Hyal-1 inhibitors from the leaves of Phyllanthus muellerianus (Kuntze) Excell.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves and twigs from Phyllanthus muellerianus Kuntze Excell are known to exert anti-inflammatory and antipyretic properties as well as wound healing properties. During a wide screening for human hyaluronidase-1 inhibitors from natural sources leaf extracts from P. muellerianus turned out to show basic anti-hyaluronidase activity. A detailed investigation of this effect should rationalize the potential anti-inflammatory activity of the extract for improved wound healing. AIM OF THE STUDY: The following study aimed to characterize the anti-Hyal-1 activity of the extract from P. muellerianus and to pinpoint the responsible natural products responsible for this bioactivity. MATERIALS AND METHODS: Using cell surface displayed human Hyal-1 on Escherichia coli, the activity of inhibitors was determined by the stains-all assay method. A hydroalcoholic extract PWE from P. muellerianus was subjected to bioactivity-guided fractionation. Active compounds were characterized by means of mass spectrometry and NMR. RESULTS: PWE exerts a concentration dependent inhibition of Hyal-1 with an IC50 of 80 µg/mL. Bioassay-guided fractionation revealed 13 compounds from the two most active fractions, mainly ellagitannins and flavonoid glycosides. The most activeHyal-1 inhibitor was found to be the ellagitannin chebulanin 10 (IC50 132 µM). This represents the first description of chebulanin in P. muellerianus. CONCLUSIONS: The hydroalcoholic extract of P. muellerianus, as well as several subfractions obtained during bioassay-guided fractionation showed strong activity against Hyal-1. The main activity can be correlated to the ellagitanin chebulanin. Additionally, also synergistic effects are observed, indicating that the traditional use of aqueous extracts of P. muellerianus is justified, rather than the use of the isolated tannins. The traditional use of the plant as an anti-inflammatory agent for improved wound-healing can be rationalized by the anti-Hyal-1 activities of its constituents.

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.