TENG KUEN WIN MONASH UNIVERSITY SUPERVISOR: DR. OLIVIA TEE

1. AN INVESTIGATION ON THE ANTI-CHOLINERGIC EFFECTS OF RADIX SAUSSUREA UPON RABBIT ILEUM IN AN ISOLATED TISSUE BATH TENG KUEN WIN MONASH UNIVERSITY SUPERVISOR: DR. OLIVIA TEE

2. Outline of Presentation • Introduction • Aims • Materials and Methods • Results • Discussion • Conclusion • Future Work • References

3. Introduction

4. Traditional Chinese Medicine and Po Chai pills • Po chai pills – treat minor diarrhoea, stomach ache, over-eating and other similar symptoms. • First introduced a century ago in China, and is still frequently used today. • However - lack of scientific evidence which indicates how this TCM works. • Thus, would be good to know how it interacts with the body to produce its effects.

5. Radix saussurea • 14 different herbs make up the Po Chai pill and contribute to its effects. • Radix saussurea is one of herbs found in the Po Chai pill and refers to the roots of the plant Saussureae lappa Clarke (木香). • Research has shown it’s use as a stomachic, an anti-helmintic, anti-viral and acts as a smooth muscle relaxant. • Main constituents of R. saussurea are costunolide, dehydrocostus lactone, alkaloid saussurine and inulin.

6. Radix saussurea

7. Agonist-antagonist relationship of ACh and Radix saussurea • Acetylcholine – acts as agonist upon Mз-receptor to cause contraction of the smooth muscle. • Radix saussurea – acts as antagonist and prevents binding of ACh to the receptor site.

8. Agonism and Antagonism • Agonist-antagonist relationship can be divided into competitive and non-competitive antagonism. • Competitive – further divided into reversible and irreversible competitive antagonism.

10. Aims

11. Aims • Investigate the anti-cholinergic effects of Radix saussurea upon rabbit ileum in an isolated tissue bath. • To establish an agonist-antagonist relationship between acetylcholine and Radix saussurea. • Determine if different extraction methods would yield varying levels of anti-cholinergic activity upon ileal muscle contraction.

12. Materials & Methods

13. Summary of extraction methods Radix saussurea powder First extraction 37°C H2O 100°C H2O 10% MeOH Second Extraction 100°C H2O 10% MeOH 37°C H2O Third Extraction 10% MeOH 100°C H2O

14. Radix saussureae powder 30g 30g +150ml dH2O @ 37°C +150ml dH2O @ 100°C 37°C H2O bath @ 30 mins 100°C H2O bath @ 30 mins Cooling down Orbital shaking overnight @ 150rpm Centrifugation @ 4000rpm, 5 minutes, 25°C Filtration Supernatant 37°C extraction ppte100°C extraction ppte ExtractExtract Overnight freezing @ -70°C w/100°C H2Ow/10% MeOH Freeze-drying Supernatant ppte Extractw/10% MeOH Rotary evaporation

15. 30g Radix saussurea powder +150ml 10% MeOH Orbital shaking overnight @ 150rpm Centrifugation @ 4000rpm, 25°C, 5 minutes Filtration Supernatant Precipitate Extract Rotary evaporation w/37°C H2O Supernatant ppte Overnight freezing @ -70°C Extractw/100°C H2O Freeze-drying

16. Animals • New Zealand male white rabbits • Weighing 2.7 – 3.3kg each • Kept at temperature of around 23 - 27°C with sufficient food and water • Starved a day before experiment, but with unlimited access to water

17. Power Lab and Tissue Bath setup

18. Results

19. 6 experiments were carried out to determine the anti-cholinergic effects of Radix saussurea upon the rabbit ileum. • Log concentration-response curves were obtained with acetylcholine in different extractions. • Data analysis and statistical studies were performed on the mean contractile responses obtained.

20. Mean anti-cholinergic effect of aqueous and methanolic extracts of Radix saussurea on acetylcholine-induced contractions of the rabbit ileum

21. Data Analysis & Statistical Studies • T-test: to determine if each extraction method has significant anti-cholinergic effect upon acetylcholine-induced contractions of the ileal muscle. • ANOVA: for the investigation of differences between the various extraction methods used.

22. T-test: Paired Two Samples for Means Both the results from the graph and the table indicate that Radix saussurea has anti-cholinergic activity upon the ileum.

23. Discussion

24. Determining the extraction method which gives the most significant anti-cholinergic effect • All p-values obtained were ≤ 0.05; thus, results can be considered to be significant. • Extract with the highest degree of anti-cholinergic effect  100°C H2O extract + 10% MeOH (p-value = 1.08 x 10-7 ) • Extract with the lowest degree of anti-cholinergic effect  37°C H2O extract (p-value = 2.56 x 10-5 )

25. Active components extracted with aqueous means within Radix saussurea have a more anti-cholinergic effect than active compounds extracted with methanolic means. • An increase in the number of extractions seems to yield a larger number of anti-cholinergic components.

26. Agonist-antagonist relationship • Radix saussureaseems to show irreversible competitive antagonism. • The log concentration-response curve shifted to the right. • There was a decrease in the maximal response.

27. Experimental limitations and shortcomings • Log concentration-response curve obtained for acetylcholine only is not always the highest; in some cases, the herbal extracts yielded a higher contractile response. • Possible explanations - condition of the ileum (freshness, time period) - contamination during extraction - volume of agonist added into the tissue bath

28. Health and physiology of the rabbit should be monitored regularly it should be ensured that a sufficient amount of food and water is available at all times.

29. Conclusion • Radix saussurea displays significant anti-cholinergic effect upon ileal muscle contractions. • Extraction method that yielded the most significant anti-cholinergic effect = 100°C H2O + 10% MeOH. • Agonist-antagonist relationship of Radix saussurea and acetylcholine is that of irreversible competitive antagonism.

30. Future work • Active components extracted from Radix saussurea can be analysed using HPLC to determine the pharmacological activity involved. • Research can be carried out on other receptors involved in contractile responses of the ileum (histamine and 5-HT receptors).

31. References • Bose, BC, Saifi, AQ, Vijayvargiya, R & Sharma, SK 1961, ‘Some Aspects of Phytochemical and Pharmalogical Study of Saussurea lappa Clarke’, Journal of Pharmaceutical Sciences, 5: 679 – 681. • Chen, HC, Chou, CK, Lee, SD, Wang, JC & Yeh, SF 1995, ‘Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells’, Antiviral Research27: 99 – 109. • Cho, JY, Baik, KU, Jung, JH & Park, MH 2000, ‘In vitro anti-inflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa’, European Journal of Pharmacology398: 399 – 407. • Eglen, RM, Hedge, SS & Watson, N 1996, ‘Muscarinic receptor subtypes and smooth muscle function’, Pharmacological Reviews 48: 531 – 565. • Health Sciences University of Hokkaido 2006, http://www.hoku-iryo-u.ac.jp/~yakusou/hyouhonen/mokkou.htm • Matsuda, H, Kageura, T, Inoue, Y, Morikawa, T & Yoshikawa, M 2000, ‘Absolute Stereostructures and Syntheses of Saussureamines A, B, C, D and E, Amino Acid-Sesquiterpene Conjugates with Gastroprotective Effect, from the Roots of Saussurea lappa’, Tetrahedron56: 7763 – 7777. • Matsuda, H, Toguchida, I, Ninomiya, K, Kageura, T, Morikawa, T & Yoshikawa, M 2003, ‘Effects of Sesquiterpenes and Amino Acid – Sesquiterpene Conjugates from the Roots of Saussurea lappa on Inducible Nitric Oxide Synthase and Heat Shock Protein in Lipopolysaccharide-Activated Macrophages’, Bioorganic & Medicinal Chemistry11: 709 – 715.

32. Monash University Malaysia 2006, PHA3801 – Pharmacology Practical Guide, School of Arts and Sciences, Malaysia. • Montgomery, DC 2001, Design and Analysis of Experiments, 5th Edition, John Wiley & Sons Inc., New York. • Neal, MJ 1987, Medical Pharmacology at a Glance, 1st Edition, Blackwell Scientific Publications, U.K. • Po Chai Pills 2006, http://www.pochaipills.com/pochaipills/index.shtml Date accessed: December 15 2006. • Rang, HP, Dale, MM, Ritter, JM, & Moore, PK 2003, Pharmacology, 5th Edition, Churchill Livingstone, U.K.

33. Thank You!