AN INVESTIGATION ON THE ANTI-CHOLINERGIC EFFECTS OF RADIX SAUSSUREA UPON RABBIT ILEUM IN AN ISOLATED...
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Transcript of AN INVESTIGATION ON THE ANTI-CHOLINERGIC EFFECTS OF RADIX SAUSSUREA UPON RABBIT ILEUM IN AN ISOLATED...
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
Outline of Presentation
Introduction Aims Materials and Methods Results Discussion Conclusion Future Work References
Introduction
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.
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.
Radix saussurea
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.
Agonism and Antagonism
Agonist-antagonist relationship can be divided into competitive and non-competitive antagonism.
Competitive – further divided into reversible and irreversible competitive antagonism.
Aims
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.
Materials & Methods
Summary of extraction methods
Radix saussurea powder
First
extraction
37°C H2O°C H2O 100°C H2O 100°C H2O 10% MeOH10% MeOH
SecondSecond
ExtractionExtraction
100°C H2O 100°C H2O 10% MeOH 10% MeOH 37°C H2O 37°C H2O
Third Third
ExtractionExtraction
10% MeOH 10% MeOH 100°C H2O 100°C H2O
Radix saussureae powder
30g 30g
+150ml dH22O @ 37°CO @ 37°C ++150ml dH2O @ 100°C2O @ 100°C
37°C 37°C H22O bath @ 30 minsO bath @ 30 mins 100°C 100°C H2O bath @ 30 mins2O bath @ 30 mins
Cooling down
Orbital shaking overnight @ 150rpm
Centrifugation @ 4000rpm, 5 minutes, 25°C°C
FiltrationFiltration
Supernatant 37°C extraction ppte°C extraction ppte 100°C extraction ppte100°C extraction ppte
ExtractExtract ExtractExtract
Overnight freezing @ -70°C Overnight freezing @ -70°C w/100°C H2Ow/100°C H2O w/10% MeOHw/10% MeOH
Freeze-drying Freeze-drying Supernatant ppte Supernatant ppte
ExtractExtract w/10% MeOHw/10% MeOH
Rotary evaporationRotary evaporation
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 ppteSupernatant ppte
Overnight freezing @ -70°C Extract w/100°C H2O
Freeze-drying
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
Power Lab and Tissue Bath setup
Results
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.
Mean anti-cholinergic effect of aqueous and methanolic extracts of Radix saussurea on acetylcholine-induced contractions of the rabbit ileum
0
1
2
3
4
5
6
7
8
9
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
log Ach concentration (M)
Mu
scle
co
ntr
acti
on
(g
mte
nsi
on
)
Acetylcholine
37°C H2O
100°C H2O
Methanol
37°C + 100°C H2O
100°C H2O + Methanol
Methanol + 37°C H2O
37°C + 100°C H2O +Methanol
Methanol + 37°C + 100°CH2O
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.
Extraction method P-value
37°C H2O 2.56 x 10-5
100°C H2O 1.81 x 10-7
10% MeOH 1.35 x 10-6
37°C H2O + 100°C H2O 3.57 x 10-7
100°C H2O + 10% MeOH 1.08 x 10-7
10% MeOH + 37°C H2O 1.04 x 10-6
37°C H2O + 100°C H2O + 10% MeOH 3.6195 x 10-7
10% MeOH + 37°C H2O + 100°C H2O 5.1762 x 10-7
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.
Discussion
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 )
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.
Agonist-antagonist relationship
Radix saussurea seems to show irreversible competitive antagonism.
The log concentration-response curve shifted to the right.
There was a decrease in the maximal response.
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
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.
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.
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).
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 Research 27: 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 Pharmacology 398: 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’, Tetrahedron 56: 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 Chemistry 11: 709 – 715.
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.