COSMETICS & PERSONAL CARE
Figure 3 - Realtime wound healing assay of 1 %, 2 % & 5 % AHE
that present multiple benefits for an overall improvement in oral health and hygiene . Our previous studies demonstrated multiple properties of Alpine Heilmoor Extract ( AHE ), including anti-inflammatory , antioxidant and skin barrier strengthening effects , for better maintenance of healthy skin . Now we have performed several in vitro and clinical studies to determine the benefits of AHE in maintaining oral health and hygiene .
Materials & methods
The anti-bacterial properties of AHE formulated at 5 % in a toothpaste , were evaluated using P . gingivalis . The colony-forming units ( CFUs ) were counted after incubation with 5 % AHE at 37 ° C for five minutes , the minutes and 24 hours . Due to the culture and growth conditions of the used test strains , the growth period was prolonged to allow CFU counting . We determined the antibacterial effects of AHE by quantifying the six bacterial species in sub-gingival pockets before and after the application of AHE using real-time polymerase chain reaction ( PCR ) on a Carpegen Perio Diagnostik . In vitro studies using HaCaT cells , normal human dermal fibroblasts , RAW cells and NIH-3T3 fibroblasts were conducted to analyse the barrier strengthening , senolytic , pro-healing , anti-inflammatory , anti-glycation and antioxidant properties of AHE .
Maintenance of the oral mucosa barrier was determined by measuring E-cadherins expression using the Western Blot technique . The anti-inflammatory effects of AHE were evaluated by the quantification of interleukin ( IL ) -1β- or lipopolysaccharide ( LPS ) -induced proinflammatory mediators ( IL-6 , IL-8 , prostaglandin E2 , IL-1β , TNF-α , monocyte chemoattractant protein 1 and matrix metallopeptidase ( MMP ) -9 . We determined the anti-glycation and antioxidant properties of AHE by measuring regulator of calcineurin 1 ( RCAN1 ), receptor for advanced glycation end-products ( RAGE ) and advanced glycation end-product ( AGE ) receptor 1 ( AGE-R1 ) gene level expression using real time quantitative PCR and by quantification of H 2
O 2
- induced ROS and LPS-induced 8-isoprostaglandin fator2α using a FluoStar Spectro-fluorometer and ELISA kits from Cayman and Biomol respectively . NO suppression was analysed using the Griess method by a sodium nitrite standard curve . Protection against senescence was determined by using a PerkinElmer Victor X5 senescence β-galactosidase ( SA β-gal ) activity assay kit . NIH-3T3 fibroblast and Incucyte 2 scratch wound assay were used to analyse the pro-healing capacities of AHE , by measurement of relative wound density . Clinical studies were performed to evaluate the benefits of AHE in improving oral health . Twenty participants completed the study , using toothpastes with 5 % AHE twice daily for 28 days . The whitening effect was evaluated with a Vita Bleachedguide 3D-Master under dermatological and dental control . Other parameters were analysed , including plaque index , gingival index and mean maximal gingiva index . The effectiveness of AHE toothpaste was also evaluated through a selfassessment questionnaire .
Anti-bacterial activity
The bacteria present in the oral cavity differ greatly between healthy and diseased subjects . Patients with periodontal disease have a greater proportion of gramnegative , proteolytic bacteria such as P . gingivalis , Tanneralla forsythia and other species in their subgingival plaques , while healthy adults mainly have gram-positive species of the Actinomyces and Streptococcus genera , and the gram-negative Veillonella . 7 P . gingivalis is highly proteolytic , with primary activity from a class of endopeptidases known as gingipains that are critical virulence factors . 8 Proteolysis actively changes the environmental conditions , notably by causing a pH increase and creating an environment more hospitable to many gram-negative anaerobes . This mediates periodontitis . 9
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