Bioactivity-guided isolation of anti-inflammatory constituents from Angelica decursiva

Abstract

Angelica, a circumboreal genus comprises of more than 60 different species, is by far the largest genus of Apeaceae in the Korean flora, and many species of this genus have long been used in traditional medicine for the treatment of many diseases. Because of the high therapeutic value of the traditionally used Angelica species, extensive research has been carried out on different species of this genus which reported wide range of bioactivities including anti-inflammatory, anti-oxidant, anti-diabetic, anticancer, neuroprotective effects. Among them, Angelica decursiva has been long used in Korean traditional medicine as an antitussive, analgesic, antipyretic, and cough remedy. In a recent study, the potential antioxidant and anti-inflammatory activities of methanol (MeOH) extract of A. decursiva and its different solvent soluble fractions have been reported. Among the fractions, the ethyl acetate (EtOAc) fraction showed strong antioxidant and anti-inflammatory activities, while the dichloromethane (CH2Cl2) fraction-derived 90% MeOH soluble fraction showed the most promising anti-inflammatory activity. Although the 90% MeOH soluble fraction was found as the most active anti-inflammatory fraction, compounds responsible for anti-inflammatory activity were not identified yet. Therefore, the present study was designed to isolate active components from 90% MeOH soluble fraction and to evaluate their anti-inflammatory activity via inhibition of nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) production as well as iNOS and COX-2 expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Repeated column chromatography of the 90% MeOH soluble fraction yielded 9 coumarins, including edulisin II (1) decursidin (2), Pd-C-III (3), 3'(S)-angeloyloxy-4'(R)-hydroxy-3',4'-dihydroxanthyletin (4),Pd-C-I (5), Pd-C-II (6), (+)-trans-decursidinol (7), umbelliferone (8), and nodakenetin (9). Among them, compound 1 was isolated for the first time from A. decursiva, while compound 4 was isolated as a new compound from natural sources. Since nodakenetin and umbelliferone showed poor NO production inhibitory activity in previous study, we have selected compounds 1 ~ 7 in the present study. Among the tested compounds, compound 1 exhibited the highest NO production inhibitory activity in a dose-dependent manner in LPS-induced NO production in RAW 264.7 cells with an IC50 value of 2.50 ± 0.16 μM. Compound 2 also showed very strong NO production inhibitory effect in LPS-stimulated RAW 264.7 cells with an IC50 value of 4.08 ± 0.09 μM, which is nearly similar to that of compound 1. In addition, compounds 3 ~ 5 showed moderate NO production inhibitory activity compared to compounds 1 and 2 with IC50 values of 15.60 ± 0.80, 26.00 ± 1.00, and 31.83 ± 0.36 μM, respectively. On the other hand, compound 6 showed weaker NO production inhibitory activity compared to compounds 1 and 2 with an IC50 value of 62.70 ± 1.86 μM. In contrast, compound 7 did not exhibit any suppressive effect on LPS-stimulated RAW 264.7 cells at the concentrations tested. Therefore, considering the NO production inhibitory activity of tested coumarins (compounds 1 ~ 7), it can be speculated that esterification of –OH at 3' or 4' position of compound 7 with senecioyl or angeloyl or acetyl group is essential for exhibiting NO production inhibitory activity by these coumarins derivatives, and the position and number of senecioyl/angeoloyl/acetyl group on these coumarins largely affects their potency. All six active coumarins (compounds 1 ~ 6) also inhibited TNF-α production and iNOS protein expression, while compounds 1 ~ 4 inhibited COX-2 protein expression in LPS-stimulated RAW 264.7 cells. These results suggest that coumarins isolated from A. decursiva might be used as potential leads for the development of therapeutic remedies for inflammation associated disorders. However, further research is needed to clarify the mechanisms of action behind this anti-inflammatory activity.