Patent Application
20080069912
The whole dry plant comes from South America or more generally from tropical countries. This plant is ground until a powdered is obtained.
The extraction of the ground plant is performed in a mixture of ethanol at 96.2° H2O (80/20); volume/volume at ambient temperature subjected to magnetic stirring and protected from light, for 6 hours.
The extract is then filtered through a nylon filter then through a cellulose membrane (to 0.22 microns). In example 2, it is then lyophilised to be used diluted to 50% in maltodextrine.
1. Objective of the Study
To assess the ability of purified spilanthol and an Acmella oleracea extract to produce a reversible blockage of muscular contractions.
2. General Methodology
Nerve-Muscle Model
Nerve-muscle co-culture is a culture model which is used to recreate human striated muscle cell innervation with spinal cord and spinal ganglion explants from rat embryos. After 21 days of culture, the muscle fibres formed contract spontaneously.
The nerve-muscle co-culture model is a model suited to studying the influence of a substance on muscle contraction frequency, as well as to studying the recuperation of contractile activity after blockage of muscle contractions by a substance.
Carisprodol at 1 mM is used as a positive control for reversible blockage of muscle contractions.
Measurement of the Frequency of Contractions
For each selected culture well, a muscle fibre showing regular contractions is referenced.
Using automated counting software, the number of contractions is counted for 30 seconds for each measurement period: before incubation (pre-incubation frequency), during incubation and during the contractile activity recuperation phase after elimination of the substance.
Interpretation of the Results
Each measure was taken in triplicate (in 3 different wells) and an activity is considered to be significant when at least 2 fibres out of 3 show the same effect according to the following modulation range:
When the frequency of contractions is greater than 120% of the pre-incubation frequency before adding the substance, we speak of an increase in the frequency of contractions, indicated by +. When the frequency becomes too high to be measurable, we speak of vibration, indicated by Vib.
When the frequency of contractions is between 80% and 120% compared to the pre-incubation frequency before adding the substance, it is not modified and is indicated by 0.
When the frequency of contractions is less than 80% of the pre-incubation frequency before adding the substance, we speak of a decrease in the frequency of contractions, indicated by—or block (blockage) if it is 0%.
We speak of complete recuperation of contractile activity after blockage when at least 2 fibres out of 3 return to a frequency of contraction greater than or equal to 80% of the pre-incubation frequency of contraction, indicated by +.
We speak of incomplete recuperation of contractile activity after blockage when at least 2 fibres out of 3 return to a frequency of contraction between 10 and 80% of the pre-incubation frequency of contraction, indicated by +/−.
3. Substances Studied
Nature and origin of the substance:
- Lyophilised extract of the aerial portions (containing flower buds) of Acmella oleracea diluted to 50% in maltodextrine.
Spilanthol purified to 97% from an Acmella oleracea extract.
4. Study Process
a/ Pure Spilanthol
The frequency of contraction is determined after 5 minutes, 1 hour and 6 hours of incubation with the substance. At 6 hours, the substance is eliminated and recuperation of contractile activity is studied 1 hour and 24 hours later.
b/ Acmella oleracea Extract
The frequency of contraction is determined (at 5 minutes, 1 hour and 6 hours) until a blockage of contractions is obtained with the substance. Once blockage is achieved, the substance is eliminated and recuperation of contractile activity is studied at 1 hour, 4 hours and 24 hours.
5. Results
Pure Spilanthol
At the concentrations (40×110% and 160×10−5%), pure spilanthol blocks muscle contractions after 5 minutes of incubation. The blockage is maintained until 6 hours and the fibres remain blocked for 24 hours after elimination of the substance.
Acmella oleracea Extract
At a concentration of 600×10−5%, the extract blocks the frequency of contraction of muscle fibres after 6 hours of incubation. After washout of the cultures, the muscle fibres totally recuperate their contractile activity in 1 hour.
At concentrations of 1,200×10−5% and 2,400×10−5%, the extract blocks the frequency of contraction of muscle fibres after 5 minutes of incubation. After washout of the cultures, the muscle fibres totally recuperate their contractile activity in 1 hour.
6. Conclusion
Under the conditions of the study, the Acmella oleracea extract and the spilanthol compound lead to a blockage of muscle contractions. Recuperation of contractile activity is observed in presence of the plant extract but not observed in presence of spilanthol. This difference should probably be attributed to a difference in the protocol since the nerve-muscle system was only incubated for 5 minutes in presence of the extract compared to 6 hours in presence of pure spilanthol.
The anti-wrinkle effect of botulinum toxin lies in its ability to inhibit subcutaneous muscle contractions considered to be responsible for expression lines (deep wrinkles); the substances tested, given their ability to inhibit contractile activity (or botox-like effect), have the same anti-wrinkle potential as botulinum toxin.
Anti-Wrinkle Day Cream
Make-Up Foundation
O/W Emulsion
W/O Emulsion
Microemulsion
W/O/W Multiple Emulsion
| Number | Date | Country | Kind |
|---|---|---|---|
| 0450093 | Jan 2004 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR05/50005 | 1/4/2005 | WO | 00 | 6/20/2006 |
