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

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