The TISTR, based in Bangkok has research and development departments looking at areas such as biotechnology, food technology, engineering, and materials technology etc. The Pharmaceutical and Natural Products Department (PNPD), looks to improve Thai medicinal plants in order to meet international standards for domestic use and exportation. The department provides a range of phytochemical & chemical analyses and biological assays for the government and private sector.

Anti-oxidative DNA damage induced by phenolic compounds

Dr. Prapaipat Klungsupya and her colleagues at PNPD have been researching anti-oxidative DNA damage induced by phenolic compounds using the Comet assay in human lymphoblastoid TK 6 cells.

The emphasis of this research is on Reactive Oxygen Species (ROS), which are normally generated as part of the defence system. These ROS are potential oxidative agents that could convert to free-radicals. If present in excess, these can lead to inactive enzymes, oxidized lipids and DNA damage, which are linked to ageing and an array of age related illnesses such as heart disease, Alzheimer and cancer. There are, however, plants that contain high quantities of naturally occurring antioxidant compounds. These antioxidant properties are expressed when the compound interacts with the ROS. In Prapaipat Klungsupia's research, 5 phenolic compounds were investigated:

• α-Tocopherol - otherwise known as Vitamin E
• Curcumin - a compound extracted from the rhizomes of the Curcuma longa plant
• Quercetin - a bioflavonoid compound, present in many plants e.g. Ginkgo biloba.
• Piperine - which comes from the Asian vine, Piper nigrum and serves as the source of the hotness of black & white pepper.
• Rutin - another bioflavonoid compound, which is contained, in high quantities, in buckwheat grain Fagopyrum sp.

Hydrogen Peroxide (H₂O₂) is a strong oxidising agent and has been used in this test to induce oxidative DNA damage and therefore represents the ROS. The single cell gel electrophoresis assay (SCGE), also known as the comet assay, was used to measure the anti-oxidative (and therefore anti-genotoxic effects) of those five compounds in the presence of H₂O₂, on the DNA of human lymphoblastoid cells (TK 6).

Cultured TK 6 cells (ATCC CRL8015) were exposed to various concentrations of each of the five phenolic compounds overnight at 37°C. Following the removal of the test compounds, the cells were treated with H₂O₂ at 4°C in darkness, to induce oxidative DNA damage. The treated cells were then harvested, washed to remove H₂O₂, embedded in low melting point agarose and layered onto slides covered with a layer of normal melting point agarose. Slides were then immersed in lysing solution, placed on an electrophoretic tray with alkaline buffer and allowed to equilibrate. Electrophoresis was performed for 20 minutes and the slides were then neutralised with Tris buffer and stained with Ethidium bromide. Using an Olympus fluorescence microscope at both100x and 400x magnification, the slides were observed for comets. The Comet Assay III image analysis system was used to randomly score at least 50 cells per slide for Tail length and Tail moment. The data obtained from this analysis was then transferred to an MS Excel spreadsheet for processing .

Results show that all 5 phenolic compounds displayed antioxidative properties at concentrations ranging from 25-100 µM when exposed to the hydrogen peroxide (H₂O₂). This indicates the inhibitory effect the compounds have on oxidative damage induced by hydrogen peroxide in human lymphoblastoid TK 6 cells.

Useful links

Thailand Institute of Scientific and Technological Research

Contact

Prapaipat Klungsupya - prapaipat@tistr.or.th