Abstract
Facial wrinkles are the predominant phenotypes of skin aging. To date, one of the most effective ways to improve wrinkles is botulinum toxin type A (BoNT/A) injection, which inhibits muscle contractions by reducing acetylcholine release from neurons. However, since BoNT/A is a hazardous neurotoxin, the injection can only be performed by medical doctors and the procedure is only possible through invasive injection, causing inconveniences such as pain. To overcome these inconveniences, we tried to find a way to reduce wrinkles non-invasively via mechanisms similar to BoNT/A. We first designed in vitro assays to test BoNT/A-like muscle contraction inhibition in two different model systems. By using the assays, we identified Zanthoxylum piperitum (Z. piperitum) fruit extract as a BoNT-like reagent (27.7% decrease of muscle contraction rates by 1000 ppm of Z. piperitum extract treatment). Next, we determined mechanisms of how Z. piperitum extract decreases muscle contraction rates and found that the extract treatment inhibits electrical signal transduction in neurons. We also showed that among known components of Z. piperitum extract, quercitrin is responsible for muscle contraction inhibition. We further identified that Z. piperitum extract has synergistic effects with acetyl hexapeptide-8 and BoNT/A light chain, which are well-known BoNT-like peptides. Finally, we showed that topical treatment of the Z. piperitum extract indeed decreases facial wrinkles and treatment of Z. piperitum extract with acetyl hexapeptide-8 has a tendency to improve wrinkles synergistically (14.5% improvement on average). The synergistic effect of the combination is expected to improve wrinkles effectively by implementing the BoNT/A mechanisms in a non-invasive way.
Highlights
A previous study showed that Botulinum toxin type A (BoNT/A) treatment could paralyze C. elegans[8], indicating that the pathways required for the BoNT/A-mediated muscle contraction inhibition are conserved in C. elegans
After we examined the muscle contraction-inhibitory effects and wrinkle-reducing effects of Z. piperitum as well as acetyl hexapeptide-8, we questioned whether these cosmetic ingredients can penetrate the skin barrier to reach sensory neurons
Among topically applied acetyl hexapeptide-8, 6.5% of samples were absorbed in the stratum corneum (SC) layer, and total 7.8% of the samples were absorbed in the epidermis and dermis layers including SC (Figure S3)
Summary
To identify compounds that inhibit muscle contraction, we first designed muscle contraction assays in two different model systems, a co-cultured cell system, and a Caenorhabditis elegans (C. elegans) system. One thousand partsper-million (ppm) of Z. piperitum extract reduced 30% of muscle contraction rates, which is similar to the effect of 10% of acetyl hexapeptide-8 treatment. This result suggests that Z. piperitum extract can act as a BoNT-like compound. We confirmed the synergistic effects of Z. piperitum extract and acetyl hexapeptide-8 on muscle contractions in different concentrations (Figure S1A) as well as in a different system (cell culture system, Figure S1B) These results suggest that Z. piperitum extract and other BoNT-like chemicals such as acetyl hexapeptide-8 and BoNT/A LC might synergistically reduce wrinkles as well. These data indicate that Z. piperitum extract treatment can improve wrinkles by inhibiting facial expression muscle contraction, and the combination of Z. piperitum extract and acetyl hexapeptide-8 might have synergistic effects on wrinkles
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