By Kenneth Tan, Marcus Tan and Ng Jing Hao Hwa Chong Institution

1. Investigating the synergism and antagonism between Traditional Chinese Medicines By Kenneth Tan, Marcus Tan and Ng Jing Hao Hwa Chong Institution Preparation of TCM Extracts Combination of TCM Extracts Test for synergy and antagonism between TCMs via observation of Daphnia sp. heart rate TEST ORGANISM ABSTRACT Traditional Chinese Medicine works on the combined effects of a variety of plant or animal materials. When certain herbs with similar characteristics are combined together, they can achieve remarkable synergistic effects. However, some combinations of medicinal materials result in the primary effect of a substance is being reduced by or nullified by another. As there is limited research to explain how TCM works, this project aims to find out the synergism and antagonism between selected TCMs. Daphnia sp., also known as water flea, has been selected as our test organism due to its use in other related studies. As it is transparent, their heart rate can be observed easily under a dissecting microscope and serves as an indicator of the extent of synergism or antagonism between the selected TCM and plant extracts in our study. Our results show that the effects for plants-based preparations on the heart rate are much stronger than the effects for animal-based preparations. The strongest antagonism was observed between the concoction of 10% Scorpion and 5% Dan-Shen while the weakest antagonism was observed between the concoction of 10% Scorpion and 15% Dang Gui. INTRODUCTION Traditional Chinese Medicine (TCM) works on the combined effects of a range of medicines (Yang et al, 2009). However, there have been many doubts about the effectiveness of many treatments using TCM, due to factors such as the environment the plant material was grown under, or probably due to certain beliefs in culture (Astin, 1998). The Chinese believe that herbs with similar characteristics, when combined together, can achieve remarkable synergistic effects. For example, the combination of Gypsum sp. and Anemarrhenae asphodeloidis enhances the effect of clearing heat from the body, and the combination of Rhizoma rhei and Mirabilitum sp. enhances the effect of purging the intestines (Wu, 2005). Some combinations of medicinal materials result in the primary effect of a substance being reduced by or nullified by the other substance, which also lead to doubts about the effectiveness of many treatments using TCM. Semen raphani is said to antagonize Radix ginseng because the former weakens the function of tonifying Qi by Radix ginseng (Wu, 2005). Hence, the objective of this project is to study the antagonism and synergy between the selected TCMs. METHOD Outline of method Animal or/and plant based preparations were weighed Preparations were boiled for 20 minutes Boiled extracts were left to cool to room temperature Extracts were centrifuged and supernatants obtained were transferred to petri dish containing cotton wool Screening extracts for synergy or antagonism by observing their effects on the heart rate of Daphnia sp. MATERIALS AND APPARATUS The heartbeat of the Daphnia sp. was monitored and recorded after 5 minutess of immersion in extracts. TCM materials were then soaked in the appropriate amount of deionised water for 30 minutes The water level in the beaker was marked Where necessary, preparations were centrifuged at 5000 RPM for 10 minutes to obtain supernatant Daphnia sp. from the beaker containing the suspension and animal/plant-based preparations were transferred to a centrifuge tube The heartbeats of 5 Daphnia sp. were counted for 1 minute. RESULTS & DISCUSSION p-value = 4.69 x 10-4 p-value = 5.34 x 10-5 p-value = 1.77 x 10-3 10% Scorpion extract has the weakest inhibitory effect. 1% Leech extract has the strongest inhibitory effect. Strongest antagonism was observed between10% Scorpion and 5%Dan-Shen. Weakest antagonism was observed between 10% Scorpion and 15% Dang Gui. Antagonism was observed between the Weak animal-based inhibitory TCM extracts. Strongest synergism was observed between the Weak plant-based inhibitory TCM extracts. Weakest synergism was observed between the Strong plant-based inhibitory TCM extracts. CONCLUSION Our results show that all the TCMs used in this study inhibit the heart rate of Daphnia sp. From the results, the strongest antagonism occurred between the 10% Scorpion and 5% Dan-Shen while the weakest antagonism was observed between 10% Scorpion and 15% Dang Gui. Synergism occurred in the treatment of highest plant extracts and lowest plant extracts, and highest animal extracts on the Daphnia sp. Antagonism was observed for the treatment of lowest animal extracts. With a p-value of less than 0.05 for all experiments, it shows that the effects of the various treatments are significantly different from one another. Findings of this study provide preliminary data for in-depth research of interactions between TCMs which can provide the basis of how they should be combined in prescriptions to improve their effectiveness in treatments of diseases. p-value = 2.39 x 10-3 5% Dan-Shen extract has the weakest inhibitory effect. 15% Dang Gui extract has the strongest inhibitory effect. Antagonism was observed between the 15% Dang Gui and5% Dan- Shen. Bulus, R.G.D. and Ronco, A.E. (1998). Acute toxicity bioassay using Daphnia obtuse as a test organism. Environmental Toxicology and Water Quality, 11, 255-258. Available online at:http://www3.interscience.wiley.com/journal/10008576/abstract?CRETRY=1&SRETRY=0[Last accessed (21 June 2009)] Cherniack, N.S. (2003). Alternative medicine for the eldery. : Springer. Hahn, S.B., Jo, Y.Y., Kyung, Y.Y., & Song. W. J. (1997). 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United States: Oxford University Press. REFERENCES FURTHER WORK • This study can be extended by carrying out the following: • Screen a broader spectrum of Plant and Animal-based preparations. • Use Animal and Plant based preparations that are used to treat nervous disorders, due to their possible interactions with the Daphnia sp. nerve cells called the cardiac ganglion that controls the heart rate of the organism. • Identify and isolate active compounds that are responsible for observed effects. ACKNOWLEDGEMENTS Our group would like to sincerely thank the following people for their guidance and constant help in this project: our mentor, Mrs. Har Hui Peng, for her invaluable advice and constant guidance and encouragement throughout the course of our project and the lab staff of HCI, especially Mdm Lim CF, for technical support.