New approach to the characterization and quantification of Antrodia cinnamomea benzenoid components utilizing HPLC-PDA, qNMR and HPLC-tandem MS: Comparing the wild fruiting bodies and its artificial cultivated commercial products

Antrodia cinnamomea (AC) is one of the Far East's treasured medicinal mushrooms and a major ingredient in different nutritional supplements targeting several serious ailments. Developing a sensitive simple quality control protocol for AC and its products is an unmet goal which we target in the current investigation through detecting its biomarkers. Among the AC components, benzenoid derivatives are considered therapeutically attractive due their anti-inflammatory and cytotoxic activities. We proposed a convenient method for concentrating the benzenoid-rich fraction (FNH), from AC wild fruiting bodies ethanolic extract (EEAC). Three benzenoids, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (1), antrocamphin A (2) and 4,7-dimethoxy-5-methyl-6-(3-methylbut-3-en-1-ynyl)-1,3-benzodioxole (3) were purified and their structures were elucidated. Different wild AC samples and the separated benzenoids (1-3) exhibited anti-inflammatory activity through inhibiting superoxide anion generation and elastase release by human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B (CB). Compound 1 exhibited the most potent activity against superoxide anion generation (IC50 5.08μg/ml) while compounds 2 and 3 were less active (IC50>10μg/ml) implying the advantageous use of 1-3 as biomarkers for AC.HPLC-tandem MS analytical protocol was developed for the quantification of the three major benzenoids from different AC commercial products. The results showed that compound 1 is a unique characteristic biomarker for AC quality control. The concentrations of compounds 2 and 3 were higher in certain sources of AC fruiting bodies and were absent in mycelia, suggesting their usefulness as biomarkers for quality control of fruiting bodies products. The HPLC-tandem MS results were compared to the results obtained by quantitative NMR.