Selenium nanoparticles (SeNPs) are worthy of attention and development for nutritional supplementation due to their health benefits in both animals and humans with low toxicity, improved bioavailability, and controlled release, being greater than the Se inorganic and organic forms. Our previous study reported that Anoectochilus burmannicus extract (ABE)-synthesized SeNPs (ABE-SeNPs) exerted antioxidant and anti-inflammatory activities. Furthermore, ABE could stabilize and preserve the biological activities of SeNPs. To promote the ABE-SeNPs as supplementary and functional foods, it was necessary to carry out a safety assessment. Cytotoxicity testing showed that SeNPs and ABE-SeNPs were harmless with no killing effect on Caco2 (intestinal epithelial cells), MRC-5 (lung fibroblasts), HEK293 (kidney cells), LX-2 (hepatic stellate cells), and 3T3-L1 (adipocytes), and were not toxic to isolated human PBMCs and RBCs. Genotoxicity assessments found that SeNPs and ABE-SeNPs did not induce mutations in Salmonella typhimurium TA98 and TA100 (Ames test) as well as in Drosophila melanogaster (somatic mutation and recombination test). Noticeably, ABE-SeNPs inhibited mutation in TA98 and TA100 induced by AF-2, and in Drosophila induced by urethane, ethyl methanesulfonate, and mitomycin c, suggesting their anti-mutagenicity ability. This study provides data that support the safety and anti-genotoxicity properties of ABE-SeNPs for the further development of SeNPs-based food supplements.

BACKGROUND: Anoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions.
OBJECTIVE: The objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety.
METHODS: The ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster.
RESULTS: The data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic.
CONCLUSIONS: Our study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1β, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.

This study investigated biological activities including antioxidative stress, anti-inflammation, and anti-insulin resistance of Anoectochilus burmannicus aqueous extract (ABE). The results showed abilities of ABE to scavenging DPPH and ABTS free radicals in a dose-dependent manner. Besides, ABE significantly reduced nitric oxide (NO) production in the lipopolysaccharide (LPS)-treated RAW 264.7 via inhibition of mRNA and protein expressions of nitric oxide synthase (iNOS). The LPS-induced mRNA expressions of cyclooxygenase-2 (COX-2) and interleukin 1β (IL-1β) were suppressed by ABE. Moreover, ABE exerted anti-insulin resistance activity as it significantly improved the glucose uptake in tumor necrosis factor (TNF)-α treated 3T3-L1 adipocytes. In addition, ABE at the concentration of up to 200 μg/mL was not toxic to human peripheral blood mononuclear cells (PBMCs) and did not induce mutations. Finally, the results of our study suggest the potential use of A. burmannicus as anti-inflammatory, anti-insulin resistance agents, or food supplement for prevention of chronic diseases.