Isoflavonoids with inhibiting effects on human hyaluronidase-1 and norneolignan clitorienolactone B from Ononis spinosa L. root extract.

Human hyaluronidase-1 (Hyal-1) is one of the main enzymes in the homeostasis of hyaluronic acid (HA), the main polysaccharide of extracellular matrix. Development of specific Hyal-1 inhibitors might be a promising target for improved wound healing, tissue regeneration, and looking at renal function for diuresis. By using surface-displayed Hyal-1 on Escherichia coli F470 cells, HA as substrate and stains-all method for quantification of undegraded HA, the respective enzyme activity can be determined easily. Based on the traditional use of extracts from the roots from Ononis spinosa L. (Restharrow root) as a weak diuretic to achieve flushing of the urinary tract and as an adjuvant in minor urinary complaints the herbal material was selected for bioactivity guided fractionation for compounds with Hyal-1 inhibition activity. Hot water and hydroalcoholic extracts showed moderate inhibiting effects (IC50 1.36 resp. 0.73 mg/mL) while dichloromethane extract exerted an IC50 of 190 μg/mL. Bioassay guided fractionation of the dichloromethane extract yielded four isoflavonoids with anti Hyal-1 activity: onogenin 1, sativanone 2, medicarpin 3 and calycosin-D 4 with inhibition rates of 25.4, 61.2, 22.4 and 23.0%, respectively at test concentration level of 250 μM. The norneolignan clitorienolactone B 5, the first time described for the genus Ononis, was inactive. The IC50 of sativanone, the most active compound was determined with 1501 μM, which was better than that of the positive control glycyrrhizinic acid (177 μM). Thus, a possible explanation for diuretic properties of Ononis spinosa L. root extract may be postulated from the results so far obtained.