Chemical characterization and biological activities of Miang extracts

Abstract

The study focused on initial investigation of fermentation impact on polyphenol contents, antioxidant and anti-inflammatory activities of Miang extracts, an ethnic fermented tea product of northern Thailand. The acetone (80%) extracts of Miang samples fermented by a nonfilamentous fungi-based process (NFP) and filamentous fungi-based process (FFP) had elevated levels of total polyphenols, total tannins and condensed tannins compared to young and mature tea leaves. The antioxidant studies also showed better the half-maximal inhibitory concentration (IC50) values for fermented leaves in both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity assays as well as improved ferric reducing antioxidant power (FRAP) compared to fresh young and mature tea leaves. Extracts of NFP and FFP samples at concentrations of 50 and 100 ppm showed better protective effects against hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species (ROS) production in HT-29 colorectal cells without exerting cytotoxicity. Additionally, lipopolysaccharide (LPS)-induced production of nitric oxide (a proinflammatory mediator as well as a reactive nitrogen species) was also inhibited by these fermented Miang extracts with an IC50 values of 17.15 µg/mL (NFP), 20.17 µg/mL (FFP), 33.96 µg/mL (young tea leaves), and 31.33 µg/mL (mature tea leaves). Therefore, both NFP-Miang and FFP-Miang showed the potential to be targeted as natural bioactive functional ingredients with preventive properties against free radical and inflammatory-mediated diseases. The ability of extracts to inhibit gastrointestinal pathogens, ameliorate oxidative stress associated complications and inflammation, is germane to potential and probable prevention and/or curation of certain cancers including colon or colorectal. Foodborne pathogens are considered as a major public health concern spanning through several decades. An investigation was done on the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica TISTR 292 and Streptococcus mutans DMST 18777. The potential of polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against Streptococcus mutans which showed bacteriostatic effects at 0.29 and 0.72 mg/mL for nonfilamentous fungi- (NFP) and filamentous-fungi-processed Miang (FFP) respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially in NFP and FFP. Characterization of the PVPP-supernatants using high performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, crystal violet-staining also showed better IC50 values for NFP (4.30 ± 0.66 mg/mL) and FFP (12.73 ± 0.11 mg/mL) against in vitro Streptococcus mutans biofilms. Homology modelling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products. NFP- and FFP-fermented Miang extracts were also able to ameliorate dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in an experimental mice model achieved through reducing colon inflammation as well as ameliorating UC associated clinical symptoms including colon length recovery, body weight recovery, inhibiting spleen enlargement, reducing the incidence of diarrhea and an overall decrease in UC disease activity index. The positive effects of both Miang extracts on intestinal relief or recovery in mouse model indicate the possibility of using Miang extract to strengthen or prevent colon cancer. This will be beneficial to the prevention or treatment of colon cancer for the medical community in the future.