## 4 Discussion
One reason for the significant skin improvement was the difference in absorption of collagen peptides. Collagen products taken orally use specific amino acids as building blocks of human collagen, such as proline, hydroxyproline, glycine, alanine, glutamic acid, and arginine [30; 31; 32]. However, the mechanisms for the re-synthesis of amino acids that are broken by digestive enzymes have not been shown. To overcome these problems, transdermal and trans-buccal routes have been investigated [33]. We previously showedthat a buccal delivery film can directly absorb a completely formed collagen peptide (2000-3000 Da) through the human buccal tissue [13]. [PERSON] et al. demonstrated that collagen peptides, such as prolyl-hydroxyproline and hydroxyprolyl-glycine, triggered the growth of fibroblasts combined with collagen gel in blood, which were associated with improved skin conditions [34]. Therefore, the buccal delivery film shows fast and strong effects on the enhancement of skin condition. However, the results in this clinical study, especially in terms of reducing the wrinkles within 2 weeks, were not completely explained by the advantages of the buccal delivery film. Therefore, we suggest that collagen peptides absorbed through the buccal mucosa had a cytokine-related homeopathic effect, similar to Botox(r). Homeopathy is a systemic therapy that is focused on health and wholeness rather than disease [35]. When taken at a higher dose, any substance can cause symptoms; however, when taken as a small dose, it can revitalize the immune system with no cell death [35]. This effect is usually applied in immunology. One of the most representative products using homeopathy in the cosmetic field is Botox(r) [36]. However, at a high dose, collagen is known to induce arthritis [37; 38; 39]. [PERSON] et al. reported that a filler injection containing collagen led to inflammatory granulomas under the eye after 7 years [40]. Therefore, according to previous studies, collagen can induce local inflammation. IL-1, an inflammation mediator, can be produced when cells are damaged [41]. Peroxynitrite (ONOO\({}^{-}\)) use can also lead to skin inflammation, arthritis, oxidation, and vascular dilatation [42; 43; 44]. According to the results of this study, total protein, relative levels of peroxynitrite (ONOO\({}^{-}\)), and IL-1\(\alpha\) production in the supernatant of RAW 264.7 cells were increased after collagen peptide use. Additionally, similar increases were seen after LPS use, which is a well-known inflammatory inducer. Because cytokine materials, such as interleukin and TNF, are proteins, the increase in total protein after the collagen treatment could be due to the production of cytokine factors. In addition, the increased levels of IL-1\(\alpha\) and peroxynitrite could be due to some damage to the cells by the collagen peptide. Many previous studies reported that collagen could produce peroxynitrite [45; 46; 47] and interleukin-1 [48; 49]. These phenomena suggest that collagen peptide delivered via buccal film enhances the reduction in wrinkles and skin hydration owing to the slight edema and vascular dilatation from IL-1\(\alpha\) and peroxynitrite, which showed no side effects in the clinical test. The results from the cytokine experiment can be the basis for future studies. Thus, collagen enhancement via buccal film delivery followed the local and temporary actions of (i) homeopathic effects induced by weak inflammation, (ii) vascular dilatation, and (iii) enhancement of interstitial fluid hydration.