Antioxidative and anti-inflammatory Activities of the Active Compounds from the Fern Cyclosorus terminans

Abstract

In this study, the three pure coumarin derivatives, interruptins A, B and C were isolated from Cyclosorus terminans with 0.20-0.84% w/w of its crude extract. As a result, interruptins A and B not only potentially scavenged intracellular reactive oxygen species (ROS) in human dermal fibroblast and human epidermal keratinocyte with % ROS scavenging as high as 76.98 %, but also induced mRNA and protein expression of antioxidant enzymes, including superoxide dismutase 1 and 2, catalase, and glutathione peroxidase. Likewise, these two coumarin derivatives remarkably suppressed ROS levels in human skin cells under ultraviolet A and B irradiation with % ROS scavenging up to 81.52%. Among the three tested isolated interruptins A-C, interruptin A exhibited the most effective DPPH proton radical scavenging capacity (IC50 = 21.79 μM) and acted as the strongest electron transferring compound to reduce ferric to ferrous ion (antioxidant value of 682.45±16.31 mmol/L ascorbic acid/mol interruptin) as well as strongly inhibited and killed a skin pathogen Propionibacterium acnes (MIC/MBC = 1.95/7.81 μg/mL). While both interruptins A and B revealed anti- inflammatory activity, interruptin B acted as the greatest anti-inflammatory agent by quenching NO radical and inhibiting NO production in LPS-induced macrophages with IC50 values as low as 67.68 μM and 0.81 μM, respectively. The molecular mechanism involved in suppression of inducible nitric oxide synthase (iNOS) gene expression which possibly due to activation of peroxisome proliferation activated receptor-y gene expression. Moreover, low concentrations of interruptins A-C presented moderate wound healing activity by accelerating human skin cell migration. Furthermore, interruptins-rich C. terminans extract developed by simple precipitation not only revealed approximately 2-folds interruptin content higher than its initial extract but also performed powerful ROS scavenging activity in human skin cells and strongly suppressed NO release from murine macrophages with 2.37 folds better than original crude extract. The prepared interruptins-rich C. terminans extract that was stored at room temperature with light protection displayed good stability over 3-months; however, extract stored in cold condition provided better stability. Finally, the high performance liquid chromatography (HPLC) with photodiode array detector was successfully validated for simultaneous analysis of interruptins A, B and C. The HPLC condition was achieved by using a reversed-phase C18 analytical column, methanol/1% aqueous acetic acid (85:15, v/v), 1 mL/min flow rate. According to the International Conference of Harmonization (ICH) guidelines, the method was validated on the basis of these parameters: linearity (R2 ≥ 0.999), range (typically 6.25-200 μg/mL), specificity, accuracy (100±10%), precision (intra-day <1%, inter-day <2%), limit of detection (LOD) and limit of quantitation (LOQ). Therefore, this model is suitable as a standard method for simultaneous examination of interruptins A, B and C in a single HPLC run. As a result, it was encouraged that the natural occurring coumarin derivatives, interruptins A and B from an edible fern C. terminans and interruptins-rich extract could be further developed as therapeutic agents or cosmeceutical products for antioxidative or anti-photooxidative, anti-inflammatory and antibacterial benefits. The validated analysis method could also provide an application for active ingredient analysis of C. terminans extract for further health product development.