The simultaneous quantification of antibacterial compounds against fish pathogens in combined extract of Angelica gigas and Artemisia iwayomogi using LC-MS/MS

In the search for antibacterial alternatives against fish pathogenic bacteria from safe and efficient medicinal plants [1], we found that a combined 50% ethanolic extract of A. gigas Nakai and A. iwayomogi Kitamura showed antibacterial activity against the fish pathogens, Streptococcus iniae and Edwardsiella tarda. Antibacterial activity-guided fractionation for the combined extract yielded the active methylene chloride (MC) fraction with minimum inhibitory concentrations (MICs) of 625, 625 and 2500 µg/ml against S. iniae, Vibrio anguillarum and E. tarda in microdilution method, respectively. From further isolations for the MC fraction, five compounds were isolated and identified as decursinol angelate (1) and decursin (2), xanthotoxin (3), demethylsuberosin (4) and 2,4-dihydroxy-6-methoxyacetophenone (5) through spectroscopic analyses such as NMR and MS [2 – 5]. Among five compounds, decursinol angelate (1) and decursin (2) showed the highest antibacterial activities showing MICs of 62.5 and 250 µg/ml against S. iniae, and MICs of 62.5 and 125 µg/ml against V. anguillarum, respectively. Xanthotoxin (3), demethylsuberosin (4) and 2,4-dihydroxy-6-methoxyacetophenone (5) were also found to be active with MICs of 500 – 1000 µg/ml for S. iniae and V. anguillarum. Therefore, contents of the most active compounds, decursinol angelate and decursin (1,2) in the combined extract were investigated using LC-MS/MS in a mode of simultaneous quantification. The average contents of decursinol angelate (1) and decursin (2) were determined as 3.68 and 6.14%, respectively. Standard curves showed good linearity (r 2 > 0.9999) within test ranges. The established method showed good precision, accuracy and specificity with intra- and inter-day variations of < 2% and average recoveries of 90.13 – 108.57%. These results suggested that the combined extract and its active compounds may be used as antibacterial alternatives for fish bacterial diseases in aquacultures.