Phytochemical analysis of propolis extracts from stingless bee tetragonula fuscobalteata and in vitro wound healing evaluation on human gingival fibroblast

Background : Propolis is one of important stingless bee’s product rich in phytochemicals compounds. But study about stingless bee propolis in Thailand was so few and lack of study on dentistry-related topic as well as oral wound healing. So this study aim to study stingless bee propolis from native specie in Thailand on its properties and activities in oral wound healing. Objective : The aim of this study was to describe chemical composition of Tetragonula fuscobalteata propolis from Chiang Mai province of Thailand, estimation of flavonoids and phenolic contents in its extract fractions, their antioxidant activities, and effects of each fraction toward cell proliferation and migration of human primary gingigval fibroblasts. Materials and Methods : Crude propolis extract was extracted with 95% Ethanol repeatly five times, pooled together and partitioned into 3 fractions based on polarity (hexane, ethyl acetate, aqueous fractions). Crude propolis extract and three propolisfractions were analyzed by gas chromatography – mass spectrometry (GC – MS) to predict compounds exist in each fractions. Each fractions were studied on estimation of total flavonoids content (TFC) with aluminum chloride assay, total phenolic content (TPC) with Folin-Ciocalteu reagent assay, and antioxidant activities with DPPH assay and FRAP assays. Biological properties of propolis fraction on primary human gingival fibroblasts, cell proliferation was analyzed with MTT cell viability assay, and migration was analyzed with scratch assay and cell migration was analyzed by blind well spontaneous migration assay. Results and Discussion : The propolis was extracted and partitioned to get hexane fraction, ethyl acetate fraction and aqueous fraction (38.88%, 19.84%, and 1.42% of pre-extracted weight). Qualitative thin layer chromatography revealed hexane fraction and ethyl acetate fraction shared most of constituents at different concentration. However, majority of aqueous fraction’s constituents were not found in other fractions. Total flavonoid assay estimated aqueous fraction to have the highest estimated flavonoids followed by hexane fraction. Total phenolic assay estimated ethyl acetate fraction to have the highest estimated phenolic compounds followed by hexane fraction. Estimation of antioxidant activities revealed hexane fraction to have the highestantioxidative activities followed by ethyl acetate fraction on both DPPH assay and FRAP assay. Results of antioxidant properties were confirmed by outcomes from biological assays as hexane fraction was the most efficient in activation of cell proliferation and migration of human primary gingival fibroblast following by ethyl acetate fraction. Gas chromatography – mass spectrometry result had also revealed composition of hexane and ethyl acetate fractions were phenolic lipids, steroid triterpenoids, pentacyclic triterpenoids, and carotenoids at different concentration. Aqueous fraction composed with variety of sugar alcohols with no flavonoid found on detectable level. Gas chromatography – mass spectrometry revealed ethyl acetate fraction had the highest level of detectable flavonoids from among three fractions, which contradicted to result from total flavonoid estimation assay. Conclusion : Thai T. fuscobalteata propolis was extracted, analyzed, and investigated on its oral wound healing activity for the first time. It contained terpenoids and phenolic lipids that were also major components in its hexane and ethyl acetate fractions of extract, many of them are antioxidative compounds, which might contributed to antioxidant and wound healing activities in this study. Hexane fraction exhibited the most efficient wound healing activation among three fractions in this study, which might be explained by high concentration of terpenoids in this fraction. However, further research is needed to isolate and identify actual active compound to study its property and application. To the best of our knowledge, the hexane fraction of Thai T. fuscobalteata propolis promotes in vitro wound healing activity. This is the strong data for applying to the in vivo study in the future.